JP7593608B2 - Method for screening specific inhibitors of Na+/H+ antiporter - Google Patents

Description

The present disclosure relates to a method for screening for a specific inhibitor of a Na ⁺ /H ⁺ antiporter, more specifically, the present disclosure relates to a method for screening for a specific inhibitor of a Na ⁺ /H ⁺ antiporter that is widely present in many eubacteria and archaea, in particular, an Mrp ( Multiple Resistance and pH adaptation)-type Na ⁺ /H ⁺ antiporter.

Cation/H ⁺ antiporters are membrane-spanning proteins that exist in the cell membrane and transport cations and protons (H ⁺ , also called hydrogen ions) in opposite directions through the membrane. For example, the Na ⁺ /H ⁺ antiporter in the cell membrane mediates the transport of ions through the cell membrane by coupling the transport of H ⁺ from outside the cell to inside the cell with the transport of sodium ions (Na ⁺ ) from inside the cell to outside the cell. Through this function, the cation/H ⁺ antiporter plays various physiological roles, including the cation tolerance of cells and intracellular pH homeostasis.

Each organism usually has multiple Na ⁺ /H ⁺ antiporter genes and expresses or has the ability to express multiple Na ⁺ /H ⁺ antiporter proteins, most of which are composed of a single polypeptide or homomultimers thereof, each encoded by a single gene.

Among them, the Mrp-type Na ⁺ /H ⁺ antiporter is unique. The Mrp-type Na ⁺ /H ⁺ antiporter is a Na ⁺ /H ⁺ antiporter belonging to the evolutionarily conserved Mrp family found in many prokaryotes (eubacteria and archaea), but in each prokaryotic species, the protein is characterized by being composed of multiple subunits encoded by multiple genes (usually seven or six) adjacent to each other in an operon. These multiple subunits are hydrophobic polypeptides with different structures, and all of these multiple subunits are required for the Na ⁺ /H ⁺ antiport activity of Mrp. In this specification, the Mrp-type Na ⁺ /H ⁺ antiporter is also simply referred to as "Mrp", but it should be noted that in past studies, Mrp-type Na ⁺ /H ⁺ antiporters of different organisms may be called by names other than Mrp. It should be noted that some members of the Mrp family transport other types of cations (eg, K ⁺ or ^{Ca2+ )} instead of or in addition to Na ⁺ .

Bacterial infections have been common diseases throughout human history. Thanks to the discovery and invention of various antibiotics, the treatment of bacterial infections has improved greatly. However, even today, serious bacterial infections accompanied by multidrug resistance are becoming evident in the form of hospital-acquired infections, and have become a social problem. For this reason, it can be said that the development of new antibiotics that target the sites of action of conventional antibiotics is beginning to show its limits, or at least is no longer able to efficiently deal with the acquisition of multidrug resistance.

It has been reported that the deficiency of Mrp in Staphylococcus aureus and Pseudomonas aeruginosa, which are typical causative bacteria of hospital-acquired infections, can lead to a reduction in the infection rate and virulence in mice, which are an animal model of infectious diseases (Non-Patent Documents 1 and 2).

Journal of Bacteriology, 2005, Vol. 187, No. 15, p. 5242-5248 Journal of Bacteriology, 2018, Vol. 200, Issue 5, e00611-17

The present inventors have focused on the possibility that Na ⁺ /H ⁺ antiporters, particularly Mrp-type Na ⁺ /H ⁺ antiporters, may be novel targets in the treatment or prevention of infectious diseases and multidrug resistance, etc. There have been no reported studies on finding inhibitors of Na ⁺ /H ⁺ antiporters by screening, and the present disclosure provides an effective screening method for searching for candidate compounds that specifically inhibit these Na ⁺ /H ⁺ antiporters.

The present disclosure encompasses at least the following embodiments.
[1]
A method for screening for a specific inhibitor of a non-Escherichia coli Na ⁺ /H ⁺ antiporter, comprising:
(a-1) culturing a test E. coli lacking an endogenous Na ⁺ /H ⁺ antiporter gene and having a non-E. coli Na ⁺ /H ⁺ antiporter gene introduced therein, in the presence of a test compound and in a first sodium ion concentration range of less than 10 mM;
(a-2) culturing the test E. coli in the presence of the test compound and under a second sodium ion concentration range of 50 to 500 mM;
(a-3) culturing the test E. coli in the first sodium ion concentration range in the absence of the test compound;
(a-4) culturing the test E. coli in the second sodium ion concentration range in the absence of the test compound, and further comprising:
(b) performing an assay for a plurality of test compounds, the assay comprising comparing the growth of the test E. coli in (a-1) with the growth of the test E. coli in (a-3), and the growth of the test E. coli in (a-2) with the growth of the test E. coli in (a-4);
method.
[2]
The method according to [1], wherein the non-Escherichia coli Na ⁺ /H ⁺ antiporter is an Mrp-type Na ⁺ /H ⁺ antiporter.
[3]
The method according to [1] or [2], wherein the test E. coli is deficient in endogenous nhaA and nhaB genes.
[4]
The method according to any one of [1] to [3], wherein the test compound is identified as a candidate for a specific inhibitor when there is no growth inhibition in the test E. coli in (a-1) compared to the growth of the test E. coli in (a-3) and there is growth inhibition in the test E. coli in (a-2) compared to the growth of the test E. coli in (a-4).
[5]
The method according to any one of [1] to [4], wherein the culturing of (a-1) to (a-4) is performed simultaneously for at least 40 types of test compounds.

1 shows growth curves of wild-type E. coli (DH5α), a KNabc strain (KNabc/pGEM) into which a pGEM7zf(+) vector has been introduced, and a KNabc strain (KNabc/pGEMmnh1) into which a vector carrying the mnh1 antiporter gene derived from Staphylococcus aureus has been introduced, in LBK media supplemented with sodium salts at different concentrations (0 to 600 mM). The growth rate was measured over time from the start of culture based on the turbidity (OD ₆₀₀ ) of the culture medium. 1 shows growth curves of wild-type E. coli (DH5α), a KNabc strain (KNabc/pGEM) into which a pGEM7zf(+) vector has been introduced, and a KNabc strain (KNabc/pGEMmnh1) into which a vector carrying the mnh1 antiporter gene derived from Staphylococcus aureus has been introduced, in LBK media supplemented with sodium salts at different concentrations (0 to 600 mM). The growth rate was measured over time from the start of culture based on the turbidity (OD ₆₀₀ ) of the culture medium. Figure 2 shows the same growth curves as in Figure 1 for each strain. The growth curves under the conditions of 300 mM NaCl addition and the absence of test compounds, used in the examples, are indicated by arrows. The NaCl addition concentration is shown in M units at the bottom of each panel. 3 is an example of a sample arrangement (plate format) of a 96-well microplate that can be used in a screening method according to one embodiment. In this example, 80 types of test compounds are assayed in a total of 80 wells in lanes 2 to 11.

In the present disclosure, the non-E. coli Na ⁺ /H ⁺ antiporter means a Na ⁺ /H ⁺ antiporter different from that possessed by natural Escherichia coli. The non-E. coli Na ⁺ /H ⁺ antiporter is typically a Na ⁺ /H ⁺ antiporter derived from a prokaryote (eubacteria or archaea) other than E. coli. The non-E. coli Na ⁺ /H ⁺ antiporter in this embodiment may be one having a corresponding ortholog in E. coli, but is more preferably a Na ⁺ /H ⁺ antiporter having no ortholog in E. coli. The non-E. coli Na ⁺ /H ⁺ antiporter may be one having no protein having 95% or more amino acid sequence identity over the entire length in natural E. coli.

In one embodiment, a particularly preferred non-E. coli Na ⁺ /H ⁺ antiporter is an Mrp-type Na ⁺ /H ⁺ antiporter. Mrp was originally discovered as a protein capable of restoring alkaline resistance to an alkaline-sensitive mutant of the alkaliphilic bacterium Bacillus halodurans C-125 (J. Bacteriol., 1990, 172, 7282-7283). Natural E. coli does not have a gene for an Mrp-type Na ⁺ /H ⁺ antiporter. In other words, E. coli does not have an ortholog of the Mrp-type Na ⁺ /H ⁺ antiporter in the evolutionary lineage. Therefore, it is considered that by performing an assay or screening in the background of E. coli as in this embodiment, it has become possible to distinguish with high sensitivity the specific effect on the externally introduced Mrp-type Na ⁺ /H ⁺ antiporter. Specific examples of Mrp-type Na ⁺ /H ⁺ antiporters include Mnh1 of Staphylococcus aureus and Sha of Pseudomonas aeruginosa. The genomes of eukaryotes, including humans, do not contain genes for Mrp-type Na ⁺ /H ⁺ antiporters.

The test E. coli used in this embodiment is based on E. coli lacking endogenous Na ⁺ /H ⁺ antiporter genes (hereinafter also referred to as "deficient strain"). Endogenous here means that the genes are inherently possessed by E. coli in the natural state. The test E. coli is preferably lacking at least nhaA and nhaB, which are the main Na ⁺ /H ⁺ antiporter genes in E. coli. It is preferable that the test E. coli is also lacking the chaA gene in addition to nhaA and nhaB, since this can further improve the sensitivity of screening.

The deletion of an endogenous gene refers to a state in which a functional gene product cannot be expressed due to the absence or mutation of a part or all of the endogenous gene. A technique for deleting any endogenous gene of E. coli is within the ordinary skill of a person skilled in the art. For example, the whole or part of the coding region and/or promoter region of the endogenous gene may simply be deleted, or the sequence may be replaced with another foreign sequence (e.g., an antibiotic resistance marker gene sequence). An example of a deletion strain suitable in this embodiment is the KNabc strain. The KNabc strain is derived from the K-12 strain, and is deleted by replacing the endogenous nhaA, nhaB, and chaA genes with the ^KmR , ^CmR , and ^ErmR genes, respectively.

The test E. coli lacks an endogenous Na ⁺ /H ⁺ antiporter gene as described above, and instead has a non-E. coli Na ⁺ /H ⁺ antiporter gene introduced therein. As described above, the non-E. coli Na ⁺ /H ⁺ antiporter is preferably an Mrp-type Na ⁺ /H ⁺ antiporter, in which case a gene complex encoding all subunits of the Mrp-type Na ⁺ /H ⁺ antiporter (typically in the form of an operon) is introduced into E. coli.

In this embodiment, introducing a non-E. coli gene into E. coli means introducing the nucleic acid of the gene into E. coli cells so that E. coli can express the product of the gene. Specifically, when the gene of the non-E. coli Na ⁺ /H ⁺ antiporter is introduced into the defective strain of E. coli to form a test E. coli, the gene product of the non-E. coli Na ⁺ /H ⁺ antiporter is expressed, so that Na ⁺ resistance is ensured even though the endogenous Na ⁺ /H ⁺ antiporter of E. coli is defective. It is understood that the non-E. coli Na ⁺ /H ⁺ antiporter in this embodiment does not necessarily have to be completely identical in sequence to that found in a natural or wild-type non-E. coli organism (e.g., a non-E. coli prokaryote), and may be a mutant type relative to the wild type.

The test E. coli used in this embodiment is preferably one that can achieve 50% or more or 75% or more of OD ₆₀₀ compared to wild-type E. coli (e.g., DH5α strain or K ^- 12 strain) cultured under the same conditions when inoculated into a medium containing 200 mM NaCl (Na + ion source) added to LBK medium so that the absorbance at 600 nm (OD ₆₀₀ ) is 0.01 and cultured with shaking at 37 ° C. for 16 hours. The wild-type E. coli referred to here is an E. coli that is not defective in the endogenous Na ⁺ /H ⁺ antiporter gene and can grow normally regardless of the addition or absence of the Na ⁺ ion source. The LBK medium is a liquid medium that is substantially free of Na ⁺ and is obtained by dissolving 10 g of tryptone, 5 g of yeast extract, and 6 g of KCl in water to make 1 L and adjusting the pH to 7.5.

Many specific methods for introducing a non-E. coli gene into E. coli are known to those skilled in the art. For example, it is preferable to introduce the non-E. coli gene into E. coli in the form of a plasmid. It is particularly preferable that the plasmid is a high-copy plasmid capable of replicating 200 or more copies per E. coli cell. One specific example of a suitable plasmid is pGEM available from Promega. The promoter for expression may be a promoter specific to the non-E. coli gene or a heterologous promoter.

The assay used in the screening method of this embodiment includes culturing a test E. coli in the presence of a test compound (a-1) and in a first sodium ion concentration range of less than 10 mM, (a-2) in the presence of the same test compound and in a second sodium ion concentration range of 50-500 mM, (a-3) in the absence of the test compound and in the first sodium ion concentration range, and (a-4) in the absence of the test compound and in the second sodium ion concentration range.

The first sodium ion concentration range is preferably 5 mM or less, more preferably 1 mM or less, and even more preferably substantially 0 mM. "Substantially 0 mM" or "substantially free of Na ⁺ " means that sodium ions are not intentionally added, and does not take into account trace amounts of sodium ion sources that may inevitably accompany the medium materials (e.g., tryptone, yeast extract, water, etc.). Therefore, when the presence or absence of addition of sodium salts or sodium ions is mentioned in this specification, it should be understood that trace amounts of sodium salts or sodium ions that may inevitably accompany the medium materials are not taken into account.

The second sodium ion concentration range is preferably in the range of 100 to 450 mM, more preferably in the range of 150 to 400 mM, and even more preferably in the range of 200 to 350 mM. Within these concentration ranges, screening can be performed with particularly good sensitivity and high reproducibility. The actual sodium ion concentrations in (a-2) and (a-4) can be made substantially the same (for example, within an error range of 10 mM between each other), and the actual sodium ion concentrations in (a-1) and (a-3) can be made substantially the same (for example, within an error range of 5 mM between each other).

The above cultures (a-3) and (a-4) are cultures that provide a reference point for the degree of growth when grown without interference in the absence of the test compound.

In order to make the comparison easier, it is preferable to make the conditions of the above cultures (a-1) to (a-4) the same except for the sodium salt concentration (sodium ion concentration) and the presence or absence of the test compound. This may mean making the basal medium and culture temperature the same. A person skilled in the art who refers to this disclosure can determine an appropriate basal medium and culture temperature for observing the effect of the sodium ion concentration and the presence or absence of the test compound on the growth (proliferation) of the test E. coli. The basal medium means a medium in which the addition of sodium salt and the test compound is omitted. The above-mentioned LBK medium is a suitable basal medium. It is considered that the comparison will be made most easily by making the basal medium and culture temperature of the cultures (a-1) to (a-4) the same, making the sodium salt concentration of (a-1) and (a-3) the same, making the sodium salt concentration of (a-2) and (a-4) the same, and making the test compound concentration of (a-1) and (a-2) the same.

In the culture of (a-1) to (a-4), particularly between (a-1) and (a-3) and between (a-2) and (a-4), and between (a-1) and (a-2) and between (a-3) and (a-4), when the addition of the test compound is omitted and the cells are inoculated so that the OD ₆₀₀ is 0.01 and cultured with shaking at 37°C for 16 hours, it is preferable to have a medium composition that can support the achievement of an OD ₆₀₀ within a range of ±25% of each other, and it is even more preferable to have a medium composition that can support the achievement of an OD ₆₀₀ of 50% or more, or 75% or more, respectively, compared to wild-type E. coli (e.g., DH5α strain or K-12 strain) cultured under the same conditions in LBK medium. Such a medium composition usually has a potassium ion (K ⁺ ) concentration of 5 to 200 mM, preferably a potassium ion concentration of 50 to 100 mM. The pH of the medium is usually 7.0 to 8.0.

The cultures (a-1) to (a-4) are each preferably carried out at a temperature of 30 to 38°C, and more preferably at a temperature of 35 to 37.5°C. The culture temperatures of (a-1) to (a-4) can be aligned to within an error range of 1°C.

The concentration of the test compound in the cultures (a-1) and (a-2) may vary depending on various factors, such as the type and potency range of the specific compound that the user intends to screen, but may be, for example, 1 μM to 1 mM, or 20 μM to 100 μM. The concentrations of the test compounds in (a-1) and (a-2) may be the same within an error range of 10%.

The assay used in the method according to the present embodiment includes, as the next step (b), comparing the growth of the test E. coli in (a-1) with that in (a-3), and comparing the growth of the test E. coli in (a-2) with that in (a-4). Through this comparison, it is possible to search for a test compound that reduces the growth of (a-2) compared to (a-4), but does not reduce the growth of (a-1) compared to (a-3), or the reduction is smaller than the above. With this system, it is possible to identify candidates for inhibitors specific to the function of the non-E. coli Na ⁺ /H ⁺ antiporter by identifying inhibition specific to sodium ion resistance dependent on the non-E. coli Na ⁺ /H ⁺ antiporter.

To make the comparison easier, it is preferable to compare the growth rates of the cultures (a-1) to (a-4) at the same time after the start of the culture. Therefore, it is preferable to start the cultures (a-1) to (a-4) simultaneously under the same conditions. The start of culture is usually understood to mean, in the case of a liquid medium, inoculating the main culture with bacterial cells or a preculture solution to start shaking culture, and in the case of a solid medium, inoculating the solid medium with bacterial cells or a preculture solution to start stationary culture.

For example, it is preferable to compare the growth degrees at 3 to 24 hours, 4 to 20 hours, or 8 to 18 hours after the start of the culture of (a-1) to (a-4). In particular, when the second sodium ion concentration range is 100 mM or less, it may be preferable to compare the growth degrees at 3 to 6 hours or 3 to 16 hours after the start of the culture. When the second sodium ion concentration is 400 mM or more, it may be preferable to compare the growth degrees at 8 to 24 hours or 14 to 24 hours after the start of the culture. The optimal comparison time point may vary depending on the specific type of non-E. coli Na ⁺ /H ⁺ antiporter to be screened, but can be appropriately determined by one skilled in the art. For example, the ^above -mentioned comparison of growth levels can be performed when ^the OD ₆₀₀ value of the culture (a-4) is 0.2 or more, preferably 0.25 or more, more preferably 0.3 or more, and even more preferably 0.4 or more higher than that of a control culture under the same conditions except that a non- E. coli Na + /H + antiporter is not introduced.

If there is no growth inhibition in the growth of the test E. coli in (a-1) compared to the growth of the test E. coli in (a-3), and there is growth inhibition in the growth of the test E. coli in (a-2) compared to the growth of the test E. coli in (a-4), the test compound can be identified as a candidate for a specific inhibitor. If growth inhibition is observed in (a-2) compared to (a-4) and also in (a-1) compared to (a-3), it is understood that growth inhibition occurs even under conditions that do not require the presence of a non-E. coli Na ⁺ /H ⁺ antiporter for growth, and therefore the test compound is considered to inhibit the growth of E. coli through another mechanism, rather than being a specific inhibitor of the non-E. coli Na ⁺ /H ⁺ antiporter.

Here, when a liquid medium is used, "no growth inhibition" means that the turbidity of the medium is not 50% or less of the comparison target, and "growth inhibition" means that the turbidity of the medium is 50% or less of the comparison target. The turbidity of the medium reflects the cell density and can be quantified, for example, as OD _600. It will be understood by those skilled in the art that a relative comparison of turbidity can be made as long as the measurements are performed using the same spectrophotometer under the same conditions. It is also well known to those skilled in the art that the medium turbidity, which is an index of growth (proliferation), is quantified as a value obtained by subtracting the absorbance of the medium itself from the absorbance of the measurement container (cuvette, plate well, etc.).

In a screening embodiment for searching for inhibitors with higher specificity and/or potency, a test compound may be identified as a candidate specific inhibitor if the turbidity of the medium in (a-1) is 75% or more, more preferably 80% or more, and even more preferably 90% or more compared to the turbidity of the medium in (a-3), and/or the turbidity of the medium in (a-2) is 25% or less, more preferably 20% or less, and even more preferably 10% or less compared to the turbidity of the medium in (a-4).

In another specific embodiment, test compounds that satisfy the conditions represented by the following formula may be identified as candidate specific inhibitors.
[Turbidity of medium in (a-1)]/[Turbidity of medium in (a-3)]×100−[Turbidity of medium in (a-2)]/[Turbidity of medium in (a-4)]×100≧x
Here, x is 50, preferably 60, more preferably 70, and even more preferably 80.

The screening method of this embodiment is preferably carried out using a liquid medium, but an embodiment using a solid medium such as an agar medium is also contemplated. When a solid medium is used, "there is no growth inhibition of the test E. coli in (a-1) compared to the growth of the test E. coli in (a-3)" means that colony formation is observed in both (a-3) and (a-1), and "there is growth inhibition of the test E. coli in (a-2) compared to the growth of the test E. coli in (a-4)" means that colony formation is observed in (a-4) but not in (a-2). The presence or absence of colony formation can be determined with the naked eye according to the conventions in this technical field. The solid medium may be the liquid medium described above to which agar has been added to form a gel.

In the screening method of this embodiment, it is understood that the same set of assays (a-1) to (a-4) and (b) is performed for multiple types of test compounds. Preferably, at least 40 types, more preferably at least 80 types of test compounds can be simultaneously cultured with (a-1) to (a-4). When a liquid medium is used, the volume of each assay, i.e., the culture of (a-1) to (a-4), is preferably 1 mL or less, more preferably 200 μL or less, and even more preferably 100 μL or less. In this screening method, a multi-well plate, for example a 96-well plate, can be used as a culture vessel and/or a measurement vessel. The cultures of (a-3) and (a-4) do not necessarily have to be performed for the number of test compounds. That is, the cultures of (a-1) and (a-2) are each performed for the number of test compounds to be screened, but the cultures of (a-3) and (a-4), which are comparison subjects not containing the test compounds, may each be provided in common for multiple test compounds.

Embodiments of the present disclosure may be characterized by a low false positive rate.Embodiments of the present disclosure may also be suitable for implementation as high-throughput screening systems, which may be directly applicable to drug discovery research or development.

In addition, the embodiments of the present disclosure can evaluate and identify candidate inhibitor compounds using the presence or absence of microbial growth or the degree of growth as an indicator, so screening can be performed at low cost using relatively simple equipment.

In this disclosure, the use of "to" to indicate a range of values means that the values before and after it are included as the lower and upper limits, respectively. When multiple possible ranges of values are separately described, such as "A-B" and "C-D," it should be understood that the ranges combining one lower limit or upper limit with the other upper limit or lower limit (e.g., "A-D," "C-B," and "B-D") are also disclosed herein. In addition, when a combination of a first element and a second element is described, and multiple possible options are described for each of the first element and the second element, it should be understood that all possible combinations of each option of the first element and each option of the second element are disclosed herein.

Here, an example of searching for an inhibitor of Mrp-type Na ⁺ /H ⁺ antiporter derived from Staphylococcus aureus is described. The operon of the mrp gene (mnh1) derived from Staphylococcus aureus was cloned into a high-copy plasmid vector of E. coli, and the plasmid was introduced into the E. coli KNabc strain by a conventional method to perform transformation. The KNabc strain is a strain in which the main Na ⁺ /H ⁺ antiporter of E. coli is deleted. Under the condition of pH 7.5, when the NaCl concentration of the medium is increased to about 50 mM, growth begins to be clearly delayed, and in the presence of 100 mM NaCl, growth is significantly inhibited, and in the presence of 200 mM or more NaCl, growth is almost completely inhibited. In contrast, wild-type E. coli and the KNabc strain expressing Mrp derived from Staphylococcus aureus can grow even in the presence of NaCl up to 600 mM (Figures 1 and 2). 1 and 2 show the degree of growth as a function of time after the start of main culture, in which the bacteria were pre-cultured as described below, then inoculated into a main culture solution, and then main cultured in the presence of NaCl at different concentrations.

Wild-type E. coli, including the K12 strain, the parent strain of KNabc, do not inherently have an Mrp-type Na ⁺ /H ⁺ antiporter. This example demonstrates that by utilizing the change in Na ⁺ resistance in test E. coli strains that lack endogenous Na ⁺ /H ⁺ antiporters and have an Mrp-type Na ⁺ /H ⁺ antiporter introduced, it is possible to very clearly distinguish inhibitor candidates that exhibit specific inhibitory activity against Mrp.

1. Reagents ・Bacto Tryptone (Difco Laboratories No. 211705)
・Bacto Yeast Extract (Difco Laboratories No. 212750)
・NaCl (special grade, Wako Pure Chemical No. 191-01665)
・KCl (special grade, Wako Pure Chemical No. 161-03541)
・Potassium hydroxide (special grade, Wako Pure Chemical No. 162-21813)

2. Strains and plasmids used: Escherichia coli KNabc strain (derived from K-12, ΔnhaA::Km ^R , ΔnhaB::Cm ^R , ΔchaA::Erm ^R )
- pGEMmnh1 plasmid (the mnh1 operon of S. aureus cloned into the pGEM7zf(+) vector available from Promega. Mnh1 is Mrp of S. aureus. pGEM7zf(+) also contains the ampicillin resistance gene.)
By introducing pGEMmnh1 into the KNabc strain by a conventional method, the KNabc strain transformed with mnh1 was obtained as a test E. coli. This test E. coli was called KNabc/pGEMmnh1.

3. Apparatus and Equipment: 96-well transparent polystyrene plate (Greiner No. 650901)
・Biotek Powerwave XS plate reader ・UV-Visible spectrophotometer UV 1800 (Shimadzu Corporation)
・15 mL polystyrene tube (AS ONE No. 34184015D)

4. Experimental procedure (1) Preparation of LBK medium (pH 7.5) and LBK+NaCl medium (pH 7.5)
10 g of Bacto Tryptone, 5 g of Bacto Yeast Extract, and 6 g of KCl were dissolved in 800 mL of MilliQ water. In the case of LBK+NaCl medium, NaCl was further added to a given final concentration (e.g., 300 mM). The pH was adjusted to 7.5 with 4M KOH. The volume was adjusted to 1 L by adding MilliQ water. The obtained liquid medium was dispensed into pressure bottles in 200 mL portions and sterilized by autoclaving (121°C, 20 min).

(2) Culture (2-1)
2 mL of LBK medium (pH 7.5) containing 2 μL each of 100 mg/mL ampicillin and kanamycin solutions was placed in a 15 mL culture tube, inoculated with the KNabc/pGEMmnh1 strain, and incubated at 37°C for 7 days while shaking at 200 rpm. Pre-incubation was carried out for ∼8 h.

(2-2)
The turbidity of the preculture was measured with a spectrophotometer, and the preculture was inoculated into the main culture so that the turbidity (OD ₆₀₀ ) was 0.01. The main culture was prepared by adding the same antibiotics as above to LBK medium or LBK+NaCl medium.

(2-3)
The inoculated culture medium was dispensed in 40 μL portions into 96-well microplates in which different test compounds dissolved in DMSO solvent were placed in separate wells to provide test compound-containing samples (final test compound concentration: 50 μM). In the lanes at both ends of each microplate, multiple samples (n≧4) were provided, including "Control" samples in which the test compound was omitted from the test compound-containing samples, i.e., only DMSO solvent was added instead of the DMSO solution of the test compound, and "Background" samples in which not only the test compound but also the test E. coli was omitted (Figure 3). Test compound-containing samples, "Control" and "Background" samples were provided for each of the LBK medium and LBK+NaCl medium.

(2-4)
The microplate was set in a Biotek Powerwave XS plate reader and cultured at 37°C for 17 hours at 570 r/min with shaking (main culture). The experiment was performed in automatic mode, measuring OD ₆₀₀ every 0.5 or 1 hour, to measure the turbidity of the medium, i.e., the growth rate of the test E. coli.

5. Data processing (1) Data quality control Based on the measured values of the "Background" and "Control" samples, which were placed on each plate, S/B, CV(Background), CV(Control), and Z' were calculated for each plate (n≧4).
Where:
・S/B = MeanC /MeanB
・CV (%) = 100 (SDB/MeanB) or 100 (SDC/MeanC)
・Z' = 1-(3SDB+3SDC)/(MeanC-MeanB)
where MeanB is the mean value of the Background, MeanC is the mean value of the Control, SDB is the standard deviation of the Background, and SDC is the standard deviation of the Control.

(2) Calculation of inhibition rate The inhibition rate (%) was calculated by subtracting the background mean value from the measured value of the sample containing the test compound (A), dividing the result by the control mean value minus the background mean value (B), and multiplying the result by 100. That is, inhibition rate (%) = (A/B) × 100. The smaller the inhibition rate number, the more the growth of the test E. coli was reduced by the test compound, i.e., the greater the inhibition.

6. Results Table 1 shows examples of the results of a screening test performed based on OD 600 measurements 16 hours after the start of main culture, with the first sodium ion concentration set to 0 mM (provided by LBK medium without NaCl) and the second sodium ion concentration set to ₃₀₀ mM (provided by LBK medium with 300 mM NaCl).

If screening of test compounds is performed under conditions where Na ⁺ resistance is ensured depending on the presence of a non-E. coli Na ⁺ /H ⁺ antiporter, positive growth inhibition is observed quite frequently, which may result in insufficient efficiency as a screening method (see the third column in Table 1). However, most of the positive compounds are found to cause growth inhibition even under low Na ⁺ concentration conditions that do not require the activity of a non-E. coli Na ⁺ /H ⁺ antiporter, and can be excluded from the list of candidates for the specific inhibitor of interest.

It was revealed that test compounds that allowed essentially normal growth at low sodium ion concentrations, such as test compounds 9, 16, and 20 in Table 1, but caused significant growth inhibition of the test E. coli at high sodium ion concentrations, could be clearly distinguished from a large number of test compounds and identified. Such a result was not necessarily expected. One of the characteristics of Mrp is the fact that the subunits predicted to be involved in ion transport have homology to NuoL, NuoM, and NuoN, which are proton transport subunits of complex I (NADH oxidoreductase) of the respiratory chain complex (Mathiesen & Hagerhall, BBA, 2002). NuoL, NuoM, and NuoN of complex I of the respiratory chain complex are also called "antiport subunits," and in 2013, Baradaran and Sazanov et al. revealed their detailed H ⁺ transport pathways through crystal structure analysis (Baradaran et al., Nature, 2013). MrpA, MrpD, NuoL, NuoM, and NuoN share a common NDH-I domain, and the charged amino acid residues predicted to be involved in ion transport are also highly conserved. Since respiratory chain complex I is naturally present in E. coli and mitochondria, it is expected that compounds that inhibit ion transport of the Mrp-type Na ⁺ /H ⁺ antiporter will also have an inhibitory effect on respiratory chain complex I. Such inhibitors may also be difficult to administer to humans. The results of this example are the first example showing that truly selective inhibitors for the Mrp-type Na ⁺ /H ⁺ antiporter may exist and that such inhibitors can be clearly identified.

Claims (4)

A method for screening for a specific inhibitor of a non-Escherichia coli Na ⁺ /H ⁺ antiporter, comprising:
(a-1) culturing a test E. coli lacking an endogenous Na ⁺ /H ⁺ antiporter gene and having a non-E. coli Na ⁺ /H ⁺ antiporter gene introduced therein, in the presence of a test compound and in a first sodium ion concentration range of less than 10 mM;
(a-2) culturing the test E. coli in the presence of the test compound and under a second sodium ion concentration range of 50 to 500 mM;
(a-3) culturing the test E. coli in the first sodium ion concentration range in the absence of the test compound;
(a-4) culturing the test E. coli in the second sodium ion concentration range in the absence of the test compound, and further comprising:
(b) performing an assay for a plurality of test compounds, the assay comprising comparing the growth of the test E. coli in (a-1) with the growth of the test E. coli in (a-3), and the growth of the test E. coli in (a-2) with the growth of the test E. coli in (a-4), and identifying the test compound as a candidate for a specific inhibitor when there is no growth inhibition of the test E. coli in (a-1) compared to the growth of the test E. coli in (a-3) and there is growth inhibition of the test E. coli in (a-2) compared to the growth of the test E. coli in (a-4).
method. The method of claim 1, wherein the non-E. coli Na ⁺ /H ⁺ antiporter is an Mrp-type Na ⁺ /H ⁺ antiporter. The method of claim 1 or 2, wherein the test E. coli is deficient in endogenous nhaA and nhaB genes. The method according to any one of claims 1 to 3 , wherein the culturing of (a-1) to (a-4) is carried out simultaneously for at least 40 types of test compounds.