AU781382B2

AU781382B2 - Fatty-acid amide hydrolase

Info

Publication number: AU781382B2
Application number: AU13606/02A
Authority: AU
Inventors: Benjamin F. Cravatt; Norton B. Gilula; Richard A. Lerner
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 1996-11-04
Filing date: 2002-01-25
Publication date: 2005-05-19
Anticipated expiration: 2017-11-04
Also published as: AU1360602A; AU781382C

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

I

Applicant(s): THE SCRIPPS RESEARCH INSTITUTE Invention Title: FATTY-ACID AMIDE HYDROLASE The following statement is a full description of this invention, including the best method of performing it known to me/us: WO 98/20119 PCT/US97/20385 FATTY-ACID AMIDE HYDROLASE

DESCRIPTION

Technical The invention relates to an enzyme which catalyzes a hydrolytic conversion between soporific fatty acid primary amides and their corresponding fatty acids and is designated a fatty-acid amide hydrolase (FAAH), to methods for enzymatically catalyzing such conversions, and to methods for inhibiting the enzymatic catalysis of such conversions. More particularly, the invention relates to FAAH protein, in either isolated or recombinant form, and to its use and inhibition.

Statement of Government Rights :This invention was made with government support under a National Institutes of Health Shared Instrumentation grant No. 1 RR07273-01. The government has certain rights in the 2 invention.

Background Sleep is a natural, periodic behavioral state during which the body rests itself and its physiological powers are restored.

It is characterized by a loss of reactivity to the environment.

During sleep, certain physiological processes of both the body and the brain function differently than they do during alert wakefulness. Normal sleep consists of at least two quite different behavioral states: synchronized sleep, during which the electroencephalogram consists of slow waves of high amplitude, and desynchronized sleep (DS) or activated sleep characterized by rapid eye movements (REM sleep), in which the electroencephalogram pattern is characterized by waves of high frequency and low amplitude. Synchronized sleep is further characterized by slow and regular respiration, by relatively WO 98/20119 PCT/US97/20385 2 constant heart rate and blood pressure, and by a predominance of delta waves. Synchronized sleep usually consists of four stages, followed by a period of activated sleep. Each cycle lasts between 80 and 120 minutes. In contrast, desynchronized sleep is further characterized by irregular heart rate and respiration, periods of involuntary muscular jerks and movements, and a higher threshold for arousal. Periods of desynchronized sleep last from 5-20 minutes and occur at about 90 minute intervals during a normal night's sleep.

0 Sleep disorders include sleep deprivation and paroxysmal sleep, narcolepsy. There has been no known pharmacological method for promoting or inhibiting the initiation of sleep or for maintaining the sleeping or waking state.

Cerebrospinal fluid (liquor cerebrosinalis) is a clear, colorless fluid that circulates within the four ventricles of the brain and the subarachnoid spaces surrounding the brain and spinal cord. Cerebrospinal fluid originates as an ultrafiltrate of the blood secreted by the choroid plexus in the lateral third and fourth ventricles. Cerebrospinal fluid is also sometimes called neurolymph. After passing through the four ventricles and the subarachnoid spaces, cerebrospinal fluid is largely resorbed into the venous system via the arachnoid villi.

Cerebrospinal fluid serves as a medium for the removal of catabolites, excretions, and waste materials from the tissues bathed by it. To date, no factor derived from cerebrospinal fluid has been reported to correlate with sleep deprivation.

What is needed is a method for analyzing cerebrospinal fluid for identifying a biochemical factor generated by subject that correlates with sleep deprivation.

Since the seminal discovery of prostaglandins, there has WO 98/20119 PCT/US97/20385 3 been increasing recognition of the role of fatty acids and their derivatives in important physiological processes, B.

Samuelsson, Les Prix Nobel 1982, pp. 153-174.

Cis-9,10-Octadecenoamide has been isolated from the cerebrospinal fluid of sleep-deprived cats and has been shown to exhibit sleep-inducing properties when injected into rats.

Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat, and man, indicating that these compounds compose a distinct family of brain lipids.

Together, these results teach that fatty acid primary amides represent a new class of biological signalling molecules that can be employed for inducing subjects to sleep. Preferred fatty acid primary amides include an alkyl chain having an unsaturation and are represented by the following formula:

NH

2 C(O) (CH 2 6

I,,,,)CH=CH(CH

2 (8,s 5

)CH

3 Preferred soporific fatty acid primary amides have an unsaturation with a cis configuration within their alkyl chain. In addition to cis- 9,10-octadecenoamide, other soporifically active fatty acid primary amides include cis-8,9-octadecenoamide, cis-11,12octadecenoamide, and cis-13,14- docosenoamide.

Deutsch et al, Biochem. Pharmacol.,46:791 (1993) has identified an amidase activity which catalyzes both the hydrolysis and synthesis of arachidonylethanolamide (anandamide) from the membrane subcellular fractions taken from neuroblastoma, glioma cells and crude homogenates of rat brain tissues. The study detected the uptake and enzymatic breakdown of arachidonylethanolamide (anandamide) to arachidonic acid (and vice versa) from the homogenates of tissues from brain, liver, kidney and lung but not from rat heart and skeletal muscles.

The active membrane fraction which displayed this amidase WO98/20119 PCT/US97/20385 4 activity was prepared by either homogenizing the desired cell line and subsequently subjecting the crude homogenate to density centrifugation or by taking the crude homogenates of rat brains and directly incubating them with anandamide.

The uptake and degradation of arachidonylethanolamide (anandamide) was assayed by incubation of ['H]-anandamide (NEN, NET-1073, 210 Ci/mmol) in the cell culture medium. It was found, by liquid scintillation counting of the aqueous and organic phases, that arachidonic acid and anandamide distributed in the organic phase. Thus, the organic extract of the cell medium was subsequently visualized using thin-layer chromatography, sprayed with a surface autoradiograph enhancer (ENIHANCE, Dupont) and exposed to X-ray film (Kodak X-OMAT AR) at -80 OC.

The serine protease inhibitor, phenylmethylsulfonyl fluoride at 1.5 mM concentration completely inhibited the amidase activity. Other inhibitors tested had little or no effect on the activity and included aprotinin, benzamidine, leupeptin, chymostatin and pepstatin.

In a second manuscript, Deusch et. Biol Chem., 1994, 269, 22937) reports the synthesis of several types of specific inhibitors of anandamide hydrolysis and their ability to inhibit anandamide breakdown in vitro. Four classes of compounds were synthesized and include fatty acyl ethanolamides, a-keto ethanolamides, a-keto ethyl esters and trifluoromethyl ketones. The most effective class of compounds were the trifluoromethyl ketones and a-keto esters. The least potent inhibitors were the a-keto amides and saturated analogs of anandamide.

As an example, when anandamide is incubated with neuroblastoma cells, it is rapidly hydrolyzed to arachidonate WO 98/20119 PCT/US97/20385 5 but in the presence of the inhibitor arachidonyl trifluoromethyl ketone, there is a 5 fold increase of anandamide levels. The study infers that polar carbonyls such as those found in trifluoromethyl ketones, may form stabilized hydrates that mimic the tetrahedral intermediates formed during the reaction between the nucleophilic residue and the carbonyl group of anandamide.

Deutsch suggests that the nucleophilic residue may be the active site of a serine hydroxyl in the hydrolytic enzyme.

This enzyme is classified as an amidase (EC where the enzyme acts on carbon nitrogen bonds other than peptide bonds.

The amidase activity is inhibited by the serine protease inhibitor, PMSF and the action of trifluoromethyl ketone inhibitors (and others) directly affect the hydrolytic activity of the enzyme. Furthermore, Deutsch suggests that anandamide is cleaved by a mechanism that involves an active site serine hydroxyl group.

What is needed is an identification of enzymes within the i: brain tissue which catalyze the degradation of soporific compound found in the cerebrospinal, for mediating the soporific activity of these compounds. What is needed is an identification of inhibitors for inhibiting the activity of enzymes which degrade soporific compounds of the type found in cerebrospinal fluid.

Brief Summary of the Invention An enzyme is disclosed herein which degrades soporific fatty acid primary amides, and is designated fatty-acid amide hydrolase, or FAAH. FAAH is one of the enzymes which mediates the activity of fatty acid primary amides, including soporific fatty acid primary amides.

As disclosed herein, FAAH is characterized by an enzymic 04/05/2005 10:41 GRIFFITH HACK +61 2 99255911 4 00262837999 N0.104 0004 -6 activity for catalyzing a conversion cis-9, octadecenoamide to oleic acid, among other substrates, as shown in Scheme 1 below, and therefor was originally identified as cis-9,10-octadecenoamidase. However, it is now shown that FAAH has activity to hydrolyse a variety of fatty acid primary amides, and therefore the anidase originally referred to as cis-9,10-octadecenoamidase is Smore appropriately referred to as FAAH.

11

NH

Cis-9,10-octadecencamidas 0 2

HO

0SCHEME 1 In a first aspect, the invention provides an isolated fatty-acid amide hydrolase (FAAH) capable of hydrolysing 25 cis-9,10-octadecenoamide, anandamide, myristic amide, palmitic amide and stearic amide, said FAAH having an amino acid residue sequence selected from the amino acid residue sequence shown in SEQ ID NO 36; (ii) the amino acid residue sequence shown in SEQ ID NO 40 from residue 3 to 581, and (iii) the amino acid residue sequence shown in SEQ ID NO 43 from residue 12 to 590.

In a second aspect, the invention provides a method for catalyzing a hydrolysis of a fatty-acid primary amide comprising the step of contacting the fatty-acid primary amide with an isolated FAAH according to the first aspect of the invention.

In a third aspect, the invention provides the COMS ID No: SBMI-01190578 Received by IP Australia: Time 10:49 Date 2005-04-05 04/05/2005 10:41 GRIFFITH HACK +61 2 99255911 4 00262837999 NO.104 0005 6a use of a FAAH according to the first aspect of the invention for the hydrolysis of anandamide, myristic amide, palmiic amide or stearic amide.

In a fourth aspect, the invention provides a method for inhibiting an enzymatically catalyzed hydrolysis of a fatty-acid primary amide by a FAAh according to the first aspect of the invention, the method comprising the step of contacting said FAAH with an inhibitor of the FAAH.

In another aspect, the invention provides a method for ascertaining the inhibitory activity of a candidate inhibitor of fatty-acid amide hydrolase (FAAH), the method comprising the following steps: Step A: forming mixture by combining FAAH according to the first aspect of the invention and a fatty-acid primary amide substrate under reaction conditions; Step B: forming mixture by combining the mixture of said Step A with the candidate inhibitor; then Step C: quantifying the conversion of said fatty-acid S 20 primary amide substrate to a hydrolysis product within 'f mixture Step D: quantifying the conversion of said fatty-acid primary amide substrate to hydrolysis product within mixture and then 25 Step E: ascertaining the inhibitory activity of the candidate inhibitor by comparing the quantifications of said Steps C and D.

f t In another aspect, the invention provides a nucleic t acid molecule encoding a fatty-acid amide hydfolase protein, said nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO SEQ ID NO 39 and SEQ ID NO 42.

In another aspect, the invention provides a method of preparing a FAAH according to the first aspect of the invention, said method comprising expressing a recombinant DNA expression vector that includes a nucleotide sequence COMS ID No: SBMI-01190578 Received by IP Australia: Time 10:49 Date 2005-04-05 04/05/2005 10:41 GRIFFITH HACK +61 2 99255911 4 00262837999 NO.104 D006 6b that encodes said FAAB, and isolating the expressed FAAH.

In another aspect, the invention provides a nucleic acid molecule encoding a portion of a fatty-acid amide hydrolase protein, said nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO 1.

In another aspect, the invention provides an isolated fatty-acid amide hydrolase (FAAH) capable of hydrolyzing cis-9,10-octadecenoamide, myristic amide, palmitic amide and stearic amide, wherein the FAAH is isolated by binding a trifluoroketone inhibitor of FAAH.

In another aspect, the invention provides a trifluoroketone inhibitor of fatty-acid amide hydrolase represented by the following structure:

O

.C (C 0. conjugated to a bead.

One aspect of the invention is directed to a purified form of FAAH. FAAH can be purified by a variety of S. 20 methods, including a chromatographic methodology.

Preferred chromatographic methodologies include affinity chromatography, electric chromatography, gel filtration chromatography, ion exchange chromatography, and partition o* chromatography. In affinity chromatography, a solid phase 25 adsorbent contains groups that bind particular proteins L* because they resemble ligands for 000.

0 :o i oo o COMS ID No: SBMI-01190578 Received by IP Australia: Time 10:49 Date 2005-04-05 WO 98/20119 PCT/US97/20385 -7which the proteins have a natural affinity. In a preferred mode, the solid phase adsorbent contains one or more FAAH inhibitors which bind the enzyme. In antibody affinity chromatography, a solid phase immunoabsorbent having antibodies with a bind specificity with respect to FAAH are employed. In electric chromatography or electrophoresis, the FAAH is separated from other molecules according to its molecular weight or isoelectric point. In gel filtration, also known as gel permeation, molecular sieve, and exclusion chromatography, the ~solid phase creates a stationary phase of gel solvent and a mobile phase of excluded solvent. The FAAH is separated according to its molecular size as it partitions between the stationary and mobile phases. The gel particles are selected to have a exclusion size in excess of FAAH. In ion exchange chromatography, a solid phase ion exchanger is employed for separating the FAAH from other molecules according to its partitioning between ionic and nonionic forces. In partition chromatography, immiscible fluids having a stationary and mobile phases are employed for separating the FAAH according to its partitioning between the two immiscible phases. Preferred chromatographic methodologies include DEAE chromatography, affinity chromatography on a solid phase having attached Hg groups derivatized with an inhibitor of FAAH such as a trifluoroketone.

In a preferred mode, a crude source of FAAH is purified in four steps. In the first step, a crude source of FAAH is purified by exchange chromatography using a DEAE chromatography column to form a first elution product. In the second step, the elution product from the first step is further purified by partitioning by with affinity chromatography to form a second elution product. In the third step, elution product from the WO 98/20119 PCT/US9720385 -8second step is further purified by partitioning with Heparin affinity chromatography to form a third elution product. In the fourth step, the elution product from the third step is further purified by partitioning with an stationary phase derivatized with a trifluoroketone inhibitor of FAAH. The eluant from the fourth step form the purified form of FAAH.

FAAH can be isolated from any of a variety of mammalian species, including rat, mouse or human, as described herein.

Fatty-acid amid hydrolase (FAAH) is characterized by .IQ. inclusion of an amino acid sequence selected from a group consisting of: GGSSGGEGALIGSGGSPLGLGTDIGGSIRFP (SEQ ID NO SPGGSSGGEGAJIGS (SEQ ID NO 6), ALIGSGGSPLGLGTD (SEQ ID NO 7), GLGTDIGGSIRFPSA (SEQ ID NO 8), RFPSAFCGICGLKPT (SEQ ID NO 9), GLKPTGNRLSKSGLK (SEQ ID NO KSGLKGCVYGQTAVQ (SEQ ID NO 11), QTAVQLSLGPMAPLV (SEQ ID NO 12), MARDVESLALCLKAL (SEQ ID NO 13), CLKALLCEHLFTLDP (SEQ ID NO 14), FTLDPTVPPFPFREE (SEQ ID NO PFREEVYRSSRPLRV (SEQ ID NO 16), in.) RPLRVGYYETDNYTM (SEQ ID NO 17), DNYTMPSPAMRRALI (SEQ ID NO 18), RRALIETKQR.EAAG (SEQ ID NO 19), LEAAGHTLIPFLPNN (SEQ ID NO FLPNNIPYALEVLSA (SEQ ID No 21), EVLSAGGLFSDGGRS (SEQ ID NO 22), 0 DGGRSFLQNFKGDFV (SEQ ID NO 23), KGDFVDPCLGDLILI (SEQ ID No 24), WO 98/20119 PCT/US97/20385 9 DLILILRLPSWFKRL (SEQ ID NO WFKRLLSLLLKPLFP (SEQ ID NO 26), KPLFPRLAAFLNSMR (SEQ ID NO 27), LNSMRPRSAEKLWKL (SEQ ID NO 28), KLWKLQHEIEMYRQS (SEQ ID NO 29), MYRQSVIAQWKAMNL (SEQ ID NO aa.) KAMNLDVLLTPMLGP (SEQ ID NO 31), and ab.) PMLGPALDLNTPGR (SEQ ID NO 32).

Another aspect of the invention is directed to a method for catalyzing the hydrolysis of a fatty acid primary amide. In this hydrolysis method, the fatty acid primary amide is combined or contacted with a catalytic amount of purified form of FAAH.

In a preferred mode, the fatty acid primary amide is of a type which includes an alkyl chain having an unsaturation or more particularly is represented by the following formula:

NH

2 C 6 an l CH=CH (CH 2 ,a ns5) CH

S.

More particularly, the unsaturation of the alkyl chain may have a cis configuration or may be identically cis-9,10octadecenoamide, cis-8,9-octadecenoamide, cis-11,12octadecenoamide, or cis-13,14- docosenoamide.

Another aspect of the invention is directed to a method for inhibiting an enzymatically catalyzed hydrolysis of fatty acid primary amides, such as cis-9,10-octadecenoamide, by FAAH. In this method, FAAH is combined or contacted with an inhibitor of FAAH. Preferred inhibitors include phenylmethylsulfonyl fluoride, HgC1 2 and a trifluoroketone having the following structure: WO 98/20119 PCT/US97/20385 10 0

F

3 C (CH2 (C HCH 3 Another aspect of the invention is directed to a method for ascertaining the inhibitory activity of a candidate inhibitor of FAAH. Thus, FAAH is admixed with a candidate FAAH inhibitor to assess inhibitory capacity in a screening method.

In a preferred method for determining inhibitory activity of a candidate FAAH inhibitor, the contemplated method comprises five steps. In the first step, a mixture is formed by combining FAAH and cis-9,10-octadecenoamide substrate under reaction conditions. In the second step, a mixture is formed by combining the mixture with the candidate inhibitor. In the third step, the conversion of cis-9,10octadecenoamide substrate to a hydrolysis product within mixture a is quantified. In the fourth step, the conversion of cis- 9 ,10-octadecenoamide substrate to hydrolysis product within mixture is quantified. In the fifth step, the inhibitory activity of the candidate inhibitor is ascertained by comparing the quantifications of steps three and four.

Another aspect of the invention is directed to a trifluoroketone inhibitor of FAAH represented by following structure:

O

F

3 C (CH 2 (CH 2 h)CH 3 Another aspect of the invention is directed to one or more nucleotide sequences the encode part or all of FAAH. The complete nucleotide sequence that encodes human, mouse or rat WO 98/20119 PCTIUS97/20385 FAAH are shown in SEQ ID Nos. 42, 39 or IS, respectively.

The partial nucleotide sequence of rat FAAH is represented as follows:

CCAGGAGGTTCCTCAGGGGGTGAGGGGGCTCTCATTGGATCTGGAGGTTCCCCT

CTGGGTTTAGGCACTGACATTGGCGGCAGCATCCGGTTCCCTTCTGCCTTCTGC

GGCATCTGTGGCCTCAGCCTACTGGCAACCGCCTCAGCAAGAGTGGCCTGAAG

GGCTGTGTCTATGGACAGACGGCAGTGCAGCTTTCTCTTGGCCCCATGGCCCGG

0 0 0009 S. GTTGAGACGCTGGGGCTATGGCTGACACTTTGAGCATGTCG

CACACTGGACCCTCTGCTCCCAATTTCCCTGAGAGGAGGTCAGG

a 0e 0 ~TCTGCGGGCGGCCTGTTCAGTGACGGTGGCCGCAGTTTTCTCCAAAC'TCAAA or*.

GGTGACTTTGTGGATCCCTGCTTGGGAGACCTGATCTTAATTCTGAGGCTGCCC

6 0 AGCTGGTTTAAAAGACTGCTGAGCCTCCTGCTGAAGCCTCTGTTTCCTCGGCTG 0 $too GCAGCCTTTCTCAACAGTATGCGTCCTCGGTCAGCTGAAAAGCTGTGGAAACTG 0 CAGCATGAGATTGAGATGTATCGCCAGTCTGTGATTGCCCAGTGGAAAGCGATG 050 CTTGGATGTGCTGCTGACCCCNATGYTNGGNCCNGCNYTNGAYYTNAYAN *0.

CCNGGNMGN (SEQ ID NO 54).

Brief Description of the Drawings Figure 1 illustrates the structures of natural agent, cis- 9,10-octadecenoamide related analogs Compound 6 is the preferred structure for naturally occurring fatty acid amide.

Figure 2 illustrates the determined partial amino acid sequence of the rat FAAI as described in Section B.4.

Figure 3 illustrates a partial purification strategy involving isolation of a plasma membrane protein fraction from rat liver using 1) a sucrose gradient of the liver membrane WO98/20119 PCT/US97/20385 12 followed by 2) a 100 mM sodium carbonate wash and 3) solubilization in trion-based buffer. The isolated liver plasma membrane is then purified by four consecutive chromatographic steps: 1) Ion exchane DEAE column, 2) Mercury inhibition column, 3) detergent exchange Heparin column followed by 4) an affinity column with a trifluoroketone inhibitor. The purified protein was determined to have a 20-30 fold enrichment of amidase activity from crude membrane protein fraction by visual comparison of the purified protein band intensity with the crude protein fraction. Estimated purified yield is 10-15% from crude liver plasma membrane protein.

Figure 4 illustrates the affinity column purification strategy (step 4, Figure 3) using a trifluoroketone inhibitor S. which is linked to a disulfide-derivatized solid support (pyridyl disulfide beads).

Figure 5 illustrates the synthetic protocol for the synthesis of the trifluoroketone inhibitor and subsequent attachment of the inhibitor to the disulfide-derivatized solid support using pyridyl disulfide beads.

Figure 6 represents an autoradiogram of a thin layer chromatography plate (SiO2, 55% ethyl acetate/hexanes) illustrating the FAAH activity of a rat brain membrane fraction with respect to the hydrolysis of radio-labelled cis-9,10octadecenoamide to oleic acid and its inhibition by phenylmethyl sulfonyl fluoride (PMSF). Lane number, content: lane 1, Cis- 9,10-octadecenoamide standard; lane 2, Cis-9,10-octadecenoamide with rat brain soluble fraction; lane 3, Cis-9,10octadecenoamide with rat brain membrane fraction; lane 4, Cis- 9,10-octadecenoamide with rat brain membrane fraction 1 mM phenylmethylsulfonyl fluoride (PMSF); lane 5, Cis-9,10octadecenoamide with rat brain membrane fraction 5 mM EDTA; WO98/20119 PCTIUS97/20385 13 lane 6, Cis-9,10-octadecenoamide with rat-pancreatic microsomes; lane 7, Cis-9,10-octadecenoamide with proteinase K (200 mg); lane 8, oleic acid standard.

Figure 7 represents an autoradiogram of a thin layer chromatography plate (SiO2, 55% ethyl acetate/hexanes) illustrating the FAAH activity of a rat brain membrane fraction with respect to the hydrolysis of radio-labelled cis-9,10octadecenoamide to oleic acid and its inhibition by mercuric chloride (HgCl 2 The optimal concentrations required for inhibition of amide hydrolysis activity lies between 50 mM and mM HgCl,. The various lanes of the TLC plate are identified as follows: lane 1, Cis-9,10-octadecenoamide standard; lane 2, Cis-9,10-octadecenoamide with rat brain soluble fraction; lane 3, Cis-9,10-octadecenoamide with rat brain membrane fraction and 500 mM HgCl,; lane 4, Cis-9,10-octadecenoamide with rat brain membrane fraction and 50 mM HgC, 2 lane 5, Cis-9,10octadecenoamide with rat brain membrane fraction and 5 mM HgCl,; lane 6, oleic acid standard. A typical HgC1, inhibition study uses a 100 mM HgC1, stock (27 mg in ImL Tris buffer (50 mM), pH 7.5) of HgCl,.

Figure 8 represents a northern blot of mRNA obtained from cloning procedures. Ribosomal markers are shown by the arrows, next to lane 1, and indicate 5kb and 2kb bands. The arrow next to lane 6 points to a 3kb band which is representative of the oleic amidase enzyme. Lane 1 is total RNA from rat brain; lane 2 is total RNA from rat lung; lane 3 is total RNA from rat kidney; lane 4 is total RNA from rat heart; lane 5 is total RNA from rat liver; lane 6 is mRNA from rat liver (mRNA loaded in lane 6 is approximately 500 ng); total respective RNA loaded in lanes 1-5 was approximately 15 Ag.

Figure 9 illustrates the deduced encoded amino acid residue WO 98/20119 PCT/US97/20385 14 sequence of rat oleamide hydrolase also referred to as a fatty acid amide hydrolase or FAAH (SEQ ID NO 36). The encoded rat FAAH is appropriately abbreviated rFAAH. Bold type indicates the putative transmembrane spanning domain as predicted by PSORT. The seven discontinuous underlined regions indicate the seven separate peptides, the designation of which is consecutive, obtained by HPLC purification of a trypsin digest of the enzyme. The double-underlined segment is the putative SH3-domain-binding sequence.

I

T

Figures 10-1 through 10-5 show the continuous doublestranded cDNA sequence for rat FAAH as described in Section D.

The encoded amino acid sequence is also indicated beginning with the ATG start site encoding methionine The stop codon is also shown as boxed. The top and bottom strands of the cDNA sequence are respectively listed in SEQ ID NOs 35 and 37 with the amino acid sequence listed with the nucleotide sequence in SEQ ID NO 35 and by itself in SEQ ID NO 36.

Figure 11 illustrates the alignment of the amidase signature sequence region of the rat FAAH (SEQ ID NO 36 from ?0 amino acid residue 215 to and including 246) with several other representative amidases as further described in Section Dl.

Residues of the signature sequence that are completely conserved among the family members are shown in bold type and the relative amino acid position of the signature sequence of each member is given by the numbers just preceding and following the sequence information. From top to bottom, the sequences have the following respective SEQ ID Nos: 36 (from residue 215 to 246); 47, 48, 49, 50, 51, 52 and 53.

Figure 12A and 12B show the respective results of Southern 0 and Northern blots as probed with an internal 800 bp fragment of rat FAAH cDNA as further described in Section D.

WO 98/20119 PCT/US97/20385 15 Figures 13-1 through 13-4 show the continuous doublestranded cDNA sequence for mouse FAAH as described in Section D2. The encoded amino acid sequence is also indicated beginning with the ATG start site encoding methionine The stop codon is also shown as boxed. The top and bottom strands of the cDNA sequence are respectively listed in SEQ ID NOs 39 and 41 with the amino acid sequence listed with the nucleotide sequence in SEQ ID NO 39 and by itself in SEQ ID NO Figures 14-1 through 14-5 show the continuous doublestranded cDNA sequence for human FAAH as described in Section D3. The encoded amino acid sequence is also indicated beginning with the ATG start site encoding methionine The stop codon is also shown as boxed. The top and bottom strands of the cDNA sequence are respectively listed in SEQ ID NOs 42 and 44 with the amino acid sequence listed with the nucleotide sequence in SEQ ID NO 42 and by itself in SEQ ID NO 43.

Figure 15A shows the expression of recombinant rat FAAH in COS-7 cells produced as described in Section E as performed by thin layer chromatography demonstrating the conversion of labeled oleamide to oleic acid as further described in Section

F.

Figure 15B shows the inhibition of recombinant rat FAAH by trifluoromethyl ketone also performed as described in Figure as further described in Section F.

Figure 15C shows the results of Western blotting of recombinant rat FAAH with antibodies generated against peptide 2.

as shown in Figure 9 as shown in the four left lanes and as competed with peptide 2 as shown in the four right lanes Samples of untransfected COS-7 cell extract are shown in lanes 4 and 8, FAAH-transfected COS-7 cell extracts are shown in lanes 3 and 7, affinity-purified rat FAAH is shown in lanes 2 WO98/20119 PCT/US97/20385 16 and 6 and a mixture of FAAH-transfected COS-7 cell extracts and affinity-purified FAAH is run in lanes 1 and 5. The proteins were probed with antibodies in the absence (lanes 1-4) or presence (lanes 5-8) of competing peptide antigen. The FAAHtransfected COS-7 cell extract but not the control contained an immunoreactive 60K-65K protein that was effectively competed away by preincubation of the antibodies with excess peptide antigen while the trace quantities of cross reactive protein observed in both transfected and untransfected COS-7 cell O. extracts were not competed by the peptide.

Figure 16 shows the ability of human recombinant expressed FAAH to hydrolyze oleamide to oleic acid, as further described in Figure 15A with thin layer chromatography and in Section F.

Figure 17 shows the results of thin layer chromatography demonstrating the conversion of labeled anandamide to arachidonic acid in rat FAAH-transfected COS-7 cells as shown in lane 3 but not in control untransfected cells (lane TLC i standards of anandamide and arachidonic acid are shown in lanes 1 and 4, respectively.

Detailed Description of the Invention A. Protocols for the Induction of Sleep Synthetic cis-9,10-octadecenoamide was injected (ip) into rats in order to test its effect on spontaneous behavior at different doses: 1 2 5 10 and 50 mg, where n number of rats tested. Rats were injected during a reversed dark period (12:12) two hours after the lights cycled off and were observed in their home cages. With the lower doses (1 and 2 mg), no overt effect on spontaneous behavior was witnessed. However, at a threshold of WO 98/20119 PCT1US9720385 17 mg and above there was a marked effect consisting of an induction of long-lasting motor quiescence associated with eyes closed, sedated behavior characteristic of normal sleep. Also as with normal sleep, the rats still responded to auditory stimuli with orienting reflex and sustained attention toward the source of stimulation. In addition, motor behavior was impaired. The latency to behavioral sedation following administration was about 4 minutes and subjects were normally active again after 1 hour (5 mg), 2 hour (10 mg), or 2.5 hour 01". (20 mg and 50 mg).

We have compared cis-9,10-octadecenoamide to vehicle and the synthetic analogs listed in Figure 1 to estimate the structural specificity of its sleep-inducing potential. Neither vehicle ethanol in saline solution) nor oleic acid showed any overt behavioral effect. Trans-9,10-octadecenoamide demonstrated similar pharmacological effects to its cis counterpart, but was much less potent as measured by the comparatively shorter duration time for its sleep-inducing properties (at 10 mg per rat, the biological effect lasted one hour for the trans isomer and two hours for the cis isomer).

When the olefin was moved either direction along the alkyl chain (to the 8,9 or 11,12 positions) or the alkyl chain length was extended to 22 carbons a substantial reduction in both the degree and duration of the pharmacological effects was observed, and while the mobility of the rats still decreased, their eyes remained open and their alertness appeared only slightly affected. Finally, polysomnographic studies on rats injected with cis-9,10-octadecenoamide show an increase in the total time of slow wave sleep (SWS) as well as in the mean duration of the SWS individual periods when compared to vehicle controls. More particularly, male Sprague-Dawley rats (300 g at WO 98/20119 PCT/US97/20385 3 18 18 the time of surgery) were implanted under halothane anesthesia with a standard set of electrodes for sleep recordings.

This included two screw electrodes placed in the parietal bone over the hippocampus to record the subjects electroencephalogram (EEG) and two wire electrodes inserted in the neck musculature to record postural tone through electromyographic activity (EMG). Rats were housed individually with at libitum access to food and water. The dark-light cycle was controlled (12:12, lights on a 10:00 One week after the surgery, rats were habituated to the recording conditions for at least three days.

Upon the completion of the habituation period, rats received 2 milliliter (ip) of either: vehicle ethanol/saline solution), cis-9,10-octadecenoamide (10 mg), or oleic acid mg). Rats were continuously recorded for four hours after the ip injection (12:00 p.m.-4:00 Rats were observed for spontaneous changes in behavior through a one-way window. Sleep recordings were visually scored and four stages were determined: wakefulness, slow-wave-sleep 1 (SWS1), slow-wave-sleep 2 (SWS2), and rapid eye movement (REM) sleep.

These increases with respect to slow wave sleep (SWS) were at the expense of waking. Distribution of REM sleep does not seem to be altered. Together, these data suggest that cis-9,10-octadecenoamide could play an important role in slow-wave sleep modulation.

The traditional view of lipid molecules as passive structural elements of cellular architecture is rapidly giving way to an ever increasing awareness of the active roles these agents play in transducing cell signals and modifying cell behavior, Liscovitch et al, Cell, 77:329 (1994). An intriguing feature of the fatty acid amides studied here is that they belong to a family of simple molecules in which a great WO 98/20119 PCT/US97/20385 19 19 deal of diversity may be generated by simply varying the length of the alkane chain and the position, stereochemistry, and number of its olefin(s). Interestingly, other neuroactive signalling molecules with amide modifications at their carboxy termini have been reported, from carboxamide terminal peptides to arachidonylethanolamide. Neuroactive signalling molecules employing carboxamide terminal peptides are disclosed by Eipper et al, Annu. Rev.'Neurosci., 15:57 (1992). Neuroactive signalling molecules employing arachidonylethanolamide is disclosed by Devane et al, Science, 258:1946 (1992). It is disclosed herein that cis-9,10-octadecenoamide is a member of a new class of biological effectors in which simple variations of a core chemical structure have unique physiological consequences.

B. Isolation and assay of integral membrane protein fraction with FAAH activity 1. Observations on Lipid Amidase Activity Lipid amidase activity has been observed in brain, liver, lung, kidney and spleen tissues, but not in heart tissue. The activity is inhibited by 1 mM PMSF (phenylmethylsulfonyl fluoride) and 50 mM HgCl, which is a test for sulfhydryl group dependency of the reaction. Since the fractions are not solubilized by 100 mM sodium carbonate (pH 11.5), the sample is apparently a membrane protein, which has been identified in nuclear, microsomal, and plasma membrane subcellular fractions, but not in the cytosol.

The enzyme catalyzed hydrolysis of cis-9,10-octadecenoamide to oleic acid by purified cis-9,10-octadecenoamide and inhibition of this enzyme by PMSF is disclosed on an WO 98/20119 PCTUS972035 20 autoradiogram of a thin layer chromatographic plate (SiO2, ethyl acetate/hexanes), illustrated in Figure 6. In each case the enzymic reaction is performed is a separate reaction vessel and the product is spotted onto a TLC plate. The various reaction conditions for the reaction vessel corresponding to each lane are identified as follows: *o lane 1: Cis-9,10-octadecenoamide lane 2: Cis-9,10-octadecenoamide fraction; lane 3: Cis-9,10-octadecenoamide fraction; lane 4: Cis-9,10-octadecenoamide fraction 1 mM PMSF; lane 5: Cis-9,10-octadecenoamide fraction 5 mM EDTA; lane 6: Cis-9,10-octadecenoamide microsomes; lane 7: Cis-9,10-octadecenoamide mg); and lane 8: oleic acid standard.

standard; with rat brain soluble with rat brain membrane with rat brain membrane with rat brain membrane with rat pancreatic with proteinase K (200 Inhibition studies of Cis-9,10-octadecenoamide hydrolysis to oleic acid with HgC1, are illustrated in Figure 7. Between mM and 5 mM HgC1, lies the optimal concentrations required for inhibition of amide hydrolysis activity. The enzyme catalyzed hydrolysis of cis-9,10-octadecenoamide to oleic acid by purified cis-9,10-octadecenoamide and inhibition of this enzyme by HgC12 is performed in a series of reaction vessels and spotted onto a thin layer chromatographic plate (SiO2, 55% ethyl acetate/hexanes). A typical HgClinhibition study uses a 100 mM WO 98/20119 WO 9820119PCT/US97/20385 21 HgCl, stock (27 mg in intL Tris buffer (50 rWi), pH 7.5) -of HgCl,.

The various reaction conditions for the reaction vessels corresponding to each lane are identified as follows: rQ~.

lane 1: Cis-9,10-octadecenoamide standard; lane 2: Cis-9,l0-octadecenoamide with rat brain fraction; lane 3: Cis-9,l0-octadecenoamide with rat brain fraction 'and 500 MM HgCl 2 lane 4: Cis-9,l0-octadecenoamide with rat brain fraction and 50 MM HgCl 2 lane 5: Cis-9,10-octadecenoamide with rat brain fraction and 5 mM HgCl 2 lane 6: oleic acid standard.

soluble membrane membrane membrane WO 98/20119 PCTIUS97/20385 22 N I Cis-9,10-octadecenoamidase phenylmethylsulfonyl fluoride (PMSF) or HgC 2

S.

S

0S@4

S

0@e*r Ho SCHEME 2 *see o f 5 0@r

S

S. S

S

SS

A unique enzymatic activity capable of degrading the putative effector molecule, cis-9,10- octadecenoamide has been identified and is disclosed herein. Rapid conversion of "C-cis-9,10- octadecenoamide to oleic acid by rat brain membrane fractions was observed by TLC. The enzymatic activity was unaffected by 5 mM EDTA, but was completely inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF). Only trace amide hydrolysis activity was observed with rat brain soluble fractions, while rat pancreatic microsomes and proteinase K showed no significant capacity to hydrolyze cis-9,10-octadecenoamide to oleic acid.

2. Synthesis of fatty acid primary amides Preferred protocols for synthesizing exemplary WO 98/20119 PCT/US97/20385 23 fatty acid primary amides are provided. The synthetic protocols differ only with respect to the chain length of the starting materials, the product yields, and the separation of the various cis and trans products. Accordingly, exemplary descriptions of synthetic protocols for the synthesis of cis-9,10octadecenoamide and several other fatty acid primary amides are provided and serve to illustrate the synthetic protocol for the entire class of fatty acid primary amides.

3. Isolation of rat integral membrane protein fraction with FAAH activity The protocol described herein is for about 5-10 g of tissue. The rat liver(s) are collected, weighed and then placed in 1mM NaHCO, on ice. Next, the liver is cut up, rinsed XS*. (2X) with 1mM NaHCO, and minced with a razor blade on a sheet of wax. It is then placed into 25 ml of 1mM sodium bicarbonate and homogenized in a tissuemizer for 2 minutes at setting 6. A dilution to 100 ml with 1mM sodium bicarbonate is subsequently performed, which is followed by a filtration through 4 layers of cheesecloth and then through 8 layers. The filtrate is then brought up to 100 ml and split into four JA-20 tubes and topped off with 1 mM sodium bicarbonate. The tubes are spun at 6,000 rpm (4500 x g) for 12 minutes at 4 0 C in the JA-20 rotor. Using a Pasteur pipette, the fat layer is sucked off and the supernatant layer is decanted and saved.

Next, the pellet is resuspended in the remaining supernatant layer with a syringe and needle. 20 mL fractions of the resuspension are then dounced 16 times with a 15 ml dounce homogenizer. The fractions are then combined into a single JA- 0 20 tube and brought up to volume with 1 mM NaHCO,. The tubes are next spun again at 6,000 rpm (4500 x g) for 15 minutes at 4 0 C in WO 98/20119 PCT/US97/20385 24 a JA-20 rotor and the supernatant is subsequently poured off and saved. The pellet is resuspended and dounced as before and then brought up to 10 ml volume with ImM sodium bicarbonate. Next, mL of 67% sucrose solution is added to a final volume of ml and the mixture is split into 2 tubes. An additional 25 mL of 30% sucrose is added to the top of each tube and spun at 27 K rpm for 1 hour 45 minutes at 4 0 C in an ultracentrifuge. The fractions are collected from the sucrose gradient and the middle band from the sucrose gradient (plasma membrane band) is placed in a capped plastic tube and filled with 1 mM sodium bicarbonate. The tube is subsequently spun at 17,000 rpm for minutes at 4 0

C.

The supernatant is discarded and the pellets are resuspended (with Douncing) in 100 mM of sodium carbonate. This solution is subsequently kept on ice for 1 hour and then spun at 100,000 g for 1 hour. The supernatant (solubilized peripheral membrane proteins) is discarded since no lipid amidase activity is present in this fraction and the pellet is resuspended (with Douncing) in 10% glycerol, 1% Triton, 0.1% phosphatidyl choline, 20 mM Hepes buffer and then stirred for two hours at 4 0

C.

Finally the solution is spun at 100,000 g for 1 hour and the supernatant thus obtained is further purified as follows.

4. Purification via 4 step column chromatocraphy process Step 1 DEAE column/ ion exchange (Figure The above solubilized supernatant batch is further purified. The supernatant batch is mixed with DEAE-Sephadex (Diethylaminoethyl-Sephadex, commercially available from Sigma chemical company) ion exchange resin for 1 hour at 4 0 C. The fraction which is bound to the DEAE resin, displays the lipid WO 98/20119 PCT/US97/20385 25 amidase activity (none in flow through). "Solubilized rat liver plasma membrane (in BI: 10% glycerol, 1% Triton X-100, 1 mM EDTA, 20 mM Hepes, pH 7.2) is passed over DEAE Fast Flow column (Pharmacia) and washed with 5 column volumes of BI, 0.2% Triton.

Then the amidase activity is eluted with 1 column volume each of mM, 100 mM, and 200 mM NaCl in BI with 0.2% Triton.

Step 2 Hg Column (Figure The above eluent from the S. DEAE exchange, is mixed with p-chloromercuric benzoic acid resin (Commercially available from BioRad chemical company) for 1 hour kgo. at 4 0 C. The fraction which is bound to the above mercury resin, displays the lipid amidase activity (none in flow through), is S washed with 5 column volumes of BI with 0.2% Triton, 5 column volumes of BI with 0.2% Triton and 150 mM NaC1, and eluted with 1.5 column volumes BI with 0.2% Triton, 150 mM NaC1, and 25 mM b-mercaptoethanol.

Step 3 Heparin column (Figure Hg-eluted amidase activity was passed over Heparin column (BioRad) and washed with column volumes of BI with 0.7% CHAPS and 150 mM NaC1 (detergent exchange). Elution was conducted with 1 column 26bi volume each of BI with 0.7% CHAPS and 300 mM, 400 mM, 500 mM, 650 mM, and 750 mM NaCl,respectively, with amidase activity eluting in the final two fractions.

Step 4 Affinity column (Figures 3 and Heparin-eluted amidase activity was mixed with Triton X-100 for a final concentration of and then passed over CF,-inhibitor linked to activated pyridyl disulphide beads (103: attachment of inhibitor to beads is described infra) and washed with 20 column volumes of BI with 0.2% Triton X-100. Elution was conducted by passing 3 column volumes of BI with 0.2% Triton and 20 mM DTT, and letting column stand at 4o C for 30 h. Then, washing column with 1.5 column volumes of BI with 0.2% Triton and 20 mM DTT WO98 2 0119 PCTIUS97/20385 26 eluted single protein of 60 kD in size.

Eluted 60 kd protein was digested with trypsin and peptides were sequenced as described infra.

The purity of the activity is then assessed after this procedure according to an assay protocol.

Assay for Fatty-Acid Amide Hydrolase Activity: The following thin layer chromatography (TLC) protocol is used for assaying cis-9,10 octadecenoamide hydrolysis activity, also referred to as fatty-acid amide hydrolase activity. Oleamide is first labeled with 1'C. To accomplish this, 4 C-Oleic acid (1-10 iM, Moravek Biochemicals, 5-50 iCi/AM) in CH2CL 2 (200 pL, 0.005-0.05 M) at 0°C was treated with excess oxalyl chloride and the reaction mixture was warmed to 25"C for 6 hours. The reaction mixture was then concentrated under a constant stream of gaseous nitrogen and the remaining residue was cooled to 0°C and treated with excess saturated aqueous ammonium hydroxide. After 5 minutes, the reaction mixture was partitioned between Et)Ac (1.5 mL) and 10% HC1 (1.0 mL). The 2 10. organic layer was then washed with water (1.0 mL) and concentrated under a constant stream of gaseous nitrogen to provide 14C-oleamide in quantitative yield as judged by TLC EtOAc in hexanes; oleamide Rf-0.2; oleic acid Rf-0.8).

Approximately 1 iCi of 4 C-oleamide (specific activity 5-50 ACi/pM) in ethanol was incubated at 37 0 C for 1-2 hours with AL of 126 mM Tris-HCl, pH 9.0 (final concentration of ethanol was The reaction mixture was then partitioned between ethyl acetate (1.0 mL) and 0.07 M Hcl (0.6 mL). The ethyl acetate layer was concentrated under a constant stream of 0 gaseous nitrogen and the remaining residue was resuspended in AL of ethanol. Approximately 3 iL of this ethanol stock was WO 98/20119 PCT/US97/20385 27 then used for TLC analysis (60% EtOAc in hexanes: oleamide Rf- 0.2; oleic acid Rf-0.8). Following exposure to solvent, TLC plates were air-dired, treated with EN 3 HANCE spray (Dupont NEN) according to manufacturer's guidelines and exposed to film at 78 0 C for 1-2 hours.

The purified protein was determined to have a 20-30 fold enrichment of amidase activity from crude membrane protein fraction by visual comparison of the purified protein band intensity with the crude protein fraction. Estimated purified ~yield is 10-15% (Figure 3).

e WO 98/20119 WO 9820119PCT/US97t20385 28 0H 3 OH__BH 3 THF (1 equiv.), 0

O

CH0CH1 3 O OH 0 -20 OC to RT, 10 h 7 suberic acid monomethyl ester

CH

3 O)L (CH 2 7 PPh 3 +Bir- 9 PPh 3 (1.5 eq), CH 3

CN,

ref lux, 20 h 85% CBr 4 (1.3 equiv.), PPh 3 (1.4 equiv), CH 2

CI

2 4 OC, 10 h, 0

CH-

3 O- Br 8 1) KHMDS (1 eq), THE, reflux, 1 hr 2) decyl aldehyde (1.2 eq), to RTfor 2hr 0 LiOH (3 eq), THF:MeOH

CH

3 0OI (CH 2 6

(CH

2 8

CH

3 HOk(CH 2 6

CH

2 )8CH 3 60-75% 8 h r, RT 60% 1) oxalyl chloride (3 eq), CH 2

CI

2 4 hr, RT 2) sat NH 4 OH, 5 min 0

H

2

N)

3 85-90% Scheme 3 WO 98/20119 PCT1US97/20385 29

H

2 NA (CH 2 7 I- -CHA) 8

O

0 12 0 CHO (C 2 8 h 3 8r 0 CHO (CH 2 P~>h 2 )aOT H3',(CH 2 7

'(OH

2 8 0TD 0 HN Ik(OH 2 7 11->CH2)OT&E HNA (CH-2) 7 =>CH2)8OH CBr 4 (1.3 eq), PPh 3 (1.4 eq),

CH

2

CI

2 10 hr PPh 3 (1.5 cq), CH.

3

CN.

reflux, 20 h 0

CH

3 OQN.-1,1-Br 13, CH130 A,(CH2)aPPh 3 +Br- 14,91%

(CH

2 )7 CH2) 8 0TBDPS 15, 76% 1) KHMDS (I eq), THF, refiux, I hr 2) 0 H Ik(CH2)aOTBDPS (1.2 eq) a a LiOH (3 eq), THF:MeOH: H 2 0 8 hr, RT 1) oxalyl chloride (3 eq), CH 2

CI

2 4 hr, RT 2) sat NH 4 0H, 5 mini TBAF (2 eq), TH-F, RT, I hr HO Ik(CH2) 7

CH

2

)OTBDPS

16, 89%

H

2 N IU(OH 2 7 I- NCHOT8DPS 17, 76% HN -fl,(CH2) 7

CH)O

18, 12, Succ Anhy (2 eq), Etc 3 N (2 eq), DMAP (A1 eq). CK 2

CI

2 10 h Scheme 4 WO 98/20119 PCTIUS97/20385 30 C. Synthetic Protocols 1. Cis-9.10-octadecenoamide Figure 1): A solution of oleic acid (1.0 g, 3.55 mmol, equiv.) in CHC1, (8.9 mL, 0.4 M) at 0 OC was treated dropwise with oxalyl chloride (5.32 mL, 2.0 M solution in CHCl 2 10.64 mmol, 3.0 equiv.). The reaction mixture was stirred at 25 °C for 4 h, concentrated under reduced pressure, cooled to 0 °C, and treated with saturated aqueous NH 4 OH (2.0 mL). The reaction mixture was then partitioned between ethyl acetate (EtOAc) (100 mL) and H,0 (100 mL) and the organic layer was dried (NaSO 4 and concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 1 as a white solid (0.810 g, 0.996 g theoretical, 1 H NMR (CDC1 3 250 MHz) 5 6.06 (bs, 1H, NH 2 5.58 (bs, 1H, NH 2 5.32 2H, CH=CH) 2.16 2H, J 7.5 Hz, CHC(0)NH,) 2.02 4H, CHCH=CHCH,), 1.61 2H, CHCHC(0)NH 2 1.29 (b s, 14H, alkyl protons), 0.87 3H, CH 3 FABHRMS (NBA/NaI m/e 282.2804 (C,,Hs 5 NO H* requires 282.2797). The regions of the spectra that distinguish between the cis and trans isomers are the olefinic protons from 5 5.3 to 5.2 and allylic protons from 5 2.0 to 1.8.

These regions identify the natural compound as cis-9,10octadecenoamide.

2. Trans-9.10-octadecenoamide Ficure 1) A solution of elaidic acid (1.0 g, 3.55 mmol, equiv.) in CHC1, (8.9 mL, 0.4 M) at 0 oC was treated dropwise with oxalyl chloride (5.32 mL, 2.0 M solution in CH,C1 2 10.64 mmol, 3.0 equiv.). The reaction mixture was stirred at 25 °C for 4 h, concentrated under reduced pressure, cooled to 0 OC, and treated with saturated aqueous NH.OH (2.0 mL). The reaction WO 98/20119 PCT/US97/20385 31 mixture was then partitioned between ethyl acetate (EtOAc) (100 mL) and H,O (100 mL), and the organic layer was dried (Na,SO.) and concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 2 as a white solid. The regions of the spectra that distinguish between the cis and trans isomers are the olefinic protons from 5.3 to 5.2 and allylic protons from 6 2.0 to 1.8. These regions identify the compound as trans-9,10-octadecenoamide.

3. Cis-8.9-octadecenoamide Figure 1): A solution of 11, synthesized infra, (0.130 g, S* 0.461 mmol, 1.0 equiv.) in CH,C1 2 (1.5 mL, 0.31 M) at 0 OC was treated dropwise with oxalyl chloride (0.69 mL, 2.0 M solution in CH,C1 2 1.38 mmol, 3.0 equiv.). The reaction mixture was stirred at 25 oC for 4 h, concentrated under reduced pressure, cooled to 0 OC, and treated with saturated aqueous NH,OH L) The reaction mixture was then partitioned between ethyl acetate (EtOAc) (100 mL) and H,O (100 mL), and the organic layer was dried (Na 2 SO,) and concentrated under reduced pressure.

Chromatography (SiO,, 5 cm x 15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 3 as a white solid. (0.105 g, 0.130 theoretical, 80.8%) 1 H NMR (CDCl,, 250 MHz) 5 5.70-5.34 4H, H,NC(0) and CH=CH), 2.21 2H, J 7.5 Hz, CH,C(0)NH,), 2.00 (m, 4H, CH,CH=CHCH,), 1.63 2H, CH,CH,C(0)NH,), 1.47-1.23 alkyl protons), 0.87 3H, RCH,); FABHRMS (NBA/CSI m/e 414.1762 (C,,H,sNO Cs' requires 414.1773) 4. Cis-11.12-octadecenoamide Figure 1): A solution of A11,12 octadecenoic acid (1.0 g, 3.55 mmol, 1.0 equiv.) in CH,C1, (8.9 mL, 0.4 M) at 0 OC was treated dropwise with oxalyl chloride (5.32 mL, 2.0 M solution WO 98/20119 PCT/US97/20385 32 in CH,C1 2 10.64 mmol, 3.0 equiv.). The reaction mixture was stirred at 25 OC for 4 h, concentrated under reduced pressure, cooled to 0 oC, and treated with saturated aqueous NHOH mL). The reaction mixture was then partitioned between ethyl acetate (EtOAc) (100 mL) and H,O (100 mL), and the organic layer was dried (Na,SO,) and concentrated under reduced pressure.

Chromatography (SiO 2 5 cm x 15 cm, 40-100% EtOAc-hexanes gradient elution) afforded 4 as a white solid.

5. Oleic acid Firure 1) Oleic acid was obtained from Aldrich chemical company, CAS #112-80-1.

6. Erucamide Figure 1) Erucamide was obtained from Aldrich Chemical Company, CAS #28,057-7.

7. Methyl-8-hydroxy-octanoate Scheme 3) A solution of suberic acid monomethyl ester g, 7.97 mmol, 1.0 equiv.) in tetrahydrofuran (THF) (32.0 mL, at -20 OC was treated dropwise with BH,.THF (1M solution in THF, 7.97 mL, 7.97 mmol, 1.0 equiv.). The reaction mixture was stirred overnight and was subsequently allowed to reach room temperature. The reaction mixture was then diluted with ethyl acetate (100 mL) and quenched with methanol (10 mL) and 10% HC1 mL). Extraction with NaHCO, (1X 20 mL), water (2X 10 mL), and brine (lX 10 mL), afforded methyl-8-hydroxy-octanoate as a crude white solid.

8. Methyl-8-bromo-octanoate Scheme 3) A solution of crude methyl-8-hydroxy-octanoate WO 98/20119 PCT/US97/20385 33 1.24 g, 7.13 mmol, 1.0 equiv.) in CH,C1, (15 mL, 0.48 M) at 0 °C was treated successively with CBr, (3.07 g, 9.27 mmol, 1.3 equiv.) and PPh, (2.61 g, 9.98 mmol, 1.4 equiv.) and the reaction mixture was stirred at 4 OC for 10 h. The reaction mixture was then concentrated under reduced pressure and washed repeatedly with Et,O (8 x 10 mL washes). The EtO washes were combined and concentrated under reduced pressure. Chromatography (SiO 2 5 cm x 15 cm, hexanes) afforded 8 as a clear, colorless oil (1.25 g, 1.69 g theoretical, 'H NMR (CDC1 3 250 MHz) 6 3.64 (s, 3H, C(0)OCH,), 3.38 2H, J 6.8 Hz, CH,Br), 2.29 2H, J=7.4 Hz CHC(O)OCH,), 1.83 2H, CH,CH,Br), 1.63 2H, CH,CHC(O)OCH,) 1.47-1.28 6H, alkyl protons).

9. Methyl-8-triphenvlphosphoranyl-octanoate-bromide Scheme 3) A solution of 8 (1.25 g, 5.23 mmol, 1.0 equiv.) in CH 3 CN (4.0 mL, 1.31 M) was treated with triphenylphosphine (1.52 g, 5.75 mmol, 1.1 equiv.) and stirred at reflux for 10 h.

Additional triphenylphosphine (0.685 g, 2.61 mmol, 0.5 equiv.) was added to the reaction mixture and stirring was continued at reflux for 5 h. The reaction mixture was concentrated under reduced pressure and washed repeatedly with EtO (5 x 10 mL washes). The remaining residue was then solubilized in the minimum volume of CH,C1, and concentrated under reduced pressure to afford 9 as a colorless foam (2.20 g, 2.61 g theoretical, 1H NMR (CDC1, 250 MHz) 6 7.82-7.51 15H, ArH), 3.70- 3.46 5H, CH, OC(O)R and CH 2 PPh) 2.13 2H, J 7.4 Hz,

CH

2

C(O)OCH

3 1.62-1.43 6H, alkyl protons), 1.30-1.02 4H, alkyl protons); FABHRMS (NBA) m/e 419.2154 (C 2

,H

3 ,BrO,P-Brrequires 419.2140).

WO 98 2 0119 PCT/US97t02385 34 Methyl-cis-8.

9 -octadecenoate (10: Scheme 3) A solution of 9 (0.71 g, 1.42 mmol, 1.0 equiv.) in THF (7.0 mL, 0.2 M) at 25 OC was treated with KHMDS (3.0 mL, M solution in THF, 1.5 mmol, 1.06 equiv.) and the reaction mixture was stirred at reflux for 1 h. The reaction mixture was then cooled to -78 OC, treated with decyl aldehyde (0.321 mL, 1.71 mmol, 1.2 equiv.) warmed to 25 OC, and stirred for an additional 30 min. The reaction mixture was then treated with saturated aqueous NH,C1 and partitioned between EtOAc (100 mL) S and H,O (100 mL). The organic layer was dried (Na,SO,) and s S concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, 0-2% EtOAc-hexanes gradient elution) afforded 10 as a colorless oil (0.290 g, 0.422 g theoretical, 68.7 'H NMR (CDC1 3 250 MHz) 6 5.34 2H, CH=CH), 3.65 3H, CHOC(O)), 2.29 2H, J 7.4 Hz, CH,C(0)OCH,), 2.00 4H, CH,CH=CHCH,), 1.61 2H, CHCH 2 C(O)OCH,), 1.29 (bs, 20 H, alkyl protons), 0.86 3H, RCH) 4* 1 1 Cis-8.9 octadecenoic acid (11: Scheme 3) 0 'e A solution of 10 (0.245 g, 0.825 mmol, equiv.) in THF-MeOH-H,O (3-1-1 ratio, 4.1 mL, 0.2 M) at 0 OC was treated with LiOH-H,O (0.104 g, 2.48 mmol, 3.0 equiv.). The reaction mixture was warmed to 25 OC, stirred for 8 h, and then partitioned between EtOAc (100 mL) and H,O (100 mL). The organic layer was washed successively with 10% aqueous HC1 (100 mL) and saturated aqueous NaCI (100 mL), dried, and concentrated under reduced pressure. Chromatography (SiO,, 5cm x 15 cm, 10-30% EtOAc-hexanes gradient elution) afforded 11 as a colorless oil (0.156 g, 0.233 g theoretical, 'H NMR (CDC1 250 MHz) 6 5.34 2H, CH=CH), 2.34 2H, J 7.4 Hz, CH,COOH), 2.01 (m, 4H, CH,CH=CHCH,), 1.61 2H, CH,CH,COOH), 1.47-1.23 20 H, WO 98/20119 PCT/US97/20385 35 alkyl protons), 0.87 3H, RCH 3 12. 1 8 -Hemisuccinate-cis-9,10-octadecenoamide (12: Scheme 4) A solution of 18 (0.047 g, 0.160 M, 1.0 equiv) in CHCl 2 -CHC1 3 1.60 mL, 0.1M) was treated successively with Et 3 N (0.045 mL, 0.320 mmol, 2.0 equiv), succinic anhydride (0.033 g, 0.320 mmol, 2.0 equiv) and DMAP (0.002 g, 0.016 mmol, 0.1 equiv), and the reaction mixture was stirred at 25 OC for 10 h.

The reaction mixture was then partitioned between CH,C1, (50 mL) and H 2 0 (50 mL) and the organic layer was washed successively with 10% aqueous HC1 (50 mL) and saturated aqueous NaCI (50 mL), dried (Na,SO4), and concentrated under reduced pressure.

Chromatography (SiO,, 3 cm x 15 cm, 0-10% MeOH-EtOAc) afforded 12 as a white solid (0.051 g, 0.063 theoretical, 1H NMR (CDC13, 250 MHz) 6 6.95 (b s, 1H, H 2 5.72 (b s, 1H, HNC(O)), 5.34 2H, CH=CH), 4.08 3H, J 6.6 Hz, CH,OC(O)R), 2.61 4H, ROC(O)CH 2 CHCOOH), 2.21 2H, J Hz, CHC(O)NH,), 2.00 4H, CHCH=CHCH,), 1.70-1.52 4H, CHCH,C(O)NH, and CHCH,OH), 1.29 (b s, 18H, alkyl protons); FABHRMS (NBA) m/e 398.2893 (C 2 Hrequires 398.2906).

13. Methyl-9-bromo-nonanoate (13: Scheme 4) A solution of methyl-9-hydroxy-nonanoate (1.1 g, 5.85 mmol, 1.0 equiv) in CH,C1 2 (30 mL, 0.2 M) at 0 OC was treated successively with CBr 4 (2.5 g, 7.54 mmol, 1.3 equiv) and PPh, (2.15 g, 8.19 mmol, 1.4 equiv) and the reaction mixture was stirred at 4 OC for 10 h. The reaction mixture was then concenctrated under reduced pressure and washed repeatedly with Et,O (8 x 10 mL washes). The EtO washes were combined and concentrated under reduced pressure. Chromatography (SiO 2 5 cm WO 98/20119 PCT/US97/20385 36 x 15 cm, hexanes) afforded 13 as a clear, colorless oil (1.02 g, 1.47 g theorectical, 69.5 'H NMR (CDC1,, 250 MHz) d 3.64 (s, 3H, C(O)OCH,), 3.38 2H, J 6.8 Hz, CH 2 Br), 2.29 2H, J 7.4 Hz CHC(O)OCH,), 1.83 2H, CHCH,Br), 1.63 2H, CHCH,C(O)OCH,) 1.47-1.28 8H, alkyl protons).

14. Methyl-9-triphenvlphosphoranyl-nonanoate-bromide (14: Scheme 4) A solution of 13 (1.02 g, 4.06 mmol, 1.0 equiv) S in CH 3 CN (3.5 mL, 1.16 M) was treated with triphenylphosphine (1.17 g, 4.47 mmol, 1.1 equiv) and stirred at reflux for 10 h.

Additional triphenylphosphine (0.532 g, 2.03 mmol, 0.5 equiv) was added to the reaction mixture and stirring was continued at reflux for 5 h. The reaction mixture was concentrated under L reduced pressure and washed repeatedly with Et,O (5 x 10 mL S washes). The remaining residue was then solubilized in the minimum volume of CH,C12 and concentrated under reduced pressure to afford 14 as a colorless foam (1.90 g, 2.08 g theoretical, IH NMR (CDC1,, 250 MHz) 6 7.82-7.51 15H, ArH), 3.70- 'o0 3.46 5H, CH,OC(O)R and CHPPh,), 2.13 2H, J 7.4 Hz, CH,C(O) OCH,), 1.62-1.02 12H, alkyl protons); FABHRMS (NBA) m/e 433.2312 (C,,H,,BrO,P Br- requires 433.2296) Methyl-18-t-butyldiphenysilyloxy-cis-9.10 octadecenoate (15: Scheme 4) A solution of 14 (1.0 g, 1.95 mmol, 1.0 equiv) in THF (6.5 mL, 0.3 M) at 25 OC was treated with KHMDS (3.9 mL, M solution in THF, 1.95 mmol, 1.0 equiv) and the reaction mixture was stirred at reflux for 1 h. The reaction mixture was 0 then cooled to -78 OC, treated with 3 (0.93 g, 2.35 mmol, 1.2 equiv), warmed to 25 OC, and stirred for an additional 30 min.

WO 98/20119 PCT/US97/20385 37 The reaction mixture was then treated with saturated aqueous NH.C1 and partitioned between EtOAc (100 mL) and H,O (100 mL).

The organic layer was dried (Na,SO,) and concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, 0-2% EtOAc-hexanes gradient elution) afforded 15 as a colorless oil (0.82 g, 1.07 g theoretical, IH NMR (CDC1 250 MHz) 7.67 4H, ArH), 7.41 6H, ArH), 5.34 2H, CH=CH), 3.65 5H, CHOC(0) and CHOTBDPS), 2.29 2H, J 7.4 Hz, CHC(0)OCH3), 2.00 4H, CHCH=CHCH,), 1.55 4H, CHCH,C(0)OCH 3 and CHCH,OTBDPS), 1.29 (b s, 18H, alkyl protons), 1.04 9H, (CH,) 3

C).

16. 18-T-butyldiphenylsilyloxy-cis-9.10-octadecenoic acid (16: Scheme 4) A solution of 5 (0.81 g, 1.47 mmol, 1.0 equiv) in S THF-MeOH-H,0 (3-1-1 ratio, 7.3 mL, 0.2 M) at 0 OC was treated S with LiOH-H,O (0.188 g, 4.48 mmol, 3.0 equiv). The reaction mixture was warmed to 25 OC, stirred for 8 h, and then S partitioned between EtOAc (100 mL) and HO (100 mL). The organic layer was washed successively with 10% aqueous HC1 (100 mL) and saturated aqueous NaCI (100 mL), dried, and concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, 10-30% EtOAc-hexanes gradient elution) afforded 16 as a colorless oil (0.700 g, 0.790 g theoretical, iH NMR (CDC1,, 250 MHz) 7.67 4H, ArH), 7.41 6H, ArH), 5.34 2H, CH=CH), 3.65 3H, J 6.5 Hz, CHOTBDPS), 2.34 2H, J 7.4 Hz, CH,COOH), 2.00 4H, CHCH=CHCH,), 1.65-1.50 4H, CHCH,COOH and CH,CHOTBDPS), 1.47-1.23 18H, alkyl protons), 1.05 (s, 9H, FABHRMS (NBA/CsI) m/e 669.2772 (C 3 ,Hs, 2 0Si Csrequires 669.2740).

WO 98 20 119 PCT/US97/20385 38 17. 18-T-butvldiphenylsilvloxy-cigs-9

O-

octadecenoamide (17: Scheme 4) A solution of 16 (0.685 g, 1.28 mmol, 1.0 equiv) in CH,C1, (4.3 mL, 0.3 M) at 0 OC was treated dropwise with oxalyl chloride (1.92 mL, 2 M solution in CH,C1,, 3.84 mmol, equiv). The reaction mixture was stirred at 25 OC for 4 h, concentrated under reduced pressure, cooled to 0 OC, and treated with saturated aqueous NH,OH (2.0 mL). The reaction mixture was then partitioned between EtOAc (100 mL) and H,O (100 mL) and the organic layer was dried (NaSOJ) and concentrated under reduced o pressure. Chromatography (SiO 2 5 cm x 15 cm, 40-100% EtOAc- Shexanes gradient elution) afforded 17 as a colorless oil (0.520 g, 0.684 g, 76.0%) 1H NMR (CDCl 3 250 MHz) 5 7.67 4H, ArH), 7.41 6H, ArH), 5.70-5.34 4H, H 2 NC(O) and CH=CH), 3.65 (t, 3H, J 6.5 Hz, CHOTBDPS), 2.21 2H, J 7.5 Hz, CHC(O)NH 2 2.00 4H, CHCH=CHCH 1.65-1.50 4H, CHCH 2 C(O)NH, and

CHCH

2 OTBDPS), 1.47-1.23 18H, alkyl protons), 1.05 9H, FABHRMS (NBA/CsI m/e 668.2929 (C 3 ,H,,O,NSi Cs" requires 668.2900) 18. 1 8 -Hvdroxy-cis-9.10-octadecenoamide (18: Scheme A solution of 17 (0.185 g, 0.345 mmol, 1.0 equiv) in THF (1.1 mL, 0.31 M) was treated with tetrabutylammoniumfluoride (0.69 mL, 1.0 M solution in THF, 0.69 mmol, 2.0 equiv) and the reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was then partitioned between EtOAc (50 mL) and H,0 (50 mL), and the organic layer was dried (Na,SO,) and concentrated under reduced pressure Chromatography (SiO 2 3 cm x 15 cm, 0-5% MeOH-EtOAc gradient elution) afforded 18 as a white solid (0.097 g, 0.103 g WO 98/20119 PCTIUS97/20385 39 theoretical, 'H NMR (CDC1,, 250 MHz) 6 5.65-5.34 4H,

H

2 NC(O) and CH=CH), 3.62 3H, J 6.5 Hz, CH 2 OH), 2.21 2H, J 7.5 Hz, CHC(0)NH,), 2.00 4H, CHCH=CHCH,), 1.65-1.50 (m, 4H, CH 2 CH,C(0)NH, and CH 2 CHOH), 1.29 (b s, 18H, alkyl protons); FABHRMS (NBA) 298.2732 H* requires 298.2746) 19. Synthesis of Compound 100 (Figure Methyl-9-t-butyldiphenylsilyloxy-nonanoate (intermediate for compound 100: Figure A solution of methyl-9-hydroxy-nonanoate (0.838 g, 4.46 mmol, 1.0 equiv: Aldrich) in CH,C1 2 (15 mL, 0.3 M) was treated successively with EtN (0.75 mL, 5.38 mmol, 1.2 equiv), t-butylchlorodiphenylsilane (1.28 mL, 4.93 mmol, 1.1 equiv), and DMAP (0.180 g, 1.48 mmol, 0.33 equiv), and the reaction mixture was stirred at 25 OC for D' 12 h. Saturated aqueous NHC1 was added to the reaction mixture and the mixture was partitioned between CH 2 C1, (100 mL) and HO (100 mL). The organic layer was dried (Na,SO) and concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, S EtOAc-hexanes gradient elution afforded the intermeidate as a 16: clear, colorless oil (1.22g, 1.831 theoretical, 'H NMR (CDC1,, 250 MHz) 6 7.66 4H, ArH), 7.38 6H, ArH), 3.67- 3.62 5H, C(O)OCH, and CHOTBDPS), 2.30 2H, J 7.4 Hz, CH,C(0)OCH 3 1.58 4H, CHCHOTBDPS and CH,CH,C(0) OCH) 1.28 (b s, 8H, alkyl protons), 1.05 9H, Methyl-9-bromo-nonanoate (intermediate for compound 100: Fiure A solution of methyl-9-hydroxy-nonanoate (1.1 g, 5.85 mmol, 1.0 equiv) in CHC1 2 (30 mL, 0.2 M) at 0 OC was treated successively with CBr. (2.5 g, 7.54 mmol, 1.3 equiv) and PPh, (2.15 g, 8.19 mmol, 1.4 equiv) and the reaction mixture was WO 98/20119 PCT/US97/20385 40 stirred at 4 OC for 10 h. The reaction mixture was then concenctrated under reduced pressure and washed repeatedly with Et,O (8 x 10 mL washes). The Et,O washes were combined and concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, hexanes) afforded the intermediate as a clear, colorless oil (1.02 g, 1.47 g theorectical, 69.5 1 H NMR (CDC1 3 250 MHz) d 3.64 3H, C(O)OCH,), 3.38 2H, J= 6.8 Hz, CHBr), 2.29 2H, J 7.4 Hz CHC(O)OCH, 2H. cH 2

CH

2 Br) 1.63 2H, CH 2

CH,C(O)OCH

3 1.47-1.28 8H, alkyl protons) 19e.

21. 9 -T-butyldiphenylsil1oxv-nonanal (intermediate S" for compound 100: Fiqure A solution of 1 (1.25 g, 2.93 mmol, 1.0 equiv) in toluene (9.80 mL, 3.0 M) at -78 OC was treated dropwise with DIBAL-H (4.40 mL, 1.0 M solution in hexanes, 4.40 mmol, equiv). The reaction mixture was stirred at -78 oC for 30 min.

The reaction mixture was then treated dropwise with MeOH (2 mL) and partitioned between EtOAc (100 mL) and H,O (100 mL). The organic layer was washed with 10 aqueous HC1 (100 mL), dried (NaSO,), and concentrated under reduced pressure.

Chromatography (SiO 5 cm x 15 cm, 0-5 EtOAc-hexanes gradient elution) afforded 3 as a colorless oil (1.1 94.9 -H NMR (CDCl 250 MHz) 6 9.76 1H, J 1.8 Hz, HC(O)R), 7.67 4H, ArH), 7.40 6H, ArH), 3.65 2H, J 6.4 Hz, CH 2

OTBDPS),

2.41 (t of d, 2H J 1.8 and 7.3 Hz, CHC(O)H), 1.58 4H, CHCH,OTBDPS and CHCH,C(0)H), 1.29 (b s, 8H, alkyl protons), 1.05 9H, FABHRMS (NBA/CsI) m/e 529.1560 (C25H3602Si Cs* requires 529.1539).

WO 98 2 0119 PCT/US97/20385 41 22. Methyl-9-triphenylphosphoranyl-nonanoate Bromide (intermediate for compound 100: Fiqure A solution of 9 -T-butyldiphenylsilyloxy-nonanal (1.02 g, 4.06 mmol, 1.0 equiv) in CH,CN (3.5 mL, 1.16 M) was treated with triphenylphosphine (1.17 g, 4.47 mmol, 1.1 equiv) and stirred at reflux for 10 h. Additional triphenylphosphine (0.532 g, 2.03 mmol, 0.5 equiv) was added to the reaction mixture and stirring was continued at reflux for 5 h. The reaction mixture was concentrated under reduced pressure and washed repeatedly with Et,O (5 x 10 mL washes). The remaining residue was then solubilized in the minimum volume of CH,C1, and concentrated under reduced pressure to afford the intermediate as a colorless foam (1.90 g, 2.08 g theoretical, 1 H NMR (CDC1,, 250 MHz) 6 7.82-7.51 15H, ArH), 3.70-3.46 CH,OC(O)R and CHPPh,) 2.13 2H, J 7.4 Hz, CHC OCH) 1.62-1.02 12H, alkyl protons); FABHRMS (NBA) m/e 433.2312

(C

2

,H

3 ,BrO,P Br- requires 433.2296) 23. Methyl-18-t-butyldiphenysilyloxy-cis-9,10octadecenoate (intermediate for compound 100: Ficure A solution of (1.0 g, 1.95 mmol, 1.0 equiv) in THF (6.5 mL, 0.3 M) at 25 OC was treated with KHMDS (3.9 mL, M solution in THF, 1.95 mmol, 1.0 equiv) and the reaction mixture was stirred at reflux for 1 h. The reaction mixture was then cooled to -78 OC, treated with 3 (0.93 g, 2.35 mmol, 1.2 equiv), warmed to 25 and stirred for an additional 30 min.

The reaction mixture was then treated with saturated aqueous NH,Cl and partitioned between EtOAc (100 mL) and H,0 (100 mL).

The organic layer was dried (Na,SO,) and concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, 0-2% WO 98/20119 PCT/US97/20385 42 EtOAc-hexanes gradient elution) afforded the intermediate as a colorless oil (0.82 g, 1.07 g theoretical, H NMR (CDC1,, 250 MHz) 6 7.67 4H, ArH), 7.41 6H, ArH), 5.34 (m, 2H, CH=CH), 3.65 5H, CH,OC(O) and CHOTBDPS), 2.29 2H, J 7.4 Hz, CH,C(O)OCH3), 2.00 4H, CHCH=CHCH,), 1.55 4H, CHCH,C(O)OCH, and CHCH,OTBDPS), 1.29 (b s, 18H, alkyl protons), 1.04 9H, (CH,) 3

C).

24. 18-T-butvldiphenylsilyloxy-cis-9.10-octadecenoic acid (compound 100: Figure A solution of Methyl-18-t-butyldiphenysilyloxycis-9,10-octadecenoate (0.81 g, 1.47 mmol, 1.0 equiv) in THF- MeOH-H 2 0 (3-1-1 ratio, 7.3 mL, 0.2 M) at 0 OC was treated with LiOH-HO (0.188 g, 4.48 mmol, 3.0 equiv). The reaction mixture was warmed to 25 OC, stirred for 8 h, and then partitioned S between EtOAc (100 mL) and H 2 0 (100 mL). The organic layer was washed successively with 10% aqueous HC1 (100 mL) and saturated .aqueous NaCl (100 mL), dried, and concentrated under reduced pressure. Chromatography (SiO,, 5 cm x 15 cm, 10-30% EtOAchexanes gradient elution) afforded 100 as a colorless oil (0.700 g, 0.790 g theoretical, 1 H NMR (CDC1 250 MHz) 5 7.67 4H, ArH), 7.41 6H, ArH), 5.34 2H, CH=CH), 3.65 (t, 3H, J 6.5 Hz, CHOTBDPS) 2.34 2H, J 7.4 Hz, CHCOOH), 2.00 4H, CHCH=CHCH,), 1.65-1.50 4H, CHCHCOOH and CHCHOTBDPS), 1.47-1.23 18H, alkyl protons), 1.05 9H, FABHRMS (NBA/CsI) m/e 669.2772 (C 3 ,,Hs0,Si Cs* requires 669.2740).

Synthesis of Compound 101 (Ficure Step 1. A solution of 100 (1.0 equiv) in CH,C1, (0.3 M) at 0 °C WO 98/20119 PCT/US97/20385 43 was treated dropwise with oxalyl chloride (4.0 equiv). The reaction mixture was stirred at 25 OC for 4 h, concentrated under reduced pressure, cooled to 0 OC, and treated with saturated aqueous NH,OH (2.0 mL). The reaction mixture was then partitioned between EtOAc (100 mL) and H 2 0 (100 mL), and the organic layer was dried (Na,SO,) and concentrated under reduced pressure.

o.

Step 2. A solution of the above step 1 intermediate compound (1.0 equiv) in ether (0.3 M) at 0 OC was treated dropwise with pyridine (8.0 equiv.) followed by trifluoroaceticanhydride equiv; Aldrich). The reaction mixture was stirred at 25 OC for 3 h, concentrated under reduced pressure, cooled to 0 OC, and treated with saturated aqueous NHOH (2.0 mL). The reaction mixture was then partitioned between EtOAc (100 mL) and HO (100 mL), and the organic layer was dried (Na,SO) and concentrated under reduced pressure.

Step 3. A solution of the above step 2 intermediate compound -26 (1.0 equiv) in THF (0.31 M) was treated with tetrabutylammoniumfluoride (1.0 M solution in THF, 3.0 equiv) and the reaction mixture was stirred at 25 OC for 3 h. The reaction mixture was then partitioned between EtOAc (50 mL) and H,O (50 mL), and the organic layer was dried (NaSO) and concentrated under reduced pressure. Product was purified by standard chromatographic conditions and yielded compound 101 in 66% overall yield for the 3 steps.

26. Synthesis of Compound 102 (Figure Step 1. A solution of 101 (1.0 equiv.) in THF (0.1 M) was WO 98/20119 PCT/ES970385 44 treated with triphenylphosphine (2.0 equiv.), followed by diethylazodicarboxylate solution (1.0 THF solution, DEAD, equiv., Aldrich) and at 0 OC for 30 minutes. The reaction mixture was concentrated under reduced pressure and washed repeatedly with EtO (5 x 10 mL washes). The remaining residue was then solubilized in the minimum volume of CH 2 C1 and concentrated under reduced pressure.

Step 2. A solution of the above step 1 compound (1.0 equiv.) in THF (0.10 M) was treated with thiolacetic acid (2.0 equiv.; Aldrich) at 0 °C for 30 minutes. The reaction mixture was concentrated under reduced pressure and washed repeatedly with EtO (5 x 10 mL washes). The remaining residue was then solubilized in the minimum volume of CH 2 C1l and concentrated under reduced pressure. Product was purified by standard chromatographic conditions and yielded compound 102 in 71% overall yield for the 2 steps.

27. Synthesis of Compound 103 (Figures 4 Step 1. A solution of 102 (1.0 equiv) in MeOH/Water (2:1 mixture, total concentration 0.20 M) at 0 OC was treated with NaOH (3.0 equiv) and stirred for 10 minutes, and then partitioned between EtOAc (100 mL) and water (100 mL). The organic layer was washed successively with 10% aqueous HC1 (100 mL) and saturated aqueous NaCl (100 mL), dried, and concentrated under reduced pressure.

Step 2. A solution of the above step 1 compound (1.0 equiv) in aqueous IN HC1 at 0 OC was stirred until the reaction mixuture achieved a pH of 7.0, and then the mixture was partitioned WO98 20119 PCTIUS97/20385 45 between EtOAc (100 mL) and water (100 mL). The organic layer was washed successively with saturated aqueous NaCI (100 mL), dried, and concentrated under reduced pressure.

Step 3. A solution of the above step 2 compound (1.0 equiv.) in aqueos ImM NaHCO 3 at 25 OC was treated with Pyridyl disulfide beads (1.1 equiv. Aldrich) and stirred for 2 hours. The beads were subsequently washed with excess saturated NaHCO 3 water (3X) and brine Standard filtration obtained the activated beads (compound 103) which were then packed into the column for affinity chromatography of the enzyme as discussed supra using *6o this CF3-inhibitor linked to activated pyridyl disulphide beads.

D. Cloning of Cis-9.10-Octadecenoamidase

CDNA

1. Cis-9.10-Octadecenoamidase cDNA Obtained from Rat Liver mRNA To obtain a cDNA clone for cis-9,10octadecenoamidase from cDNA library generated from rat liver mRNA, degenerate oligonucleotide primers were designed based on the amino acid residue sequence of cis-9,10-octadecenoamidase polypeptide fragment obtained from a trypsin digest. Briefly, the cis-9,10-octadecenoamidase, purified as described above, was subjected to a trypsin digest to form internal polypeptide fragments as performed by Worchester Foundation, Worchester,

PA.

The resultant polypeptide fragments were purified by HPLC and seven HPLC fractions showing discrete peptide masses as measured by Matrix-Assisted-Laser-Desorption-Ionization with Time-of- Flight (MALDI TOF, PerSeptive Biosystems Linear Instrument) mass spectrometry were selected for microsequencing. Seven WO 98/20119 PCT/US97/20385 46 polypeptide fragments were microsequenced having lengths ranging from 12 to 25 amino acid residues as indicated in Figure 9 indicated by seven discontinuous singly underlined regions in the complete rat cis-9,10-octadecenoamidase amino acid residue sequence. Each peptide possessed the required lysine or arginine residue at its C-terminus indicating that the tryptic digest proceeded with the anticipated selectivity.

The degenerate oligonucleotide primers were designed to incorporate a unique restriction site into the 5' ends of the S primers that functioned as either forward and the backward primers. The forward primers are also referred to as upstream, sense or 5' primers. The backward primers are also referred to as downstream, anti-sense or 3' primers. The restriction sites were incorporated into the polymerase chain reaction (PCR) products to allow for insertion into the multiple cloning site of a sequencing vector as described below.

OOSSoe The synthesized 5' and 3' degenerate oligonucleotides were designed respectively corresponding to portions of sequenced peptides 1 and 2 as shown in Figure 9 as indicated by the first two discontinuous singly underlined amino acid residue sequences. The degenerate nucleotides are indicated by IUPAC codes N A, C, G or T and R A or G. The nucleotide sequence of the 5' degenerate primer corresponding to peptide 1 was 5'CGGAATTCGGNGGNGARGGNGC3' (SEQ ID NO 3) incorporating an EcoRI restriction site and translating into the amino acid sequence GGEGA (SEQ ID NO The nucleotide sequence of the 3' degenerate primer that corresponded to peptide 2 was 5'CGGGATCCGGCATNGTRTARTTRTC3' (SEQ ID NO 33) incorporating an BamHI restriction site and translating into the amino acid sequence DNYTMP (SEQ ID NO 34).

WO 98/20119 PCT/US97/20385 47 To amplify regions of cDNA encoding cis-9,10octadecenoamidase, rat liver mRNA was reversed transcribed into cDNA for use a template in PCR with selected pairs of degenerate oligonucleotide primers described above. PCR was performed under conditions well known to one of ordinary skill in the art with each cycle of 40 total cycles having the temperatures 94°C for 30 seconds, 60 0 C for 45 seconds and 72 0 C for 60 seconds.

o Of the cloned PCR fragments, three were selected for sequencing. The three PCR fragments were 350 base pairs (bp), S 400 bp and 750 bp. Sequencing of these cis-9,10octadecenoamidase-encoding cDNA fragments showed that the 750 bp fragment contained the sequences of both the 350 and 400 bp fragments.

The 350 bp cDNA fragment obtained by PCR was then labeled internally and used as a probe for Northern analysis on electrophoresed rat liver mRNA. The probe hybridized to a fragment approximately 2.5 to 3.0 kilobases (kb) in length, S which is the expected size of the cis-9,10-octadecenoamidase mRNA that encodes a 60 kDa protein.

To isolate a cDNA clone encoding the complete cis-9,10octadecenoamidase protein, the 350 bp probe was then internally labeled with 3 "P used to screen a Xgtll cDNA library from rat liver mRNA obtained from Clontech (Palo Alto, CA). For screening, the amplified 350 bp fragment was first digested with EcoRI and BamHI for directional cloning into a similarly digested pBluescript II SK(-)(Stratagene, La Jolla, CA). The resultant sequence indicated that the 350 bp fragment encoded the peptides 1 and 2 from which the degenerate oligonucleotide primers were designed confirming the accuracy of the PCR and amplification of the desired clone. The methods for cloning the WO 98/20119 PCT/US97/20385 48 cis-9,10-octadecenoamidase cDNA of this invention are techniques well known to one of ordinary skill in the art and are described, for example, in "Current Protocols in Molecular Biology", eds. Ausebel et al., Wiley Sons, Inc., New York (1989), the disclosures of which are hereby incorporated by reference.

Four positive clones were identified from a screening of 4.5 X 10 s plaques-. Two clones of 2.7 kb in length and 1 of kb in length, were obtained. The partial sequence of one of the 2.7 kb clones, designated p60, indicates that the clone does contain cis-9,10-octadecenoamidase-specific sequences.

The rat liver cDNA clone designated p60 obtained above has been deposited with American Type Culture Collection (ATCC) on or before June 12, 1996 and has been assigned the ATCC accession number 97605. This deposit was made under the provisions of the i Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures S maintenance of a viable plasmid for 30 years from the date of each deposit. The plasmid will be made available by ATCC under the terms of the Budapest Treaty which assures permanent and unrestricted availability of the progeny of the plasmid to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638). The assignee of the present application has agreed that if the plasmid deposit should die or be lost or destroyed when cultivated under WO 98/20119 PCT/US97/20385 49 suitable conditions, it will be promptly replaced on notification with a viable specimen of the same plasmid.

Availability of the deposit is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

A partial nucleotide sequence of the top strand of the cDNA clone containing 780 nucleotides described above is listed in SEQ ID NO 1 along with the deduced amino acid residue sequence. The encoded amino acid residue sequence is listed separately in SEQ ID NO 2. In order to show the amino acid residue encoded by each triplet codon in the Sequence Listing, a stop codon, TAA, was added at positions 781 to 783 to allow for the coding sequence (CDS) function in the Patentin program used to prepare the Sequence Listing. In other words, the stop codon is artificially inserted into the nucleotide sequence shown in SEQ ID NO 1 to facilitate the translation of the cDNA coding sequence into an amino acid sequence.

The actual position of the cis-9,10-octadecenoamidase nucleotide position within a complete cDNA clone is evident from the complete cDNA sequence as described below.

The two largest positive cDNA clones were then cloned into pBluescript II and sequenced. One clone encoded a partially processed transcript containing the full coding sequence of the oleamide amidase with an additional 200 bp of intronic sequence. The other clone encoded a fully processed oleamide amidase transcript but fused to the 5' end of the clone was a 300 bp fragment encoding rRNA. Fusion of the two clones through an internal overlapping HindIII restriction site generated the full-length rat cis-9,10-octadecenoamidase also referred to as fatty acid amide hydrolase abbreviated as FAAH.

WO 98/20119 PCTIUS97/20385 50 The clone was sequenced with sequencing primers that were synthesized on a Beckman Oligol000M Synthesizer.

The resultant full length rat cDNA FAAH clone, also referred to as rFAAH cDNA, contained 2473 bp, which contained a single 1.73 kb open reading frame encoding 63.3 kDa of protein sequence as shown in Figures 10-1 to 10-5. The double-stranded rat FAAH cDNA sequence is available by GenBank with Accession Number U72497. The encoded rat FAAH protein is also referred to as rFAAH protein. The clone contained 50 bp of sequence 5' to the first ATG designation the start of the open reading frame.

The clone also contained 685 bp of 3' untranslated region between the first stop codon indicating the end of the open reading frame and the poly A tail.

In Figures 10-1 through 10-5, the encoded amino acid residue is positioned directly underneath the second nucleotide of a triplet codon. For example, at the initiation site where ATG encodes methionine the A nucleotide begins at nucleotide position 50 and the G nucleotide is 52. The encoded M is located underneath the T nucleotide at nucleotide position 51. As presented in the figure, thus, the indicated triplet codons are not as indicated. The top and bottom strands of the cDNA sequence are also respectively listed as SEQ ID NOs 35 and 37. The encoded amino acid sequence is shown with the top strand in SEQ ID NO 35 and again by itself in SEQ ID NO 36.

Although the 50 bases of nucleotide sequence upstream of the first ATG did not possess an in-frame stop codon, the following several lines of evidence supported the 2.47 kb cDNA encoding the complete oleamide hydrolase protein sequence: 1) The size of the cDNA matched closely the predicted size of the mRNA transcript as estimated by Northern blot (Figure 12B as discussed below); 2) The sequence surrounding the first ATG WO 98/20119 PCT/US97/20385 51 possessed the required consensus sequence for eukaryotic translation initiation sites, in particular, an A is present at the -3 position and a G is present at the +4 position; and 3) When transiently transfected with oleamide hydrolase cDNA, COS-7 cells translated a functional protein product that comigrated with affinity isolated oleamide hydrolase on SDS-PAGE (Figure 12B, lane 1 and discussed below).

Database searches with the oleamide amidase protein sequence (FAAH) identified strong homology to several amidase :1i enzyme sequences from organisms as divergent as Agrobacterium tumefaciens (Klee et al., Proc. Natl. Acad. Sci.. USA, 81:1728- 1732, 1984), Pseudomonas savastanoi (Yamada et al., Proc. Natl, Acad. Sci.. USA, 82:6522-6526, 1985); Aspergillus nidulans (Corrick et al., Gene, 53:63-71, 1987), Saccharomyces cerevisiae (Chang et al., Nuc. Acids Res., 18:7180, 1990), Caenorhabditis elegans (Wilson et al., Nature, 368:32-38, 1994), and Gallus domesticus (Ettinger et al., Arch, Biochem. Biophys., 316:14-19, 1995). These amidases collectively compose a recently defined enzyme family (Mayaux et al., J. Bacteriol., 172:6764-6773, 1990) whose members all share a common signature sequence as shown in Figure 11. The encoded amino acids beginning at position 215 and extending through 246 of the rat fatty acid amide hydrolase (oleamide hydrolase or FAAH) contain residues that are found in a family of amidases. The sequence in the cis-9,10-octadecenoamidase rat protein of this invention has is GGSSGGEGALIGSGGSPLGLGTDIGGSIRFPS as shown in SEQ ID NO 36 at amino acid positions 215 to 246. The alignment over the amidase signature sequence region of the rat FAAH with several other representative amidases reveals that the signature sequence is completely conserved among the amidase family members. Those amino acids are shown in bold faced type in the figure and the WO 98/20119 PCT/US97/20385 52 relative amino acid position of the signature sequence in each amidase is given by the numbers just preceding and following the sequence information. The assigned SEQ ID NOs for each of the sequences are listed in the legend to the Figure in Brief Description of the Figures.

To our knowledge, an oleamide amidase also referred to as FAAH is the first mammalian member of this enzyme family to have been molecularly characterized.

Hydropathicity plot and transmembrane domain searches (TMpred and PSORT programs) of the rat FAAH sequence were conducted, and each search indicated a strong putative transmembrane domain from amino acids 13-29 (bold type in Figure The 50 amino acid region surrounding and encompassing the putative transmembrane domain of rat FAAH shares no homology with protein sequences of other amidase family members, indicating that one of the unique modifications of the rat amidase may be its integration into the membrane.

Interestingly, additional analysis of the FAAH sequence revealed a polyproline segment, amino acids 307-315 (double underlined in Figure that contains a precise match from positions 310 to 315 to the consensus class II SH3 domain binding sequence, PPLPXR (SEQ ID NO 38) Feng et al, Science, 266:1241-1246, 1994), suggesting that other proteins may interact with FAAH to regulate its activity (Pawson, Nature, 373:573-580, 1995) and/or subcellular localization (Rotin et al, EMBO 13:4440-4450, 1994).

Southern and Northern blot analyses were conducted with an internal 800 bp fragment of the rat FAAH cDNA to evaluate the genomic copy number and tissue distribution of FAAH, respectively.

For the Southern blot, 10 pg of rat genomic DNA was WO 98/20119 PCT/US97/20385 53 digested with the indicated restriction enzymes (100 units each) for 12 hours and then run on a 0.8% agarose gel. Rat genomic DNA was first isolated from rat liver as follows: approximately 500 mg of rat liver was shaken overnight at 55'C in 2 ml of 100 mM Tris (pH 0.2% SDS, 200 mM NaC1, and 0.2 mg/ml of proteinase K. The mixture was then spun at 15,000 rpm for minutes and the supernatant was removed and treated with an equal volume of isopropanol. The precipitated genomic DNA was removed, partially dried, and resuspended in water by heating at 1*8 55C for 4 hours. 10 Ag of the DNA was digested with the indicated restriction enzymes (100 units each) for 12 hours, and then run on a 0.8% agarose gel. The DNA was then transferred under capillary pressure to a GeneScreenPlus hybridization transfer membrane (DuPont NEN) for use in Southern blot analysis. The blot was handled according to manufacturer's (Clontech) guidelines and subjected to the following S post-hybridization washes: one 20 minute wash in a solution of 1% SDS and 0.2 X SSC (30 mM NaC1, 3.0 mM sodium citrate, pH at 25'C, followed by two 20 minute washes in a solution of 0.1% SDS and 0.2 X SSC at 65'C and one additional post-hybridization wash SDS, 0.1 X SSC, pH 7.0) at 65 0 C for 1 hour. The blot was then exposed to X-ray film for 12 hours at -78'C.

Southern blot studies showed that the FAAH probe hybridized primarily to single DNA fragments using several different restriction digests of the rat genome (Figure 12A). As expected, two hybridizing bands were observed in the HindIII digested DNA, as the FAAH probe contained an internal HindIII site. These results are most consistent with the FAAH gene being a single copy gene.

For Northern analyses, blots obtained from Clontech were WO 98/20119 PCT/US97/20385 54 handled according to manufacturer's guidelines, except that an additional post-hybridization wash with a solution of 0.1% SDS and 0.1 X SSC (15 mM NaC1, 1.5 mM sodium citrate, pH 7.0) at 0 C for 1 hour was conducted to ensure removal of nonspecific hybridization. The resulting blot was exposed to X-ray film for 6 hours at -78 0

C.

Northern blot analysis with the FAAH probe identified a single major mRNA transcript of approximately 2.5 kb in size that is most abundant in liver and brain, with lesser amounts present in spleen, lung, kidney, and testes (Figure 12B). This transcript was not detectable in either heart or skeletal muscle, consistent with previously reported biochemical studies identifying no anandamide hydrolase activity in these two tissues (Deutsch et al, Biochem. Pharmacol., 46:791-796, 1993).

1S' The Northern blot also contained low level hybridization of the FAAH cDNA probe to a few larger transcripts present only in those tissues expressing the 2.5 kb transcript as well. These transcripts may be either unprocessed or alternatively spliced forms of the 2.5 kb mRNA. In addition, the regional distribution of the rat FAAH transcript in the rat brain was examined by Northern analysis revealing highest level of the hippocampus and thalamus with lower levels of transcript detectable in other regions of the brain, including olfactory bulb, cortex, cerebellum and pituitary. Preliminary in situ hybridization analysis of rat brain slices has also identified high expression levels for rat FAAH in both hippocampus and hypothalamus. Lastly, Northern analysis of mouse FAAH expression levels at various stages in mouse embryonic development was performed where the mouse FAAH was first 0 observed between days 11 and 15 with levels continuing to increase dramatically from day 15 to 17.

WO 98/20119 PCT/US97/20385 55 2. Cis-9.10-Octadecenoamidase cDNA Obtained from Mouse Liver mRNA The mouse homolog of the rat cis-9,10- Octadecenoamidase cDNA was obtained from screening a mouse liver 5'-stretch plus cDNA library (Clontech) using the same conditions as described above for obtaining the rat cDNA with the one exception that the entire rat cDNA (Figure 10-1 through 10-5) was used as the labeled probe.

The resultant mouse double-stranded 1959 bp cDNA homolog and encoded amino acid residue is shown in Figure 13-1 through 13-4 with the ATG start site beginning at nucleotide position 7 indicated with the boxed methionine residue. The stop codon, TGA, is similarly boxed as shown on Figure 13-4 at nucleotide positions 1744 to 1746 followed by the 3' untranslated region. The top and bottom strands of the cDNA sequence are also respectively listed as SEQ ID Nos 39 and 41.

The encoded amino acid sequence is shown in with the top strand in SEQ ID NO 39 and again by itself in SEQ ID NO 3. Cis-9.10-Octadecenoamidase cDNA Obtained from Human Liver mRNA A cDNA clone for the human homolog of cis-9,10octadecenoamidase was similarly obtained as described above for the rat by 'screening a human liver 5' stretch plus cDNA library (Clontech) with the exception that the entire rat cDNA prepared above was used as the labeled probed and less stringent hybridization (25% instead of 50% formamide in the manufacturer's recommended hybridization buffer) was employed.

Washing conditions also included 2X SSC containing 0.1% SDS at 50°C instead of 1 X SSC containing 0.1% SDS at 65 0

C.

The resultant human double-stranded 2045 bp cDNA homolog WO 98/20119 PCT/US97/20385 56 and encoded amino acid residue is shown in Figures 14-1 through 14-5 with the ATG start site beginning at nucleotide position 36 indicated with the boxed methionine residue. The stop codon, TGA, is similarly boxed as shown on Figure 14-4 at nucleotide positions 1773 to 1775 followed by the 3' untranslated region. The top and bottom strands of the cDNA sequence are also respectively listed as SEQ ID Nos 42 and 44.

The encoded amino acid sequence is shown in with the top strand in SEQ ID NO 42 and again by itself in SEQ ID NO 43.

E. Preparation of Expressed Recombinant the Fatty Acid Amide Hydrolase Cis-9.10-Octadecenoamidase: For preparing recombinant FAAH proteins for use in this invention, the rat, mouse and human cDNAs obtained above were separately cloned into the eukaryotic expression vector pcDNA3 for transient expression studies in COS-7 cells.

*.For preparing the rat, mouse and human FAAH recombinant protein, the corresponding FAAH cDNAs were excised from the Bluescript II vectors and separately ligated into the eukaryotic expression vector, pcDNA3 (Invitrogen, San Diego, CA). 100 mm dishes of COS-7 cells were grown at 37 0 C to 70% confluency in complete medium (DMEM with L-glutamine, non-essential amino acids, sodium pyruvate and fetal bovine serum). The COS-7 cells were then washed with serum-free medium and treated with 5 ml of transfection solution (5-6 Ag of FAAH-pcDNA3 vector were preincubated with transfectamine (Gibco-BRL) for 30 minutes in 1 ml of serum free medium, then diluted to a final volume of 5 ml with serum free medium). The COS-7 cells were incubated at 37 0

C

for 5 hours, at which point 10 ml of complete medium was added to the cells and incubation was continued at 37 0 C for 12 hours.

The transfection solution was then aspirated away from the COS-7 WO 98/20119 PCT/US97/20385 57 cells, and the cells were incubated in a fresh batch of complete medium for another 24 hours. The COS-7 cells were harvested with a cell scraper, pelleted at low speed, washed twice with 1 mM NaHCO 3 and resuspended in 200 pl of 1 mM NaHCO 3 The resuspended COS-7 cells were dounce homogenized 12 times and pl of the resulting cell extract was used to assay for oleamide hydrolase activity (assay is detailed above in Section B6) with the results as described below in Section F. Control COS-7 9. ~cells were prepared identically except that the pcDNA3 vector used for transfection contained the FAAH cDNA in reverse orientation.

The resultant expressed recombinant FAAH proteins for rat, human and mouse are then used as described below to assess specificity and enzymatic activity.

Fatty Acid Amide Hydrolase Specifificty and Activity of the Expressed Recombinant Fatty Acid Amide Hydrolases As described above, the transfected COS-7 cells were lysed to generate a cell extract for each of the recombinant expressed rat, mouse and human FAAH proteins of this invention.

While untransfected COS-7 cells contained negligible amounts of oleamide hydrolase activity, COS-7 cells transfected with the rat FAAH cDNA expressed high levels of oleamide hydrolase activity (Figure 15A). The assay was performed as described in Section B where by TLC the conversion of oleamide to oleic acid was assessed. As shown in Figure 15A, COS-7 cells transiently tranfected with rat oleamide hydrolase cDNA in expression vector pcDNA 3 shown in lane 3 but not in untransfected COS-7 cells (lane 1) or control transfected cells WO 98/20119 PCTIUS9720385 58 (lane 2, transfected with pcDNA3 containing the oleamide hydrolase cDNA in reverse orientation), were effective at converting labeled oleamide to oleic acid. Similar results were obtained with COS-7 cells transiently transfected with human oleamide hydrolase as shown in Figure 16 where the conversion to oleic acid is seen only in lane 2 as compared to control COS-7 cells in lane 1.

This enzyme activity, like the rat liver plasma membrane oleamide hydrolase activity, was inhibited by trifluoromethyl ketone as evidenced in Figure 15B as shown in lane 2 of the figure the rat oleamide hydrolase-transfected COS-7 cells in the presence of 50 AM trifluoromethyl ketone as compared to the untreated extract in lane 1.

To confirm specificity of the expressed recombinant proteins, Western blot analyses with anti-FAAH polyclonal antibodies alone or in the presence of competing peptides were S. performed. Samples of cell extract from rat FAAH-transfected and untransfected COS-7 cells with approximately equal protein amounts were heated to 65 0 C for 10 minutes in loading buffer 0" with 2% SDS and 5% P-mercaptoethanol. The samples indicated above were then run on an 8-16% polyacrylamide gradient Tris-glycine gel, and transferred to nitrocellulose for Western blotting. The nitrocellulose blot was blocked with 5% Blotto in TBS-Tween overnight at 4 0 C, and then incubated with polyclonal antibodies generated against peptide 2 as previously described gg/ml in TBS-Tween) generated against an internal

FAAH

peptide sequence for 2 hours at 25 0 C. The blot was then washed in TBS-Tween incubated with a secondary antibody-horseradish peroxidase conjugate for 30 minutes at OC, washed again in TBS-Tween, and developed with Stable Peroxide Solution and Luminol/Enhancer Solution (Pierce). Peptide WO98 2 0119 PCT/US97/20385 59 competition experiments were conducted by preincubating 1000-fold molar excess of the peptide antigen corresponding to peptide 2 as previously described with polyclonal antibodies for minutes prior to addition of antibodies to the blot.

Western blotting of the rat cDNA transfected COS-7 cell extract with polyclonal antibodies generated against the internal peptide 2 sequence of FAAH showed a 60-65 kDa immunoreactive band that comigrated with affinity-isolated

FAAH

on SDS-PAGE (Figure 15C). Untransfected COS-7 cell extract P" contained no detectable immunoreactive protein band of this .ooo o size. Additionally, the immunoreactivity of the 60-65 kDa protein was effectively competed away by preincubation of the antibodies with excess peptide antigen (Figure 15C), while the *o "l trace quantities of cross reactive protein observed in both the transfected and untransfected COS-7 cell extracts were not competed by this peptide.

Previous work suggested that the enzyme activity that hydrolyzes oleamide may be the same activity that converts S anandamide (arachidonyl ethanolamide) to arachidonic acid.

Therefore, COS-7 cells transfected with the rat FAAH cDNA were assayed for anandamide hydrolase activity. To assess the enzymatic activity of the expressed recombinant fatty acid amide hydrolases of this invention on labeled anandamide, the following enzymatic assay was performed. "C-anandamide was synthesized as follows: 12.5 iCi (specific activity of ACi/jiM) of 14 C arachidonic acid (Moravek Biochemicals) was dissolved in 100 Al CH 2 Cl 2 cooled to 0°C, and treated with excess oxalyl chloride. The reaction mixture was stirred at 25 0

C

for 6 hours, after which time the solvent was evaporated. The remaining residue was cooled to 0 C, treated with a large excess of ethanolamine, and stirred at 25 0 C for 15 minutes. The WO 9820119 PCT/US97/20385 60 reaction mixture was then partitioned between ethyl acetate and 2 M HC1, and the organic layer was washed with water and then evaporated to dryness. The resulting "C-anandamide was diluted with unlabeled anandamide to a final specific activity of pCi/IM in ethanol. Approximately 1 gCi of "C-anandamide and Al of dounce homogenized COS-7 cell extract were used for each anandamide hydrolase assay as detailed above for the oleamide hydrolase assays. Briefly, FAAH hydrolysis assays were conducted in triplicate with 100 iM substrate, 35 Ag of rat transfected COS-7 cell protein for 5 minutes at 37 0 C (except in the case of stearic amide, where due to low solubility, 20 iM substrate comparison to oleamide were conducted). Products were separated on TLC as described previously, scraped into •g ~scintillation fluid, and radioactivity was quantitated by scintillation counting. Substrate hydrolysis in the presence of equal amounts of untransfected COS-7 cell protein extract served as background control in all cases and was substracted from FAAH hydrolysis rates to give the data as presented below.

The results of the anandamide assays showed that while untransfected COS-7 cells contained negligible quantities of anandamide hydrolase activity, transfected COS-7 cells produced high levels of anandamide hydrolase activity-(Figure 17). Thus, FAAH has the capacity to hydrolyze both oleamide and anandamide, indicating that the amidase may act as a general degradative enzyme for the fatty acid amide family of signaling molecules.

The substrate promiscuity of FAAH is reminiscent of the monoamine oxidase enzymes which serve to oxidize a variety of amine-containing neurotransmitters.

To further assess the substrate specificity spectrum of enyzmatic hydolytic activity of the recombinant expressed proteins of this invention, other 4 C-labeled fatty acid amides WO 98/20119 PCT/US97/20385 61 were synthesized as described in Section B6 and above for 4

C-

oleamide, with the exception of anandamide as described.

The results showed that while recombinant expressed rat FAAH catalyzes the hydrolysis of oleamide and anandamide at approximately equal rates, FAAH does discriminate among fatty acid amides, as FAAH hydroylzes other representative fatty acid amides, including myristic amide, palmitic amide and stearic amide at a significantly reduced rate as compared to that seen with oleamide or anandamide as shown in Table 1 below. Where b indicated in the table the anandamide and oleamide hydrolysis rates are considered to be 100% of FAAH activity to which other fatty acid amide hydrolysis rates are compared.

Table 1 Substrate Rate of Hydrolysis* Anandamide (100 pM) 333 30 100 Oleamide (100 iM) 242 20 72.6 Myristic Amide (100 AM) 81 7 24.3 Palmitic Amide (100 AM) 33 2 9.9 Oleamide (20 AM) 41 2 100 Stearic Amide (20 AM) 2.3 1 5.8 Rate is measured in nmol/min/mg for each Comparable assays are performed with the mouse and human recombinant homologs to the rat enzyme as used above.

Thus, as shown above, the rat FAAH enzyme was not without substrate preference, albeit it did exhibit activity against a number of amide substrates. The degree to which FAAH showed substrate selectivity is best exemplified by the nearly twenty fold rate difference between the enzyme's hydrolysis of oleamide WO 9820119 PCT/US97/20385 62 and steric amide, two compounds that only differ by a single degree of unsaturation at the A9 position. This pattern was also confirmed with assays with the inhibitor trifluoromethyl ketone that was a twenty fold stronger inhibitor of FAAH than for the corresponding trifluoromethyl ketone analog of stearic amide. Thus, FAAH significantly favors the bent alkyl chain of oleamide over the straight alkyl chain of stearic amide.

o*o. A deletion mutant for generating a soluble form of the FAAH molecules of this invention was also prepared. A construct was created in which the putative transmembrane domain was deleted resulting in a truncated FAAH beginning at amino acid residue of the encoded protein rather than 1. To prepare this construct, the following primers were designed for PCR amplification of the 5' end of rat FAAH cDNA lacking the first 140 bp encoding the amino terminal 30 amino acids of FAAH. The 5' and 3' primers had the respective nucleotide sequences 5'GCGGTACCATGCGATGGACCGGGCGC3' (SEQ ID NO 45) encoding amino acids 30-35 and containing a KpnI site and an artificial stop codon and 5'GGTCTGGCCAAAGAGAGG3' (SEQ ID NO 46) where its reverse complement encodes amino acids 199-204.

The amplified transmembrane deleted rat FAAH cDNA fragment was then digested with the appropriate restriction enzymes (KpnI and HindIII) and cloned into the similarly digested FAAHpBluescript vector replacing the original cDNA 5' end. The deleted construct was confirmed by sequencing and then excised and transferred to pcDNA3 for expression studies as described herein.

For expression, the transfected COS-7 cell extract was separated into soluble and membrane fractions as follows: the extract was spun at 2500 rpm for 5 minutes at 25 0 C and the supernatant was transferred to an airfuge tube and spun in an 63 ultracentrifuge (30 psi for 40 minutes at 4 0 C) for preparing soluble supernatant. The pellet contained the membrane bound fraction that was then resuspended in a volume of 1 mM NaHCO 3 equal to the volume of the supernatant.

The transmembrane-deleted expressed recombinant FAAH was functional in COS-7 cell expression assays as described above. The mouse and human transmembrane truncation homologs of the rat cDNA are similarly prepared and used in practicing this invention.

Given the increasing number of studies demonstrating biological activities for various members of the fatty acid amide family of signaling molecules, the discovery of a family of fatty acid amide hydrolases (FAAH) having homology between rat, mouse and human as described herein provides a valuable invention for ongoing studies dedicated to understanding the regulation, mechanism, and pharmacology of the metabolic process that inactivates the fatty acid amides. In addition, the cloned FAAH gene in conjunction with potent FAAH inhibitors provides the ability in both elucidating the physiological pathways affected by the fatty acid amide family and developing systemic approaches towards the pharmacological intervention of these biological processes.

25 In the claims which follow and in the preceding description of the invention, except where the context '-".requires otherwise due to express language or necessary implication, the word "comprise: or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition or further features in various embodiments of the invention.

It is to be understood that a reference herein to a prior art publication does not constitute an admission that 35 the publication forms a part of the common general knowledge in the art in Australia, or any other country.

WO 98/20119 PCT/US97/20385 64 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: The Scripps Research Institute STREET: 10550 North Torrey Pines Road CITY: La Jolla STATE: -California _e COUNTRY: US ZIP: 92037 TELEPHONE: (619) 784-2937 TELEFAX: (619) 784-9399 (ii) TITLE OF INVENTION: FATTY-ACID AMIDE HYDROLASE (iii) NUMBER OF SEQUENCES: 54 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US97/ FILING DATE: 04-NOV-1997 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/743,168 FILING DATE: 04-NOV-1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/489,535 FILING DATE: 12-JUN-1995 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 783 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO WO 98/20119 WO 9820119PCT1US97/20385 65 (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .783 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i:

AGC

Ser 1

CCA

Pro OGA GOT Cly Gly TOC TCA 0CC GGT Ser Ser Gly Gly GAG CCC Oiu Gly 10 GCT CTC ATT GGA TCT OGA Ala Leu Ile Gly Ser Oly *1.12.* OCT TCC CCT CTG Cly Ser Pro Leu COT TTA Gly Leu CCC ACT Cly Thr

CAC

Asp 25 ATT CCC CCC AOC Ile Gly Gly Ser ATC CCC TTO Ile Arg Phe CCT TCT CC Pro Ser Ala TTC TCC CCC ATC TOT CCC CTC AAG Phe Cys Cly Ile Cys Gly Leu Lys 40 COT ACT CCC AAC CC Pro Thr Gly Asn Arg OTO AGC Leu Ser AAG ACT CCC OTO AAC Lys Ser Cly Leu Lys 55 CCC TOT CTC TAT GGA CAC ACO OCA OTO Gly Cys Val Tyr Gly Gin Thr Ala Val 192 240

CAG

Gin 65

TOC

Cys

OTO

Val CTT TOT CTT CCC CCC Leu Ser Leu Cly Pro 70 CTG AAA OCT CTA CTC Leu Lys Ala Leu Leu ATO CCC CCC OAT OTO Met Ala Arg Asp Val 75 TOT GAG CAC TTC TTC Cys Glu His Leu Phe 90 GAO AGO CTC CC CTA Glu Ser Leu Ala Leu ACC TTC GAO OCT ACC Thr Leu Asp Pro Thr OCT CCC TTT Pro Pro Phe 100 CCC TTO AGA GAO GAG Pro Phe Arg Ciu Clii 105 GTC TAT AGA ACT TOT AGA COO Val Tyr Arg Ser Ser Arg Pro 110

OTO

Leu CT GTG Arg Val 115 CCC TAO TAT GAG ACT Gly Tyr Tyr Giu Thr 120 GAO AAC TAT ACC ATO Asp Asn Tyr Thr Met 125 CCC AGO OCA Pro Ser Pro 384 WO 98/20119 WO 9820119PCT/US97/20385 66 GGT ATG Ala Met 130 GGC GAG Gly His 145

AGG

Arg AGO GCT CTG ATA Arg Ala Leu Ile 135 GAG ACC AAG GAG AGA CTT GAG GCT OCT Giu Thr Lys Gin Arg Leu Giu Ala Ala 140 432 480 ACG GTG ATT CCC Thr Leu Ile Pro 150

TTC

Phe

TTA

Leu CCC AAC AAC ATA CCC TACG CC GTG Pro Asn Asn Ile Pro Tyr Ala Leu 155 160 1~

C

GAG GTC GTG TCT GG Glu Val Leu Ser Ala 165 CTG CAA AAG TTC AAA Leu Gin Asn Phe Lys 180 ATC TTA ATT GTG AGG Ile Leu Ile Leu Arg 195 GCC GOC GTG TTC ACT Gly Gly Leu Phe Ser 170 GGT GAG TTT GTG GAT Gly Asp Phe Val Asp 185 GAG GGT GGC Asp Gly Gly CGC AGT TTT Arg Ser Phe 175 CCC TGC TTG GGA GAG GTG Pro Gys Leu Gly Asp Leu 190 CTG CCC Leu Pro AGC TGG TTT AAA AGA GTG CTG AOC GTG Ser Trp Phe Lys Arg Leu Leu Ser Leu 200 205 CCC CTO OCA CCC TTT CTG AAC AGT ATG Arg Leu Ala Ala Phe Leu Asn Ser Met 220 GTG CTG Leu Leu 210 AAO CCT CTG TTT CCT Lys Pro Leu Phe Pro 215 GGT GCT CCC Arg Pro Arg 225 TCA GGT GAA AAO Ser Ala Giu Lys 230 CTG TGC AAA CTG Leu Trp Lys Leu 235 GAG CAT GAG ATT GAG Gin His Giu Ile Giu 240 ATG TAT CCCGAG TOT Met Tyr Arg Gin Ser 245 GTG ATT GGC GAG Val Ile Ala Gin TGG AAA GCG ATO AAG TTG GAT Trp Lys Ala Met Asn Leu Asp 250 255 768 GTG GTG CTG AGO TAA Val Leu Leu Thr 260 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: WO098/20119 -67- LENGTH: 260 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: PCTIUS97/20385 Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ser Gly Gly Ser Pro Pro Ser Ala Leu Gly Leu Gly Thr Asp Ile 25 Gly Gly Ser Ile Arg Phe Gly Asn Arg Phe Cys Gly Ile Cys 40 Gly Leu Lys Pro Thr Leu Ser Lys Ser Gly Leu Lys 55 Gly Cys Vai Tyr Gly Gin Thr Ala Val Gin Leu Ser Leu Gly Pro 70 Met Ala Arg Asp Val 75 Giu Ser Leu Ala Leu Cys Leu Lys Ala Leu Cys Glu His Leu Ph e Thr Leu Asp Pro Thr Val Pro Pro Leu Arg Val 115 Phe 100 Pro Phe Arg Glu C iu 105 Val Tyr Arg Ser Ser Arg Pro 1 L0 Pro Ser Pro Gly Tyr Tyr Giu Thr 120 Asp Asn Tyr Thr Met 125 Ala Met 130 Arg Arg Ala Leu Ile 135 Giu Thr Lys Gin Leu Pro Asn Asn 155 Arg 140 Leu Giu Ala Ala Gly 145 His Thr Leu Ile Pro Phe 150 Ile Pro Tyr Ala Giu Val Leu Ser. Ala Gly Gly Leu Phe Ser Asp Gly Gly Arg Ser Phe 165 170 175 WO 98/20119 PCT/US97/20385 68 Leu Gln Asn Phe Lys Gly Asp Phe 180 Val 185 Asp Pro Cys Leu Gly Asp Leu 190 Ile Leu Ile 195 Leu Arg Leu Pro Ser 200 Trp Phe Lys Arg Leu 205 Leu Ser Leu Leu Leu 210 Lys Pro Leu Phe Pro 215 Arg Leu Ala Ala Phe 220 Leu Asn Ser Met .t.

Arg 225 Pro Arg Ser Ala Glu 230 Lys Leu Trp Lys Leu 235 Gin His Glu Ile Glu 240 Met Tyr Arg Gin Ser 245 Val Ile Ala Gin Lys Ala Met Asn Leu Asp 255 eo Val Leu Leu Thr 260 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CGGAATTCGG NGGNGARGGN GC INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 98/20119 PCTUS9M20385 69 FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Gly Gly Glu Gly Ala 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH; 31 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu lie Gly Ser Gly Gly Ser '1 5 10 Pro Leu Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe Pro 20 25 INFORMATION FOR SEQ ID NO:6: SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Gly Ser 1 5 10 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear WO 98 2 0119 PCT/US97/20385 70 (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ala Leu Ile Gly Ser Gly Gly Ser Pro Leu Gly Leu Gly Thr Asp 1 5 10 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Gly Leu Gly Thr Asp Ile Gly Gly Ser Ile Arg Phe Pro Ser Ala 1 5 10 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear 5 (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Arg Phe Pro Ser Ala Phe Cys Gly Ile Cys Gly Leu Lys Pro Thr 0 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal WO 98/20119 PCTYUS97/20385 71 (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Leu Lys Pro Thr Gly Asn Arg Leu Ser Lys Ser Gly Leu Lys 1 5 10 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly Gin Thr Ala Val Gin 1 5 10 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Gln Thr Ala Val Gln Leu Ser Leu Gly Pro Met Ala Arg Asp Val 1 5 10 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: WO 98/20119 PCT/US97/20385 72 Met Ala Arg Asp Val Glu Ser Leu Ala Leu Cys Leu Lys Ala Leu 1 5 10 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Cys Leu Lys Ala Leu Leu Cys Glu His Leu Phe Thr Leu Asp Pro 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid 2 TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Phe Thr Leu Asp Pro Thr Val Pro Pro Phe Pro Phe Arg Glu Glu 1 5 10 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Pro Phe Arg Glu Glu Val Tyr Arg Ser Ser Arg Pro Leu Arg Val 1 5 10 WO 98/20119 PCT/US97/20385 73 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Arg Pro Leu Arg Val Gly Tyr Tyr Glu Thr Asp Asn Tyr Thr Met 1 5 10 I" INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Asp Asn Tyr Thr Met Pro Ser Pro Ala Met Arg Arg Ala Leu Ile S1 5 10 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Arg Arg Ala Leu Ile Glu Thr Lys Gln Arg Leu Glu Ala Ala Gly 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: WO 98/20119 PCT/US97/20385 74 LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Glu Ala Ala Gly His Thr Leu Ile Pro Phe Leu Pro Asn Asn 1 5 10 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Phe Leu Pro Asn Asn Ile Pro Tyr Ala Leu Glu Val Leu Ser Ala

S

1 5 10 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Glu Val Leu Ser Ala Gly Gly Leu Phe Ser Asp Gly Gly Arg Ser 1 5 10 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid WO 98/20119 PCT/US97/20385 75 TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Asp Gly Gly Arg Ser Phe Leu Gin Asn Phe Lys Gly Asp Phe Val 1 5 10 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Lys Gly Asp Phe Val Asp Pro Cys Leu Gly Asp Leu lie Leu lle 1 5 10 *o INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Leu lie Leu lie Leu Arg Leu Pro Ser Trp Phe Lys Arg Leu 1 5 10 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 98/20119 PCT/US97/20385 76 FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Trp Phe Lys Arg Leu Leu Ser Leu Leu Leu Lys Pro Leu Phe Pro 1 5 10 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Lys Pro Leu Phe Pro Arg Leu Ala Ala Phe Leu Asn Ser Met Arg 1 5 10 INFORMATION FOR SEQ ID NO:28: 2b*' SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids S. TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Leu Asn Ser Met Arg Pro Arg Ser Ala Glu Lys Leu Trp Lys Leu 1 5 10 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: WO 98/20119 PCT/US97/20385 77 Lys Leu Trp Lys Leu Gin His Glu Ile Glu Met Tyr Arg Gin Ser 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Met Tyr Arg Gin Ser Val Ile Ala Gin Trp Lys Ala Met Asn Leu 1 5 10 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Lys Ala Met Asn Leu Asp Val Leu Leu Thr Pro Met Leu Gly Pro 1 5 10 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Pro Met Leu Gly Pro Ala Leu Asp Leu Asn Thr Pro Gly Arg 1 5 WO 98/20119 PCT/US97/20385 78 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:-NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CGGGATCCGG CATNGTRTAR TTRTC INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Asp Asn Tyr Thr Met Pro 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2472 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 50..1789 (xi) SEQUENCE DESCRIPTION: SEQ ID WO 98/20119 WO 9820119PCTIUS97/20385 79 GGTTTCTGCG AGCGAGTTC TCTCGGGTOG CGOTOGGCTG CAGOAGATO ATG GTG Met Val CTO AGO GAA GTO TOG ACG AG CTO TOT 000 OTO TOC 000 OTT TG OTA 103 Leu Ser Glu Val Trp Thr Thr Leu Ser Cly Val Set Gly Val Gys Leu 0CC Ala

TG

Gys AOC TTO Set Leu TTG TGG0CC Leu Set Ala 25 0CC Ala OTG GTC CTO CGA TOO ACC 000 CG Val Val Leu Arg Trp Thr Gly Arg GAG AAG 0CC COG 0CC OCO Gin Lys Ala Arg Gly Ala 35 40 AGO OTO GAG AGC ATO GAG Set Leu Clu Thr Met Asp GGT GAG CTO GAG TCG GAG Pro Asp Leu Asp Set Clu 000 ACG AGO 000 CG Ala Thr Arg Ala Arg 45 CG AAG CG CA 000 Gin Lys Gin Arg Ala TTG GOGCGTG GAG AAT Phe Arg Leu Gin Asn AAO 000 OTO Lys Ala Val

GAG

Gin 60 151 199 247 295 343 GGG TTG CTO Ala Leu Leu

OTA

Val CG AAC Oln Lys TTA GAG ACT OCA GAG CTO Leu Gin Ser Gly Ciu Leu 90 ACC CTG CCC CTA GTC GAA GTG Thr Leu Pro Leu Leu Gin Leu TOG CCA GAO OCT OTO TTG TTT Set Pro Giu Ala Val Phe Phe

ACT

Th r

TAG

Tyr 100 GTG OCA PIAG 0CC TOG OAA Leu Cly Lys Ala Trp Oiu 105 OTO AAG AAA GO AGO AAG TOO OTO Val Asn Lys Gly Thr Asn Cys Val 110 391

ACC

Thr 115 TOG TAT CG ACCGCAG Set Tyr Leu Thr Asp 120 TOT GAG ACT CG CTO Gys Glu Thr Gin Leu 125 TCG CAG GCC Ser Cln Ala CCA CCC Pro Arg 130 439 CG GC CTO GTG.TAT COT OTC GCT OTO AOC GTG AAG OAA TG TTC AC Gin Cly Leu Leu Tyr Cly Val Pro Val Set Leu Lys Clu Gys Phe Set 487 WO 98/20119 P~U9I08 PCTIUS97/20385 80 135 140 TAG AAG GGC CAG GAG TGG AGA GTG Asp Ser Thr Leu Tyr Lys Gly His 150

GGG

Gly 155

GTG

Val TTG AGG GTG AAT GAG GGG ATG Leu Ser Leu Asn Giu Gly Met 160 535 GGA TGG GAA TGT GAG TOT Pro Ser Glu Ser Asp Gys 165 GTG GTG Val Val 170

GAA

Gin GTG TTG AAG GTG GAG GGA Val Leu Lys Leu Gin Gly 175 2.

GGT GTO Ala Val 180 GGG TTT OTG Pro Phe Val

GAT

His

AGG

Thr 185

AAT

Asn GTG GGG GAG TGG ATG TTA AGG TTT Val Pro Gin Ser Met Leu Ser Phe 190 583 631 679

GAG

Asp 195 TGG ACT AAG GGT GTG Cys Ser Asn Pro Leu 200 TTT GG GAG AGG ATO Phe Gly Gin Thr Met 205 AAG GGA TGG AAO TC Asn Pro Trp Lys Ser 210 dOG GCT GTG ATT GGA Gly Ala Leu Ile Gly 225 TGG AAO Ser Lys AGG GGA GGA Ser Pro Gly 215 GGT TGG TGA Gly Ser Ser GGG GOT Gly Gly 220 TGT GGA GGT TC Ser Oly Oly Ser 230

GGT

Pro

GTO

Leu TTA GGG Leu Gly 235 AGT GAG ATT Thr Asp Ile GGCGC AGG ATG Gly Gly Ser Ile 240 AAO GGT AGT GG Lys Pro Thr Gly 255 GGG TTG GGT Arg Phe Pro 245 TGT GGG TTG Ser Ala Phe TG dG Gys Oly 250 ATG TGT GGC GTG Ile Gys Oly Leu AAG GG Asn Arg 260 GTG AGC AAG Leu Ser Lys AGT GGG Ser Gly 265 GTG AAO dG TGT GTG TAT GGA GAG AG Leu Lys Giy Gys Val Tyr Oly Gin Thr 270

GGA

Ala 275 GTO GAG GTT TGT OTT GCG Val Gin Leu Ser Leu Oly 280 GGG ATG GGG GGG Pro Met Ala Arg 285 OAT GTO Asp Val GAG AGG Giu Ser

GTG

Leu 290 919 WO 98/20119 PCTLUS97/20385 81 GC CTA TGC CTG AAA GOT CTA CTG TOT Cys Ala Leu Cys Leu COT ACC GTG OCT Pro Thr Val Pro 310 Ala Leu Leu

GAG

Ciu 300 GAO TTG TTC AGO TTO GAO His Leu Phe Thr Leu Asp 305 967 1015 COG TTG CCC TTC AGA Pro Leu Pro Phe Arg 315 GAO GAG OTO TAT AGA ACT TOT Glu Oiu Val Tyr Arg Ser Ser 320

AGA

Arg CCC OTO Pro Leu 325 CT GTO GO TAO Arg Val Cly Tyr

TAT

Tyr 330 GAO ACT GAG AAC TAT Ciu Thr Asp Asn Tyr 335 AGO ATG Thr Met 1063 1111 AGO OCA Ser Pro 340 GOT ATO AGO AOG OCT Ala Met Arg Arg Ala 345 OTO ATA GAG AGO AAG CG AGA OTT GAG Leu Ile Ciu Thr Lys Gin Arg Leu Olu 350

GOT

Ala 355 CT CCC GAG AGO Ala Gly His Thr OTO ATT COG TTO TTA CCC Leu Ile Pro Phe Leu Pro 360 365 TOT CO GC GGG OTO TTG Ser Ala Oly Gly Leu Phe 380 AAG AAG ATA CCC TAO Asn Asn Ile Pro Tyr 370 CO OTO GAG CTC OTG Ala Leu Ciu Val Leu 375 ACT TTT OTO CAA AAG Ser Phe Leu Gin Asn 390 ACT GAG GOT Ser Asp Cly GC C00 Cly Arg 385 1159 1207 1255 TTG AAA GGT GAG TTT GTC CAT Phe Lys Oly Asp Phe Val Asp 395 COG TG TTC GGA Pro Gys Leu Oly 400

GAG

Asp OTG ATO Leu Ile 405

TTA

Leu ATT OTO AGO Ile Leu Arg OTO CCC AGO TOG TTT AAA AGA OTO OTO Leu Pro Ser Trp Phe Lys Arg Leu Leu 410 415 TTT OCT 000 OTO GA CG TTT CTO AAO Phe Pro Arg Leu Ala Ala Phe Leu Asn 430 AGO OTO Ser Leu 420 OTO CTO AAO Leu Leu Lys COTT CCC Arg Pro Arg COT OTO Pro Leu 425 1303 1351 1399

ACT

Ser

ATO

Met TOA OCT GAA AAGCOTO TOO AAA OTO GAG CAT GAG Ser Ala Clu Lys Leu Trp Lys Leu Gin His Oiu WO 98/20119 WO 9820119PCT1US97/20385 82 435 440 445 450 ATT GAG Ile Giu ATG TAT CC CGAG TCT GTG ATT GC CAC TGG AAA CCG Gin Trp Lys Ala Met Tyr Arg 455 Gin Ser Val Ile Aia 460 ATG AAG Met Asn 465 1447 1495 TTG GAT GTG Leu Asp Val

GTG

Leu 470 CG ACC CCC ATO TTG Leu Thr Pro Met Leu 475 GC CC T GCT OTO GAT TTO AAG Cly Pro Ala Leu Asp Leu Asn 480

ACA

Thr CCC GCC Pro Giy 485 AGA CCC ACA CCC OCT Arg Ala Thr Gly Ala 490 ATC AC TAG AGO GTT Ile Ser Tyr Thr Val 495 CTC TAG AAC Leu Tyr Asn TG CTO Gys Leu 500 GC TTC GGT C Asp Phe Pro Ala

CCC

Gly 505

GAA

Giu OTO GTG OCT OTC AGO ACT CTG ACCG CC Val Val Pro Val Thr Thr Val Thr Ala 510

GAG

Giu 515 GACGOAT GGG CG ATC Asp Asp Ala Cln Met 520 CTC TAG AAA GOC TAG TTT Leu Tyr Lys Oly Tyr Phe 525 COO OAT ATC Giy Asp Ile 530 1543 1591 1639 1687 1735 TOO GC ATC ATG CTO Trp Asp Ile Ile Leu 535 OTO OCT OTO CG TG Val Ala Val Cmn Cys 550 AAO AAO CCC ATO AAA Lys Lys Ala Met Lys 540 OTO OCT OTO COG TOO Val Ala Leu Pro Trp 555 AAT ACT GC GOT CTO OCT Asn Ser Val Cly Leu Pro 545 CG OAA GAG CTO TOT CG Gin Clu Olu Leu Cys Leu 560 AGO TTG Arg Phe ATO CCC Met Arg 565 GAG OTO GAA CG Clu Val Olu Cmn 570 CG ATO AGO OCT CAA AAO CG CCA Leu Met Thr Pro Cmn Lys Gin Pro 575 1783

TCG

Ser TCACGTCCT TCATCCOCGA GCCTGGAOC AGGTAAOCC CATGCOCTOT 1836 580 WO 98/20119 WO 9820119PCTIUS97/20385 83

GCACTGTAGC

GACGTGTCTA

CC CGTTAT CC

CGTGGCCCAA

CATTAGGCCC

TAAC CACATC

ACGGACATGT

CTTGATCGAC

TTTATGGCTC

GACCTCACTC

CATTAAAGGC

CCCATCTATT

CCTGCCCTCC

TCTACTTTCC

CGATCACCAA

CTCCGAACCA

ACTCTCCTC

CCTTCCCACT

CCTCCCCCTC

CTCTATTTCT

TCTACATGAG

GTATGCCACC

CAGGAGCCAC

CCTGGACTCC

ATCCTGATTC

CATTCAAAAA

GACTCCTGGC

TCCAAAGCCT

TGACTCCTGT

CACTTCCTTC

TGTCGACACA

CCTGGCTTTC

ACAAAGAAAA

CACCCACGAC

TGCAGCCACA

CCTGCTTTTT

CAATGCGTTT

AAGCCTGTCC

CCCTACTTCT

CCTTCCTTTC

CTCAGTCCAC

ACCTTTCTCT

CCAACCCCCA

ACCAAGTCTC

ATGGCAGCCA

ATCTATTTTC

AGACCCTCCA

GTCACCCACA

TTATTCAGAT

CTCTCTGCCG

CAGTAGCCCT

GCACAGCGAA

GACCTTCCTC

GCAGGAATGA

TGGGTATCTC

GAGCTGCCTG

AGATAGACAC

TGACCCCAGC

ACACGCCCTT

GGCTGTCCAG

1896 1956 2016 2076 2136 2196 2256 2316 2376 2436 2472 AACTCACAAC GCTGCCTCCC TGGGTGCTGG

AAAAAA

INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 579 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36-: Met Val Leu Ser Clu Val Trp Thr Thr Leu Ser Gly Val Ser Gly Val 1 5 10 Cys Leu Ala Cys Ser Leu Leu Ser Ala Ala Val Val Leu Arg Trp Thr 25 Gly Arg Cln Lys Ala Arg Gly Ala Ala Thr Arg Ala Arg Gln Lys Gln 40 WO 98/20119 PTU9108 PCTIUS97/20385 84 Arg Ala Ser Leu Glu Thr Met Asp Lys Ala Val Gin Arg Phe Arg Leu Gin Asn Pro Asp Leu Asp 70 Ser Glu Ala Leu Leu 75 Thr Leu Pro Leu Leu Gin Leu Val Gin Leu Gin Ser Gly Glu Leu Ser Pro Glu Al a Val Phe Phe Thr Gys Val Thr 115 Tyr 100 Leu Gly Lys Ala Trp 105 Giu Val Asn Lys Gly Thr Asn 110 Ser Gin Ala Ser Tyr Leu Thr Asp 120 Cys Glu Thr Gin Leu 125 Pro Arg 130 Gin Gly Leu Leu Tyr 135 Gly Val Pro Val Ser 140 Leu Lys Glu Gys Ser Tyr Lys Gly His 150 Asp Ser Thr Leu Gly 155 Leu Ser Leu As-n Gly Met Pro Ser Giu 165 Ser Asp Cys Val Val Gin Val Leu Lys Leu 175 Gin Gly Ala Ser Phe Asp 195 Val 180 Pro Phe Val His Thr 185 Asn Val Pro Gin Ser Met Leu 190 Asn Pro Trp Gys Ser Asn Pro Leu 200 Phe Gly Gin Thr Met 205 Lys Ser 210 Ser Lys Ser Pro Gly 215 Gly Ser Ser Gly Gly Giu Gly Ala Leu 220 Ile 225 Gly Ser Gly Gly Ser 230 Pro Leu Gly Leu Gly 235 Thr Asp Ile Gly Gly 240 Ser Ile Arg Phe Pro 245 Ser Ala Phe Gys Gly 250 Ile Gys Gly Leu Lys Pro 255 WO 98/20119 WO 9820119PCT[US97/20385 8S Thr Gly Asn Arg Leu Ser Lys Ser 260 C ly 265 Leu Lys Gly Cys Val Tyr Gly 270 Asp Val Glu Gin Thr Ala 275 Val Gin Leu Ser Leu 280 Gly Pro Met Ala Arg 285 Ser Leu 290 Ala Leu Cys Leu Lys 295 Ala Leu Leu Cys Glu 300 His Leu Phe Thr Leu 305 Ser Asp Pro Thr Val Ser Arg Pro Leu 325 Pro Leu Pro Phe Arg 315 Giu Glu Val Tyr Arg 320 Arg Val Gly Tyr Tyr 330 Glu Thr Asp Asn Tyr Thr .335 Met Pro Ser Pro Ala Met Arg 340 Ala Gly His Thr Arg Ala 345 Leu Ile Glu Thr Lys Gin Arg 350 Asn Asn Ile Leu Giu Ala 355 Leu 360 Ile Pro Phe Leu Pro 365 Pro Tyr 370 Al a Leu Glu Val Leu 375 Ser Ala Gly Gly Leu 380 Phe Ser Asp Gly Gly 385 Arg Ser Phe Leu Gin 390 Asn Phe Lys Gly Asp 395 Phe Val Asp Pro Cys 400 Leu Gly Asp Leu Ile 405 Leu Ile Leu Arg Leu 410 Pro Ser Trp Phe Lys Arg 415 Leu Leu Ser Leu 420 Leu Leu Lys Pro Leu 425 Phe Pro Arg Leu Ala Ala Phe 430 Leu Asn Ser Met Arg Pro Arg 435 Ser 440 Ala Giu Lys Leu Trp Lys Leu Gin 445 His Glu 450 Ile Glu. Met Tyr Arg 455 Gin Ser Val Ile Ala 460 Gin Trp Lys Ala WO 98/20119 PCT/US97/20385 86 Met Asn Leu Asp Val Leu Leu Thr Pro Met Leu 475 Gly Pro Ala 465 470 Leu Asp 480 Val Leu 495 Leu Asn Thr Pro Gly 485 Arg Ala Thr Gly Ala 490 Ile Ser Tyr Thr Tyr Asn Cys Thr Ala Glu 515 Leu 500 Asp Phe Pro Ala Gly 505 Val Val Pro Val Thr Thr Val 510 Tyr Phe Gly Asp Asp Ala Gin Met '520 Glu Leu Tyr Lys Gly 525 Asp Ile 530 Trp Asp Ile Ile Leu 535 Lys Lys Ala Met Lys 540 Asn Ser Val Gly Leu 545 Pro Val Ala Val Gin 550 Cys Val Ala Leu Pro 555 Trp Gin Glu Glu Leu 560 Cys Leu Arg Phe Met 565 Arg Glu Val Glu Gin 570 Leu Met Thr Pro Gin Lys 575 Gin Pro Ser INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 2472 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

TTTTTTTTTT

TGAGTTGAAA

AAACCTTGTC

ACTGAGGAAG

CTTTGTGGTG GCATACGCCT TTAATCCCAG CACCCAGGCA GGCAGCCTTG GCCAGCCTCA TCTACAGAGT GAGGTCCTGG ACAGCCAGGG CTACTGAGAG TCCACAACAA ATAGAGGAGC CATAAAAAGG GCGTGTCGGC AGAGAGGTGG GAAGTGCAGG GGCAGGGTCC ATCAAGGCTG GGGTCAATCT GAATAAGAAA 120 180 240 WO 98/20119 WO 9820119PCT1US97/20385 87

GGAAGGACAC

CTAGGGAGGC

AGCCTTCCCA

GCATTGTTTI

AGCAGGCAkI

GCTGCAGGAG

TGGGTGGTGG

CAGAGGTGGC

ACCTCGGCA

GCGACAGGCA

CCAAAGTAGC

GGCACCACGC

GTGGGTCTGC

TCCAAGTTCA

TGCAGTTTCC

CGAGGAAACA

AGAATTAAGA

AAACTGCGGC

ATGTTGTTGG

TCTATCAGAG

CCCACAGA

GTAGGGTCCA

TCCACATCCC

GCCTTCAGGC

AAGGCAGAAG

CCAGATCCAA

CATGGGTTCA

GGGACATTGG

CAGTGAGATT

TTGTAGCTGA

GCCTGGGACA

TTCACTTCCC

CTCTGTAACT

TCAGGATTGT

TGCTTCTGCC

ACCACGGCCG

GCCACACTT

CTCGCACAAA

CAGTCAAGT(

TTTGGAGCA(

LGCACTCTCGG

TGAATGTTG(

CAGGATGGA/

TCCAGGGGAC

CTCCTGAATP

GGATGAACGP

TGAACCTCAC

GAGAGACI

GTTTGTAGAG

CCGCAGGGAA

CCGGTGTGTT

TCGCTTTCCA

ACAGCTTTTC

GAGGGTTCAG

TCAGGTGTCC

CAGCGTCAGT

GTAAGAAGGG

CCCTCCTCAT

GGGGTCTAGA

AGGTGAACAA

GGGCCATGGG

CACTCTTGCT

GGAACCGAT

TGAGAGCCC

TGGTCTGGCC

TATGCACAAA

CCGATGGCAT

AGCATTGGTT

GCTGAGTCTC

AGGCCTTTCG

TGTGTAGGAG

GCAGCCGGAA

GCGCCCTGGT

CCGACAACAA

CGCTOCAGA

cc 3GCAAGGACAI 3GAGAGTGATC 7TCCCACGGCC

TGATCCTTGC

AGTAGACCAT

GGCAGGTAGA

LCATGGGCCTA

CCCTCACGAT

ACACAGCTCT

ATTTTTCATG

TTCCATCTGG

GTCCAGGCAG

GAAATGCAGA

CTGGGCAATC

AGCTGACCGA

CAGGAGGCTC

CAAGGAGGGA

GAAGAGGCCG

AATCAGCGTG

AGCTGGGGTG

AGTTCTATAG

GTGCTCACAC

GCCAAGAGAA

GAGGGGTTG

GCTGGCGCCA

CTCACCCCCT

AAAGAGAGGG

GGGGACAGCT

GCCCTCATTG

GAGGCTCACA

ACAGTGGGTC

CAGGTAAGTA

TTGGAGTAGG

GCGCTGGAGCC

CGCGGCGCCC

GGTGGAGGCT

GTCCCTGTGT

TGGTTACAGG

CTAATGGAGA

CCCACGTCAT

AACGGGGAGG

GAG CTCTTC C

CAGTGCACAG

GGCTGCTTTT

TCCTGCCAGG

GCCTTCTTCA

GCATGGTCCT

TTGTAGAGAA

GCAGGGCCCA

ACAGACTGGC

GGACGCATAG

ACCAGTCTTT

TCCACAAAGT

CCGGCAGAGA

TGGCCAGCAG

GGCATGGTAT

ACCTCCTCTC

AGTAGAGCTT

AGCTGCACTG

CCAGTAGGCT

ATGTCAGTGC

GAGGAACCTC

TTACTGCAGT

CCCTGCAGCT

AGGCTCAAGC

OGGACACCAT

AGATAGGAGG

AAGAACACAG

GGCAGGGTCA

GCCTTGTCCA

CGGGCCTTCT

AGGCAAACCC

CTATCTTGTG

CAGCTCTGGA

TACCCAGAAA

TCCTGCTGC

AAGGTCCAGA

CTGTGCTGGG

CGCATGGGGC

GAGGGGTCAT

GCAGAGCCAC

GGATGATGTC

CGGCGGTCAC

CGGTGTAGCT

ACATGGGGGT

GATACATCTC

TGTTGAGAAA

TAAACCAGCT

GACCTTTGAA

GGACCTCCAG

CCTAAGTCT

AGTTGTCAGT

TGAAGGGCAA

TCAGGCATAG

GCGTCTGTCC

TGAGGCCACA

CTAAACCCAG

CTGGGCTCTT

CAAAGCTTAA

TCAACACTTG

CCACTGTGGA

AGAGCAGGCC

TCACGCAGTT

CCTCTGGGGA

GCAAGGCCTC

EGGTCTCCAG

3GCGCCCGGT

'GGAGACCCC

GGTGACAGAA

GGGTCTGGAC

ATAGATAAAC

TGCCATAAAA

CTTGGTTGTC

CGTTCCTCG

TTAGGTCCTC

CAGCTGTTCC

GCACTGGACA

CCAGATATCC

AGTGGTGACA

GATAGCCCCT

CAGGACCACA

AATCTCATGC

GGCTGCCAGG

GGGGAGCGTC

GTTTTGGAGA

GGCGTAGGGT

CTGCTTGGTC

CTCATAGTAC

GGGAGGCACG

CGCGAGGCTG

ATAGACACAG

GATGCCGCAG

AGGGGAACCT

GGAGGACTTC

CATGGACTOG

CACCACCACA

GTCGTGGCCC

CTGCCGTGGG

GGTCCGTTTG

CAGCTCTCCA

,GAGTCCAGG

3CTGGCTCGG

'CATCGCAGG

kGACAGCGTG 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2472 GATGATCTCC TGCAGCCGAC CGGCACCCGA 'GAGAACTGG WO 98/20119 PCT/US97/20385 88 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Pro Pro Leu Pro Xaa Arg 1 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 1959 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1746 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: TGG GTC ATG GTG CTG AGC GAA GTG TGG ACC GCG CTG TCT GGA CTC TCC 48 Trp Val Met Val Leu Ser Glu Val Trp Thr Ala Leu Ser Gly Leu Ser 1 5 10 GGG GTT TGC CTA GCC TGC AGC TTG CTG TCG GCG GCG GTG GTC CTG CGA 96 Gly Val Cys Leu Ala Cys Ser Leu Leu Ser Ala Ala Val Val Leu Arg 25 TGG ACC AGG AGC CAG ACC GCC CGG GGC GCG GTG ACC AGG GCG CGG CAG 144 Trp Thr Arg Ser Gin Thr Ala Arg Gly Ala Val Thr Arg Ala Arg Gin 40 WO 98/20119 WO 9820119PCT/US97/20385 89 AAG CAG CGA GCC GGC CTG GAG ACC ATG GAG AAG GCG GTG GAG CGC TTC Lys Gin Arg Ala Gly Leu Glu Thr Met Asp Lys 55 Ala Val Gin Arg Phe

CGG

Arg CTG GAG Leu Gin AAT GCT Asn Pro

GAG

Asp 70 CTG GAT TCA GAG GGG Leu Asp Ser Glu Ala 75 TTG GTG Leu Leu GGT GTG GGG Ala Leu Pro 240 915 GTG GTG CAA GTG GTA GAG AAG Leu Leu Gin Leu Val Gin Lys TTA GAG AGT Leu Gin Ser 90 GGG GAA GTG TGG GGA GAA Gly Glu Leu Ser Pro Glu 288 336 GGT GTG GTG TTT Ala Val Leu Phe 100 AGG TAG GTG GGA AAG Thr Tyr Leu Gly Lys 105 CCC TOG GAA GTG Ala Trp Glu Val AAG AAA CG Asn Lys Oly 110 GAG CG TGG Gin Leu Ser a a 2

AGG

Thr AAG TOT Asn Gys 115 GTO AGG TGG TAT GTG Val Thr Ser Tyr Leu 120 AGT GAG TGT GAG AGT Thr Asp Gys Giu Thr 125 384 GAG GGG Gin Ala 130 CGA CCC GAG GGC GTG Pro Arg Gin Cly Leu 135 GTG TAT CCC GTG GGC GTO AGG GTG AAC Leu Tyr Gly Val Pro Val Ser Leu Lys 140

CAA

Ciu 145

MGC

Asn

MAG

Lys TGG TTC AC TAG MAG Gys Phe Ser Tyr Lys 150 GAG COT CTG AGA TG Glu Gly Val Thr Ser 165 GTG GAG GGA GGT GTG Leu Gin Gly Ala Val 180 GGC GAT GGT TGG AGA Gly His Ala Ser Thr 155 GTG GGG TTA ACT TTG Leu Oly Leu Ser Leu 160 432 480 528 GAG AGT GAG TGT Clu Ser Asp Gys 170 OTO GTG Val Val GTG GAG GTA GTG Val Gin Val Leu 175 GGC TTT GTG GAG AGG MGC Pro Phe Val His Thr Asn 185

GTG

Val1 GOG GAG TGG Pro Gin Ser 190 576 ATG GTA Met Leu

AGC

Ser TAT -GAG Tyr Asp TG AGT AAC GGG CTG TTT GGG Cys Ser Asn Pro Leu Phe Gly GAG AGG ATG MGC Gin Thr Met Asn WO 98/20119 WO 9820119PCT1US97/20385 90 195 200 CCG TG Pro Trp 210 AAG CCC TCC AAG AGT Lys Pro Ser Lys Ser 215 CCA OGA GOT TGC TCA GOG GOT GAG GGG Pro Gly Gly Ser Ser Oly Gly Giu Gly 220

GCT

Ala 225 CTC ATT GGA TGT Leu Ile Oly Ser

GGA

Oly '230 GGC TCC CCT Gly Ser Pro 5 GGG GG AGC ATG CGO Oly Gly Ser Ile Arg 245 TTC CCT TCT GCC Phe Pro Ser Ala CTG GOT TTA GGG ACT GAG ATG Leu Gly Leu Gly Thr Asp Ile 235 240 TTC TGT GGC ATC TGT GGC CTG Phe Gys Oly Ile Gys Gly Leu 250 255 AGT GGC GTG AAG AGG TOT GTT Ser Oly Leu Lys Ser Gys Val 270 720 768 816 864 AAO COT ACT Lys Pro Thr TAT OGA CG Tyr Oly Gin 275 000 Oly 260 AAG CG CTC Asn Arg Leu AGC AAG Ser Lys 265 AGA OCA GTG GAG CTT Thr Ala Val Gin Leu 280 TCT OTT GOC CCC ATO GA COG OAT Ser Vai Oly Pro Met Aia Arg Asp 285 OTO OAT Vai Asp 290 AGG CTG OCA TTG TG ATO AAA GCC Ser Leu Aia Leu Gys Met Lys Ala 295 CTA GTT TOT GAO OAT TTO Leu Leu Gys Oiu Asp Leu 300 TTG CG TTG GAG TGG AGG Phe Arg Leu Asp Ser Thr 305 310 TAG AGA AGT TOT CGA GGG Tyr Arg Ser Ser Arg Pro 325 ATO CCC GGC TTO COG Ile Pro Pro Leu Pro 315 GTT GT OTO OGA TAG Leu Arg Val Oly Tyr 330 TTC AGO GAO GAO ATG Phe Arg Olu Glu Ile 320 TAT OAA ACT GAG AAC Tyr Giu Thr Asp Asn 335 960 1008

TAG

Tyr ACC ATO CCC Thr Met Pro 34.0 ACT OCA GO ATO AGO AGO OCT OTG ATO GAG ACG AAO Thr Pro Ala Met Arg Arg Ala Val Met Giu Thr Lys 1056 WO 98/20119 WO 9820119PCT/US97/20385 91 GAG ACT Gin Ser

CTC

Leu 355 GAG GGT GGT CGC Giu Ala Ala Gly

GAG

His 360 ACG CTG GTC Thr Leu Val CCC TTC TTA CCA AAC Pro Phe Leu Pro Asn 365 1104 AAC ATA Asn Ile 370 CCT TAT Pro Tyr GGC CTG GAG Ala Leu Giu 375 GTG CTG TCG GCA GGT GGC CTG TTC ACT Val Leu Ser Ala Cly Gly Leu Phe Ser 380 1152 GGT GGG TGC Cly Gly Gys

S

TCT TTT Ser Phe 390

CTC

Leu

CAA

Gin AAC TTC AAA Asn Phe Lys 395 GGC GAG TTT GTG CAT Cly Asp Phe Val Asp 400 1200 1248 GCC TCC TTG CCC GAG Pro Gys Leu Cly Asp 405 GTC GTG TTA GTG GTG Leu Val Leu Val Leu 410 AAG GTG GGC ACG TGG TTT Lys Leu Pro Arg Trp Phe 415 AAA AAA Lys Lys CTG CTG Leu Leu 420 AGC TTG CTG CTG Ser Phe Leu Leu AAG GGT GTG TTT GGT CGG CG CA Lys Pro Leu Phe Pro Arg Leu Ala 425 430 GGG TCA CCC CAA AAC CTG TGG CAA Arg Ser Ala Ciu Lys Leu Trp Glu 445

GCC

Ala TTT GTG Phe Leu 435 AAC ACT ATG TCT Asn Ser Met Gys

GGT

Pro 440 1296 1344 1392 CTG GAG Leu Gin 450 CAT GAG ATT GAG ATG His Giu Ile Glu Met 455 TAT CCC GAG TCC GTC ATT CCC GAG TGG Tyr Arg Gin Ser Val Ile Ala Gin Trp 460

AAG

Lys 465 CCA ATG AAC TTG GAG Ala Met Asn Leu Asp 470 GTG CTC CTA ACC CCC Val Val Leu Thr Pro 475 GCC AGA CCC ACA CCC Cly Arg Ala Thr Gly 490 ATG CTG GGT CCT GCT Met Leu Gly Pro Ala 480 GCT ATC AGC TAG ACT Ala Ile Ser Tyr Thr 495 1440 1488 GTG CAT TTC AAG ACA Leu Asp Leu Asn Thr 485 GTT CTC TAT AAC.TGC Val Leu Tyr Asn Gys GTG GAG TTC GCT CC CCC CTG GTG GGT CTC ACC Leu Asp Phe Pro Ala Cly Val Val Pro Val Thr 1536 WO 98/20119 WO 9820119PCTIUS97/2O385 92 510 ACT GTG ACC Thr Val Thr 515

OCT

Ala GAG GAG GAT Glu Asp Asp 0CC Ala 520 CG ATG GAA GAG TAG AAA CCC TAG Gin Met Glu His Tyr Lys Gly Tyr 525 1584

TTT

Phe OAT ATG TOG GAG AAC Asp Met Trp Asp Asn 535 ATT CG AAO AAO OGC ATO AAA AAG GOT Ile Leu Lys Lys Gly Met Lys Lys Oly 540 1632 10 2O~

ATA

Ile 545

GAG

Glu GC OTO OCT OTO GT Gly Leu Pro Val Ala 550 CTG TOT CTO COG TTG Leu Gys Leu Arg Phe 565 OTG CG TG OTO GCT Val Gin Cys Val Ala 555 ATO COO GAO OTO GAA Met Arg Glu Val Olu 570 OTO COG TOO CG GAA Leu Pro Trp Gin Olu 560 COO OTO ATO AGO OCT Arg Leu Met Thr Pro 575 GAA AAO Glu Lys CGG CGA TGT Arg Pro Ser 580 TOAGOOTOAT TGATCGCCG AGTCTGOAO GACCTAAGGG 1680 1728 1783 1843 1903 1959 CCATGCGTC TOCACTGAG CCCCATCTAT TCAOOATCGT OCCAGGCATO AOOAOATGCG CAGOACGGA AOAOOCAACG AGCTOCGGTC GGCTOOAGTC CTACAGAAAG GOAGOACATO CCCTCATAA~ GCAAOTTGG AGOAGOTCC GCGGAATTGC TOGAGCCOG OGGATC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 581 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Trp Val Met Val..Leu Ser Olu Val Trp Thr Ala Leu Ser Oly Leu Ser 1 5 10 WO 98/20119 WO 9820119PCT/US97/20385 93 Gly Val Gys Leu Ala Gys Ser Leu Leu Ser Ala Ala Val Val Leu Arg Trp Thr Arg Ser Gin Thr Ala Arg 40 Gly Ala Val Thr Arg Ala Arg Gin Lys Gin Arg Ala Gly Leu Glu Thr Met Asp Lys 55 Ala Val Gin Arg Phe V000 0*0 Ar g Leu Gin Asn Pro Asp 70 Leu Asp Ser Glu Ala 75 Leu Leu Ala Leu Pro Leu Leu Gin Leu Val Gin Lys Leu Gin Ser Gly Giu Leu Ser Pro Giu Ala Val Leu Phe 100 Thr Tyr Leu Gly Ala Trp Giu Val Asn Lys Gly 110 Gin Leu Ser Thr Asn Cys 115 Val Thr Ser Tyr Leu 120 Thr Asp Gys Giu Thr 125 Gin Ala 130 Pro Arg Gin Gly Leu 135 Leu Tyr Gly Val Pro 140 Val Ser Leu Lys Glu 145 Gys Phe Ser Tyr Lys 150 Gly His Ala Ser Leu Gly Leu Ser Leu 160 Asn Glu Gly Val Thr 165 Ser Glu Ser Asp Gys 170 Val Val Val Gin Val Leu 175 Lys Leu Gin Met Leu Ser 195 Gly 180 Ala Val Pro Phe His Thr Asn Val Pro Gin Ser 190 Thr Met Asn Tyr Asp Cys Ser Asn 200 Pro Leu Phe Gly Gin 205 Pro Trp 210 Lys Pro. Ser Lys Ser 215 Pro Gly Gly Ser Ser 220 Gly Gly Glu Gly WO 98/20119 WO 9820119PCT1US97/20385 94- Ala Leu Ile Gly Ser Gly Gly Ser Pro Leu 225 230 Gly 235 Leu Gly Thr Asp Ile 240 Gly Giy Ser IleArg 245 Phe Pro Ser Ala Phe 250 Cys Gly Ile Gys Gly Leu 255 Lys Pro Thr Tyr Gly Gin 275 Gly 260 Asn Arg Leu Ser Lys 265 Ser Gly Leu Lys Ser Gys Vai 270 Aia Arg Asp 0 eve* Goes 2 S 0 Thr Aia Vai Gin Leu '280 Ser Vai Giy Pro Met 285 Vai Asp 290 Ser Leu Ala Leu Met Lys Ala Leu Leu 300 Cys Giu Asp Leu Phe 305 Arg Leu Asp Ser Thr 310O Ile Pro Pro Leu Pro 315 Phe Arg Giu Giu Tyr Arg Ser Ser Arg 325 Pro Leu Arg Vai Giy 330 Tyr Tyr Giu Thr Asp Asn 335 Tyr Thr Met Gin Ser Leu 355 Thr Pro Ala Met Arg 345 Arg Aia Val Met Giu Thr Lys 350 Leu Pro Asn Giu Ala Ala Gly His 360 Thr Leu Val Pro Phe 365 Asn Ile 370 Pro Tyr Ala Leu Giu 375 Val Leu Ser Ala G ly 380 Gly Leu Phe Ser Asp 385 Gly Giy Gys Ser Phe 390 Leu Gin Asn Phe Gly Asp Phe Vai Asp 400 Pro Gys Leu Gly Asp 405 Leu Val Leu Val Leu Lys Leu Pro 410 Arg Trp Phe 415 Arg Leu Ala 430 Lys Lys Leu Leu 420 Ser Phe Leu Leu Lys 425 Pro Leu Phe Pro WO 98/20119 WO 9820119PCTIUS97/20385 95 Ala Phe Leu Asn Ser Met Cys Pro Arg Ser Ala Glu Lys 445 Leu Trp Glu 435 440 Leu Gin 450 His Giu Ile Glu Met 455 Tyr Arg Gin Ser Val 460 Ile Ala Gin Trp Lys 465 Ala Met Asn Leu Asp 470 Vai Val Leu Thr Pro 475 Met Leu Cly Pro Ala 480 ro.

Leu Asp Leu Asn Thr 485 Pro Gly Arg Ala Gly Ala Ile Ser Tyr Thr 495 Val Leu Tyr Thr Vai Thr 515 Asn 500 Gys Leu Asp Phe Ala Gly Val Val Pro Val Thr 510 Lys Gly Tyr Ala Giu Asp Asp Ala 520 Gin Met Giu His Tyr 525 Phe Giy 530 Asp Met Trp Asp Asn 535 Ile Leu Lys Lys Gly 540 Met Lys Lys Gly Gly Leu Pro Val Ala 550 Val Gin Cys Val Ala 555 Leu Pro Trp Gin G lu 560 Gl u Leu Cys Leu Arg 565 Phe Met Arg Glu Val 570 Glu Arg Leu Met Thr Pro 575 Glu Lys Arg Pro Ser 580 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 1959 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA WO 98/20119 PCTIUS97/20385 96 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 10 0.

GATCCCCCC

TCCTCCGTI

TCTCCTCAl

TACCTCCTC

CAGCCGTTC

CCACTCCAC

CCACATATC

AGTCGTCAC,

GATACCCC

TACCACCAQi

AATCTCAT(

GGCTGCCAG(

CCCCAGCTT(

GTTTTCGAGY

CGCATAAGG]

CTGCTTGGTC

TTCATATAI

GCGGGGGATC

TGCCAGGCTA

ATAAACACAG

GATOCCACAC

AGCCACCCT

CGAGGGCTTC

CATGGACTGG

CACCACCACA

AGCATCGCCC

CTGCCGTGCC

GGTCCCTTTC

CACTTCCCCA

TGAATCCACC

GCCCGCTCC

CCATCCCACC

AGACAGCGCG

'G GCTCCAGGAA 'T CTGTAGGACT CG CGTGCCAGGA C ACAGCTGGCC C ACCTCCCGCA A GCCACACGCA C CCAAAGTAC A GGCACCACCC r CTGCCTCTGC 3 TCCAAGTTCA

TGCAGTTCCC

3CGACGAAACA 3AGCACTAAGA

SAAAGACCAGC

ATGTTGTTTG

TCCATCACAG

CCCACACGAA

CTGGAGTCCA

TCCACATCCC

CTCTTCAGC

AACGCAGAAGC

CCAGATCCAA I1 CACGGGTTCA I GCGACCTTGG TI CAGTCACTCT C TTCTAGCTGA A GCCTGGGACA C TTCACTTCCC A CTCTGTAACT T TCAGGATTCT G TGCTTCTGCC G ACCACCCCCG C CTCCACACTT C'

TTCCGGGCGA

CCAGGCAGG

TCCTGAATAG

AGATGAATGA

TGAACCGCAC

GGCCTATACC

CTTTGTAGTG

CCGCAGGGAA

CCGGTGTGTT

TTGCCTTCCA

ACAGCTTTTC

CACCCTTCAG

CCAGGTCCCC

CACCATCACT

GTAAGAAGG

CCCTCCTCAT

GCGGTCGAGA

kGCGGAACAA I ,TCCCATGGG C ,ACTCTTGCT C ~GAACCGGAT

G

GAGAGCCCC C

GGTCTGCC

GTGCACAAA C :CCATCTCAC A GCATTCCTT

G

CTCAGTCTC

A

CCCCTTTCC

C.

CTGTACCAG CAGCCGGAA

G'

CGCCCTGGT

C~

CGACAGCAA

G(

CCTCAGCAC

C~

GCTGGTCCAG

CCAGGTGGTT

ATGCCGCTGC

CCCTCAAGAT

ACACAGCTCT

CTTTTTCATG

TTCCATCTCC

GTCCAGGCAG

CAAATCCAGA

CTGGGCAATC

CGCTGACCGA

CAGGAAGCTC

CAAGCAGGGA

GAACAGCCCA

GACCAGCTG

CGCTGGAGTC

kCTTCTGTAG ~TCCTCACAA I CCAACAGAA t AGCCCGTTC C ~CTGCCCC

A

:TCACCCCCT

G

AAGACGGCG

T

GGCACAGCT C

CCCTCCTTCA

AGCCTCACG G CACTCACTC

A

AGGTAGCTA A GCGAGCAGC G' CCCTGCACC Gi kCCGCGCCC CC

ACTTGGTTAT

CCCTCTTCCC

AGTGCAGAC

GGCCGCTTTT

TCCTCCCAGG

CCCTTCTTCA

GCATCGTCCT

TTATAGACAA

CCACGACCCA

ACGGACTGC

GCACACATAC

AGCAGTTTTT

TCCACAAAGT

CCCCGACA

rGGCCACCAC

;GCATGGTCT

kTCTCCTCCC

~GTAGGGCTT

LCCTGCACTG

;CAGTAGGCT

~TGTCAGTGC(

AGGAACCTC C TACTCCAGT C CCTGCAGCT TI AACTTAACC C GCACGCCAT A GATAGGACG T AGAGCACAG C GCAGAGCCA C CCTTGTCCA T GGGCGGTCT G

GCAGGGCATG

OTOCTGOCA

CCATGGCCCT

CACGGCTCAT

CCACACCCAC

GAATGTTCTC

CAGCCGTCAC

CAGTGTACCT

CCATGGGGGT

GATACATCTC

TCTTCAGAAA

TAAACCACCT

CCCCTTTGAA

GGACCTCCAG

CCTCGAGACT

AGTTGTCAGT

TGAACGGCAA

rCATGCACAA

CTGTCTGTCC

CAGCcCACA

TAAACCCAG

~TCGACTCTT

~ATAGCTTAG

'CAGTACCTG

:CAGTCTCGA

~GAGCAGGCC

CACACACTT

TTCTCGA

CAACGCCTC

GCTCTCCAG

GCTCCTGCT

GGAGAGTCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1959 CTGCAGGCT ACGCAAACCC kTCACCCA WO 98/20119 PCTUS97/20385 97- INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 2045 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(ix) FEATURE: NAME/KEY: CDS LOCATION: 1775 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: TG CCG GCC OGT AGG GAG GAG GAG OCT GAA CCC ATC ATG Pro Gly Oly Arg Oln Cln Cln Ala Clu Cly Ile Met 1 5 10 a o..

C

CTC GAG TAC Val Cln Tyr GAG GTG TGC CCC CG CGCOCT CCC CCC TOO CCC CTC CCC CTG CCC TOO lu Leu Trp Ala Ala Leu Pro Cly Ala Ser Oly C C20 25 Val Ala Leu Ala Cys *TG TTCGOTO Gys Phe Val CC CC CC Ala Ala Ala OTG CG CTG CCC TOG TGG CCC Val Ala Leu Arg Trp Ser Gly CCC CCC AGO Arg Arg Thr 000 Ala CCC GC Arg Gly C OTO OTO Ala Val Val CCC CC OGA GAG AAC GAG OGA CC GC OTO Arg Ala Arg Gln Lys Gln Arg Ala Gly Leu 55 GAG AAC ATO GAG AGO CCC CCC GAG COO TTC CCC OTO GAG AAC OCA GAG Glu Asn Met Asp Arg Ala Ala Oln 70 Arg Phe Arg Leu Cln Asn Pro Asp CG GAG TOA GAO CG OTO OTA CO CTC CCC OTO OCT GAG OTO OTO CG Leu Asp Ser Clu Ala Leu Leu Ala Leu Pro Leu Pro Oln Leu Val Oln 85 90 WO 98/20119 PCTIUS97/20385 98 AAC TTA GAG ACT AGA GAG CTG GOC OCT GAG GOC GTG CTC TTC ACC TAT 335 Lys Leu His Ser Arg Giu Leu Ala Pro Glu Ala Val Leu Phe Thr Tyr 110 ACC TCC Thr Ser GTG OGA AAO GCC TGG Val Gly Lys Ala Trp 115 GAA GTG AAC AAA COG ACC AAC Oly Thr Asn Glu Val Asn Lys 120 TOT GTG Gys Val 125 383 .1Q.

C

TAT CTC Tyr Leu

CTGCOTC

Leu Leu 145 OCT GAG Ala Asp 130 TOT GAG ACT GAG CG TCT CG CCC CCA AGO GAG GO Gys Oiu Thr Gin Leu Ser Gin Ala Pro Arg Gin Gly 135 140 431 479 TAT CCC GC CCT OTO Tyr Gly Val Pro Val 150 AOC CTC AAG GAO TG Ser Leu Lys Olu Cys 155 TTG AGO TAG AAC Phe Thr Tyr Lys 0GCCGAG GAG TOG AG Gly Gin Asp Ser Thr 160 GAG TG GAG AGO OTA Oiu Gys Asp Ser Val 180 CCC TTCGOTO GAG AGO Pro Phe Val His Thr 195

OTO

Leu 165 GG TTG AGO GTG Oly Leu Ser Leu AAT CAA COG OTO CCC CC Asn Oiu Gly Val Pro Ala 170 175 AAG OTG GAG OCT CCC OTO Lys Leu Gin Oly Ala Val 190 OTO OTG Val Val

CAT

His OTO GTG Val Leu 185 527 575 623 AAT OTT OCA CG Asn Val Pro Gin 200 TOG ATG TTG AGO TAT GAG TGC Ser Met Phe Ser Tyr Asp Gys 205 ACT AAG CCC Ser Asn Pro 210 AGO OCA 000 Ser Pro Oly 225 OTO TTT GC CG Leu Phe Gly Gin 000 TOG TGA 000 Gly Ser Ser Gly 230 AGO OTO AAC OCA TOG AAG TOG TOG AAA Thr Val Asn Pro Trp Lys Ser Ser Lys 215 220 OCT OAA CCC 000 OTO ATCGG00 TOT OGA Gly Glu Gly Ala Leu Ile Gly Ser Gly 235 671 719 CCC TOG Gly Ser CCC OTO CCC TTA GCG ACT OAT ATO OGA GC AGO ATG CG TTG Pro Leu Oly Leu Oly Thr Asp Ile Gly Oly Ser Ile Arg Phe WO 98/20119 PCT/US97/20385 99 240 CCC TCG TCC TTC TGC Pro Ser Ser Phe Cys 260 245 250 GGC ATC TGC GGC CTC Gly Ile Cys Gly Leu AAG CCC ACA GGG AAC CC Lys Pro Thr Gly Asn Arg 270 815 CTC AGG AAG Leu Ser Lys

AGT

Ser 275 CCC CTG AAG COG TOT Gly Leu Lys Gly Gys 280 GTC TAT GGA GAG Val Tyr Gly Gin GAG GCA GTG Giu Ala Val 285

CGT

Arg CTG TCG Leu Ser 290 GTG GGC 0CC ATG CC Val Gly Pro Met Ala 295 CGC GAG GTG GAG AGC CTC GGA CTG Arg Asp Val Ciu Ser Leu Ala Leu 300 GAG ATG TTC CGC TTG GAG CCC ACT Asp Met Phe Arg Leu Asp Pro Thr 315 863 911 959 TG CTG Cys Leu 305 CGA CCC CTG GTG TGC Arg Ala Leu Leu Cys 310

GAG

Giu

GTG

Val 320 CCT CCC TTG CCC Pro Pro Leu Pro

TTC

Phe 325

AGA

Arg GAA GAG GTC TAG Giu Glu Val Tyr 330

ACC

Thr AGC TCT GAG Ser Ser Gin

CCC

Pro 335 1007 2S CTO GT GTG COG TAG TAT Leu Arg Val Oly Tyr Tyr 340 GAG ACT Giu Thr GAC AAC Asp Asn 345 TAT ACC ATG CCC TCC CCC Tyr Thr Met Pro Ser Pro 350 1055 CCC ATG AGG CCC Ala Met Arg Arg 355 CCC GTG CTG GAG ACC Ala Val Leu Olu Thr 360 AAA GAG AGC Lys Gin Ser CTT GAG CCT CC Leu Ciu Ala Ala 365 1103

GCC

C ly CAC ACG His Thr 370 CTC GTT CCC TTC Leu Val Pro Phe TTG CCA Leu Pro 375 AC AAG ATA CCC CAT OCT CTO 1151 Ser Asn Ile His Ala Leu GAG ACC CTG TCA AGA GOT COG CTC TTC ACT GAT GCT GGG GAG ACC TTC 1199 Giu Thr 385 Leu Ser Thr Gly Gly Leu Phe Ser Asp Cly Gly His Thr Phe 390 395 WO 98120119 PCT/US97/20385 100 CTA GAG AAC TTC AAA GGT OAT TTC GTG GAG CCC TGC GTG GO GAG CTG 1247 Leu 400 Gin Asn Phe Lys dly 405 Asp Phe Val Asp Pro 410 Gys Leu Gly Asp Leu 415 GTC TCA ATT Val Ser Ile CTO AAG Leu Lys 420 CTT CCC CAA TOG CTT Leu Pro Gin Trp Leu 425 AAA GOA CTG Lys Gly Leu 070 0CC TTC Leu Aia Phe 430 1295 CTG GTG AAG OCT Leu Val Lys Pro 435

GTG

Leu CTG GGA Leu Pro GGA AAA Gly Lys AGO CTG Arg Leu 440 TCA GCT TTC CTC AGG Ser Ala Phe Leu Ser 445 AAG ATO Asn Met AAO TGT GT Lys Ser Arg 450 TGG OCT Ser Ala

GTG

Leu 455 TOG GAA CG GAG GAG GAG ATC GAG Trp Giu Leu Gin His Glu Ile Glu 460 1343 1391 1439 070 TAG Val Tyr 465 CG AAA ACC OTG Arg Lys Thr Vai

ATT

Ile 470 0CC GAG TOG AGO OCO GTG GAG CTG OAT Ala Gin Trp Arg Ala Leu Asp Leu Asp 475 070 Val 480 070 CTG AGC CCC ATO Val Leu Thr Pro Met 485 CT G0CC CCT OCT GTG Leu Ala Pro Ala Leu 490 GAG TTO AAT 0CC CGA Asp Leu Asn Ala Pro 495 CTO TAG AAG TG GTG Leu Tyr Asn Gys Leu 510 GCC AGO 0CC ACA 000 Gly Arg Ala Thr Gly 500 0CC GTG Ala Val AGC TAG ACT Ser Tyr Thr 505 1487 1535 1583 GAG TTC OCT Asp Phe Pro OCA 000 Ala Gly 515 OTO 070 CCT 070 Val Val Pro Val 520 AGC AG OTO ACT OCT GAG GAG Thr Thr Vai Thr Ala Giu Asp 525 GAG 0CC GAG Giu Ala Gln 530 ATO GAA CAT TAG AGO Met Giu His Tyr Arg 535 GOC TAG 777 000 Gly Tyr Phe Oly

OAT

Asp 540 ATG TOG GAG Ile Trp Asp 1631 AAG ATO CTG CG AAG 000 ATO AAO AAO AOT 070 000 070 G 070 0CC Lys Met Leu Gin Lys Gly Met Lys Lys Ser Val Gly Leu Pro Val Ala 1679 WO 9&120119 WO 9820119PCTIUS97/20385 101

GTG

Val 560

ATG

Met ;21. Q.

545 GAG TGT GTG GCT CTG Gin Cys Val Ala Leu 565 CGG GAG GTG GAG GGA Arg Glu Val Glu Arg 580 CGAGAG GACGTGAGAC TC CTGCGA GAGCAAGGAA AT CGCTGC AAGAAGGGC GA ~TGGTG CTGATCGTC CA kAGTAA CAGCCCCGGA AT 550

GGG

Pro TGG GAA GAA Trp Gin Glu

GAG

Glu 570

GGGT

CCTG

AACC'

TGTC'

GCCC

555

TTG

Leu

NAG

Lys CTG ATO AGC COT CAA Leu Met Thr Pro Glu 585 ACACTCTC TGCAGCGCAG GTGGTGA TGGGGCAGAG CTGGGTGA GTCTGGACCT CCGGGATG TGGGAGCGGA

GOTA

GOTTI

CGAT

TGGG

TOT GTG GG TTG Cys Leu Arg Phe 575 GAG TCA TGC TGATGGCTGT Gin Ser Ser 590 GTGAGG GGAGAGCTGG if CGGTGT CCTCTCCCG 19 CCGTGG TGTGGTGGCC 19 rATGAG ATAGCGAAG 20 20 727 1782 ~42 ~02 62 '22

T

INFORMATION FOR SEQ ID NO:43: SEQUENGE CHARAGTERISTICS: LENGTH: 590 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENGE DESCRIPTION: SEQ ID NO:43: Pro Gly Gly Arg Gin Gin Gin Ala Glu Gly Ile Met Val 1 5 10 Leu Trp Ala Ala Leu Pro Gly Ala Ser Gly Val Ala Leu 25 Phe Val Ala Ala Ala Val Ala Leu Arg Trp Ser Gly Arg 40 Arg Gly Ala Val Val Arg Ala Arg Gln Lys Gin Arg Ala Gin Tyr Glu Ala Cys Cys Arg Thr Ala Gly Leu Glu WO 98/20119 WO 9820119PCT11JS97/20385 102- Asn Met Asp Arg Ala Ala Gin Arg 70 Phe Arg Leu 75 Gin Asn Pro Asp Leu Asp Ser Glu Ala Leu Leu Ala Leu Pro Leu 90 Pro Gin Leu Val Gin Lys Leu His Ser Gly Lys Ala 115 Arg 100 Giu Leu Ala Pro Giu 105 Ala Val Leu Phe Thr Tyr Val 110 Thr Ser Tyr Trp Glu Val Asn Lys 120 Gly Thr Asn Gys Val1 125 Leu Ala Asp Gys Giu Thr Gin Leu Ser Gin Ala Arg Gin Gly Leu 130 135 Leu 145 Tyr Gly Val Pro Val 150 Ser Leu Lys Clii Phe Thr Tyr Lys G ly 160 Gin Asp Ser Thr Leu 165 Gly Leu Ser Leu Asn 170 Glu Gly Val Pro Ala Glu 175 Gys Asp Ser Val 180 Val Val His Val Leu 185 Lys Leu Gin Gly Ala Val Pro 190 Asp Gys Ser Phe Val His 195 Thr Asn Val Pro Gin 200 Ser Met Phe Ser Tyr 205 Asn Pro 210 Leu Phe Gly Gin Gly Ser Ser Gly 230 Thr 215 Val Asn Pro Trp Lys Ser Ser Lys 220 Ile Gly Ser Gly Ser Pro 225 Gly Gly Glu Gly Ala Leu 235 Gly 240 Ser Pro Leu Gly Leu Gly Thr Asp Ile C ly 250 Gly Ser Ile Arg Phe Pro 255 WO 98/20119 PCTUS97/20385 103 Ser Ser Phe Cys 260 Gly Ile Cys cay Leu Lys 265 Pro Thr Gly Asn Arg Leu 270 Ser Lys Ser Gly Leu Lys Gly Cys Val Tyr Gly Gin 275 280 Glu Ala Val Arg 285 Leu Ala Leu Cys Leu Ser 290 Val Gly Pro Met Ala 295 Arg Asp Val Glu Ser 300 Leu 305 Arg Ala Leu Leu Glu Asp Met Phe Arg 315 Leu Asp Pro Thr Pro Pro Leu Pro Arg Giu Giu Val Tyr 330 Thr Ser Ser Gin Pro 335 Arg Val Gly Tyr 340 Tyr Glu Thr Asp Asn 345 Tyr Thr Met Pro Ser Pro Ala 350 Ala Ala Gly Met Arg Arg 355 Ala Val Leu Glu Lys Gin Ser Leu Glu 365 His Thr 370 Leu Val Pro Phe Leu 375 Pro Ser Asn Ile His Ala Leu Glu Leu Ser Thr Gly Gly 390 Leu Phe Ser Asp Gly 395 Gly His Thr Phe Leu 400 Gin Asn Phe Lys Gly 405 Asp Phe Val Asp Pro 410 Cys Leu Gly Asp Leu Val 415 Ser Ile Leu Lys 420 Leu Pro Gin Trp Leu Lys Gly Leu Leu 425 Ala Phe Leu 430 Val Lys Pro 435 Leu Leu Pro Arg Leu 440 Ser Ala Phe Leu Ser Asn Met Lys 445 Ser Arg 450 Ser Ala Giy Lys Leu Trp Giu Leu Gin His Giu Ile Giu Val 455 460 WO 98/20119 PCT/US97120385 104 Tyr Arg Lys Thr Val Ile Ala Gin Trp Arg Ala Leu Asp Leu Asp Val 465 470 475 480 Val Leu Thr Pro Met 485 Leu Ala Pro Ala Leu 490 Asp Leu Asn Ala Pro Gly 495 Arg Ala Thr Phe Pro Ala 515 Gly 500 Ala Val Ser Tyr Thr 505 Met Leu Tyr Asn Gys Leu Asp 510 L 0..

Gly Val Val Pro Val 520 Thr Thr Val Thr Ala 525 Glu Asp Giu Ala Gin 530 Met Glu His Tyr Arg 535 Gly Tyr Phe Gly Asp 540 Ile Trp Asp Lys Met 545 Leu Gin Lys Gly Met 550 Lys Lys Ser Val C ly 555 Leu Pro Val Ala Val 560 Gin Gys Val Ala Leu Pro Trp Gin Giu 565 Giu 570 Leu Cys Leu Arg Phe Met 575 Arg Glu Val Giu Arg Leu Met Thr 580 Pro 585 Glu Lys Gin Ser Ser 590 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 2045 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: AATTCCGGGG CTGTTAGTTG GGCCTTGGCC TATGTCATAC CCATGGGCTG

CCACATGGGG

GTGGAGGGAT CAGGACGAAG AGAGGGGACC AGAGCAGGGA TGGAGGTCCA

GACTCAGGGA

120 WO 98/20119 PCTIUS97/20385 105 CTCGGCGCTT CTTGCACCCC OTTGGOGAG AGOACACGGA ACCCTCTC'CC

CCATGCAGGA

0..

a a

CATTTCCT'

TGAGTCTC)

TCCCTCCA(

CTGCACCGG

GATATCCCC

CGTGACACG

CGCCCCTGl

CACCACATC

CTCCTGCTG

TGACACCT

AACCTTCAC,

CTGTAGGAAi

ATCGGCTAT(

TTTGGTCTC(

ATAGTACCC(

AGGCACACTC

CAGGCTCTCC

GACACAGCCC

GCCCCAGAAC

GGAGCCTCCA

GGACTTCCAT

GGACTGTCCA

CACTACGCTC

CTGGCCCTTC

CCTTCGCC

CCCTTTGTTC

CTCTCTACTC

GTCCAGGTCT

CGCTCCCTGC

SCG CAGCC

CAGCCGGCC

CGGCA

rG CTGTCGCAGC kG CTCCTCTGCA ;C TCCCCCATGA X ACCCCCAGCC 'A AACTACCCC C ACCACCCCTG CG CCCCTGCCTC C AGGTCCAGCG C AGTTCCCACA T GGCACCAGAG h. ATTGAGACCA 3 GTGTGCCCAC 3 TTCCTTGGCA 3 ACCACGGCCC 3ACACGCAGG GGTCCAAGcC ACGTCCCCGG

C

TTCAGCCCAC

'I

GACCAGGCGA

A

GACCCGATGA

C

GGGTTCACGG

T

ACATTGCTGT C TCGCACTCCC

C

TAGGTGAAGC

A

TCAGACAGCT

G~

ACTTCCCACG C4 TGTAACTTCT

G(

GGCTTCTCGA

G(

TTCTCTCCCG

CC

ACCCCCGCCG

CC

AGGCCAGCTC

GCCAGAGCCA

ACCGCAGACA

CCACACTCTT

TGTAATGTTC

CAGCAACTC

GCGCATTCAA

CCCTCCACTG

GTTTTCCAC

GCTTCACCAG

GCTCCCCCAG

CATCACTGAA

'\GAAGGGAAC(

GCCTCATGGCC

3CTCACAGCT

C

GAACATGTC

C

~CATCGGGCC

C

CTTGCTGAC

C

~GCGGATCCT

C

CCCCCCTTC

A

'CTGCCCAAA C CACGAAGGG C, CCGCACCCC

T'

CTCCTTGAC

GI

AGTCTCACA CG CTTTCCCAC A CACCAGCTG

AC

,CCGAAGCG Cl 3CGCACCAC

CC

ACGAAGCA C

TGCCCTGACT

TCAGGATGAC

CAACTCTTCT

CTTCATGCCC

CATCTGCGCC

CAGCCAGTTG

GTCCACACCA

GGCAATCAcC

CGAACGACAC

GAACCCCAGC

GCAGCGGTCC

3ACCCCACCTC

,ACCCTGTC

GCCGCAGGCc i GTGTACACC I TCGCACACC A ACCGAGAGA C CCGTTCCCT

C

CCTCCGATA

T

CCCCCTGAC C AGGGGTTA

C

ACGGCACCC Ti rGATTCAGC

C

CTCACAGGC

A(

I'CAGCCAGA Ti CAGCTCAAG

AC

GCAGCGC

AC

'GCGCCCC CTV CCCCCCGC C :ACGCCACG

C

AGGCTGGGCT

TGCTTTTCAC

TGCCAGGGCA

TTCTGCAGCA

TCGTCCTCAG

TACGCATAG

GGCGCCACCA

CTTTTCCCT

TTCATCTTC

ACTCCTTTAA

k.CGAAATCAC

;TTGACAGCG

3CCCCAGCCT ~TCCTATAGT I1 CTTCTCTCA

P

~GGCCTCCCA

C

CCACTGCCT

C

TGGGCTTGA

G

CAGTGCCTA

A

AGCCCCCTC

C

TCCAGTCAT

A

GCAGCTTCA

G

rCAAGCCCA Gi CGCCATACA

G(

kGCACCTCA Cl CACCGCCT CY GCCTACCA

C

*CTCCATGT

TC

:CGTCCCc

C

CACCCCCG AG

OCAGACAGTO

GCCTCATCAC

GACCCACACA

TOTTOTOCCA

CAGCACCCT

TCTACCTGAC

TGCCCGTCAG

ACACCTCCAT

TGAGCAAAGC

GCCATTCGGGG

CTTTGAACTT

C'CTCCAGAC

;AAGCCTCTG

.GTCAGTCTC

LCCGCAAGCC

CCACACTGC

:CTGTCCATA

GCCCCACAT

G CC GAG CC

GCTTTTGCA

GCTGAACAT

CACATCCAC

CGTCCACTC

CAGGCCCTG

kCAGTTGCT

GCCCGCCAG

CCCTCTCA.

TCCAGGCC

:CCGGACCA

180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2045 CACACCTCCT ACTGCACCAT CATCCCTTCA GCCTGCTCCT

GCCTACCCC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid WO 98/20119 PCT/US97/20385 106 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GCGGTACCAT GCGATGGACC GGGCGC 26 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: SGGTCTGGCCA AAGAGAGG 18 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu Ile Ala Gly Gly Gly Ser 1 5 10 Leu Gly Ile Gly Ser Asp Val Ala Gly Ser Ile Arg Leu Pro Ser 25 WO 98/20119 PCT/US97/20385 107 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 0: Gly Gly Ser Ser Gly Gly Glu Gly Ala Leu lie Gly Ala Gly Gly Ser 1 5 10 Leu Ile Gly Ile Gly Thr Asp Val Gly Gly Ser Val Arg Ile Pro Cys 20 25 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: Gly Gly Ser Ser Gly Gly Glu Ser Ala Leu Ile Ser Ala Asp Gly Ser 1 5 10 Leu Leu Gly Ile Gly Gly Asp Val Gly Gly Ser Ile Arg Ile Pro Cys 20 25 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 98/20119 PCT/US97/20385 108 (v) (xi) FRAGMENT TYPE: internal SEQUENCE DESCRIPTION: SEQ ID Gly Gly Ser Ser Gly Gly Glu Gly Ser Leu Ile Gly Ala Gly Ser Ile Arg His Gly Ser Ile Pro Ser Leu Leu Gly Leu Gly Thr Asp Ile Gly 25 .1 a a~ a.

a. a a INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Gly Gly Ser Ser Gly Gly Glu Gly Ala Ile Val Gly Ile Arg Gly Gly Val Pro Ala Val Ile Gly Val Gly Thr Asp Ile 20 Gly 25 Gly Ser Ile Asp INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: Gly Gly Ser Ser Gly Gly Val Ala Ala Ala Val Ala Ser Arg Leu Met 1 5 10 WO 98/20119 WO 9820119PCrTIUS97/20385 109- Leu Gly Gly Ile Gly Thr Asp Thr Gly Ala Ser Val Arg Leu Pro Ala 25 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: i~.

S. S. OS

S

0*

*SSS

S.

Gly 1 Gly Ser Ser Gly Gly Val Ala Ala 5 Ala Val Ala Ser 10 Gly Ser Ile Arg Gly Ile Val Ile Pro Ala Pro Leu Ser Val Gly Thr Asp Thr 20 Gly 25

S.

S

S. S S INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 819 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

CCAGGAGGTT

TTAGGCACTG

CTCAAGCCTA

ACGGCAGTGC

CTGAAAG CT C

TTCAGAGAGG

AACTATACCA

CCTCAGGGGG

ACATTGGGG

CTGGCAACCG

AGCTTTCTCT

TACTGTGTGA

AGGTCTATAG

TGCCCAGCCC

TGAGGGGGCT

CAGCATCCGG

CCTCAGCAAG

TGGCCCCATG

GCACTTGTTC

AAGTTCTAGA

ACTATGAGG

CTCATTGGAT

TTCCCTTCTG

AGTGGCCTGA

GCCCGGGATG

ACCTTGGACC

CCCCTGCGTG

AGGGCTCTGA

CTGGAGGTTC

CCTTCTGCGG

AGGGCTGTGT

TGGAGAGCCT

CTACCGTGCC

TGGGGTACTA

TAGAGACCAA

CCCTCTGGGT

CATCTGTGGC

CTATGGACAG

GGCGCTATGC

TCCCTTTCCC

TGAGACTGAC

GCAGAGACTT

120 180 240 300 360 420 WO 98/20119 PCTIUS97/20385 110

GAGGCTGCTG

GTCCTGTCTG

GGTGACTTTG

TTTAAAAGAC

AAGAGTATGC

TATCGCCAGT

ATGYTNGGNC

OCCACACGCT

CGGGCGGCCT

TGGATCCCTG

TGCTGAGCCT

GTCCTCGGTC

CTGTGATTC

CNGCNYTNGA

GATTCCCTTC

GTTCAGTGAC

CTTGGGAGAC

CCTGCTGAAG

AGCTGAAAAG

CCAGTGGAAA

'YrNAAYACN

TTACCCAACA

GGTGGCCGGA

CTGATCTTAA

CCTCTGTTTC

CTGTGGAAAC

GCGATGAACT

CCNGGNMGN

ACATACCCTA

GTTTCTCCA

TTCTGAGGCT

CTCGGCTGGC

TGCACCATGA

TGGATGTGCT

CGCCCTGGAG

AAACTTCAAA

GCCCAGCTGG

AGCCTTTCTC

GATTCAGATG

GCTGACCCCN

480 540 600 660 720 780 819 0@ 0 S @000

S

S.

5 0

SOS.

05 @5 S

S

OSSSO@

S

S. 9

SS

5.55 55.5 @55555 9 .55.

5.55 5S S. S 0 5* 5* 5

Claims

1. An isolated fatty-acid amide hydrolase (FAAH) capable of hydrolysing cis-9,10-octadecenoamide, anandamide, myristic amide, palmitic amide and stearic amide, said FAAH having an amino acid residue sequence selected from the amino acid residue sequence shown in SEQ ID NO 36; (ii) the amino acid residue sequence shown in SEQ ID NO from residue 3 to 581, and (iii) the amino acid residue sequence shown in SEQ ID NO 43 from residue 12 to 590.

2. The FAAH of claim 1, having the amino acid residue sequence shown in SEQ ID NO 36. 15 3. The FAAH of claim 1, having the amino acid residue sequence shown in SEQ ID NO 40 from residue 3 to 581. o j

4. The FAAH of claim 1, having the amino acid residue sequence shown in SEQ ID NO 43 from residue 12 to 590.

5. A method for catalyzing a hydrolysis of a fatty-acid primary amide comprising the step of contacting the fatty- acid primary amide with an isolated FAAH according to any preceding claim.

6. The method for catalyzing a hydrolysis of a fatty- acid primary amide according to claim 5 wherein the fatty- acid primary amide includes an alkyl chain having an unsaturation, wherein the unsaturation is in an alkyl chain having a cis configuration.

7. The method for catalyzing a hydrolysis of a fatty- acid primary amide according to claim 5 wherein the fatty- COMS ID No: SBMI-01190578 Received by IP Australia: Time 10:49 Date 2005-04-05 04/05/2005 10:41 GRIFFITH HACK +61 2 99255911 00262937999 NO. 112 acid primary amide is selected from the group consisting of cis-9,10-octadecenoamide, cis-8,9-octadecenoamide, cis- 11,12-octadecenoamide, cis-13,14-docosenoamide, and a fatty-acid primary amide having the formula: NH 2 C (CH2) (6<n<ii)CH=CH (CH 2 >n>S)CH 3

8. The use of a FAAH according to claim 1 for the hydrolysis of anandamide, myristic amide, palmitic amide or stearic amide.

9. A method for inhibiting an enzymatically catalyzed hydrolysis of a fatty-acid primary amide by a FAAH of claim 1, the method comprising the step of contacting said 15 FAAH with an inhibitor of the FAAH. The method of claim 9 wherein said fatty-acid primary 0 S. amide substrate is selected from the group consisting of cis-9,10-octadecenoamide, anandamide, myristic amide, 20 palmitic amide and stearic amide. e

11. The method according to claim 9 wherein said fatty- Ss** acid primary amide is cis-9,10-octadecenoamide. *0

12. The method of claim 9 wherein said inhibitor of FAAH is selected from the group consisting of phenylmethylsulfcnyl fluoride, HgC12, and a trifluoroketone having the following structure: o Jj__ r~rrN .AzK% 104 008 COMS ID No: SBMI-01190578 Received by IP Australia: Time 10:49 Date 2005-04-05 04/05/2005 10:41 GRIFFITH HICK +61 2 99255911 4 00262937999 NO.104 0009 113

13. A method for ascertaining the inhibitory activity of a candidate inhibitor of fatty-acid amide hydrolase (FAAH), the method comprising the following steps: Step A: forming mixture by combining FAAH according to claim 1 and a fatty-acid primary amide substrate under reaction conditions; Step B: forming mixture by combining the mixture of said Step A with the candidate inhibitor; then Step C: quantifying the conversion of said fatty-acid primary amide substrate to a hydrolysis product within mixture Step D: quantifying the conversion of said fatty-acid primary amide substrate to hydrolysis product within mixture and then Step E: ascertaining the inhibitory activity of the candidate inhibitor by comparing the quantifications of said Steps C and D.

14. The method of claim 13 wherein said fatty-acid S 20 primary amide substrate is selected from the group consisting of cis-9,10-octadecenoamide, anandamide, myristic amide, palmitic amide and stearic amide.

15. A nucleic acid molecule encoding a fatty-acid amide 25 hydrolase protein, said nucleic acid molecule having a nucleotide sequence selected from the group consisting of 9. SEQ ID NO 35, SEQ ID NO 39 and SEQ ID NO 42.

16. A method of preparing a FAAH according to claim 1, said method comprising expressing a recombinant DNA expression vector that includes a nucleotide sequence that encodes said FAAH, and isolating the expressed FAAH. COMS ID No: SBMI-01190578 Received by IP Australia: Time 10:49 Date 2005-04-05 04/05/2005 10:41 GRIFFITH HACK +61 2 99255911 4 00262837999 NO.104 P010 114

17. The method of claim 16 wherein said FAAH is isolated by purification by a chromatographic method selected from affinity chrcmatography, electric chromatography, gel filtration chromatography, ion exchange chromatography, and partition chromatography.

18. The method of claim 17 wherein said affinity chromatography employs a solid phase adsorbant derivatized with a trifluoroketone inhibitor of FAAH for adsorbing the FAAH.

19. The method of claim 18 wherein said FAAH is isolated by purification as follows: Step A: a crude source of FAAH is purified by exchange chromatography using a DEAE chromatography column to form a first elution product; then Step B: the first elution product of said Step A is further purified by elution on an Hg affinity •e chromatography column to form a second elution product; 20 then Step C: the second elution product of said Step B is further purified by elution on a Heparin affinity chromatography column to form a third elution product; and then 25 Step D; the elution product of said Step C is further purified by elution on an affinity chromatography column derivatized with a trifluoroketone inhibitor of FAAH to oot: form the purified form of FAAH.

20. A trifluoroketone inhibitor of fatty-acid amide hydrolase represented by the following structure: COMS ID No: SBMI-01190578 Received by IP Australia: Time 10:49 Date 2005-04-05 04/05/2005 10:41 GRIFFITH HACK +61 2 99255911 00262837999 NO.104 0011 115 0 F3 C (C(CH C H 3 conjugated to a bead.

21. A nucleic acid molecule encoding a portion of a fatty-acid amide hydrolase protein, said nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO 1.

22. An isolated fatty-acid amide hydrolase (FAAH) capable of hydrolysing cis-9,10-octadecenoamide, myristic amide, palmitic amide and stearic amide, wherein the FAAH is isolated by binding a trifluoroketone inhibitor of FAAH. Dated this 4 t h day of April 2005 THE SCRIPPS RESEARCH INSTITUTE By their Patent Attorneys GRIFFITH HACK 6' 0 IS o oooo COMS ID No: SBMI-01190578 Received by IP Australia: Time 10:49 Date 2005-04-05