WO2020136298A2 - Caspase 1 inhibitors for the treatment of anaemia - Google Patents

Abstract

The present invention refers to a composition comprising at least a caspase-1 inhibitor for use in a method of treatment of a disease selected from the list consisting of anemia-associated to chronic diseases, chemotherapy-induced anemia and Diamond-Blackfan anemia.

Description

Caspase 1 inhibitors for the treatment of anemia.

Field of the Invention

The present invention relates to the field of medicine, in particular to the treatment of anemia with caspase-1 inhibitors.

Background of the Invention

Hematopoiesis is the process of blood cell formation that occurs during embryonic development and adulthood to produce the blood system (Jagannathan-Bogdan and Zon, 2013). In vertebrates, blood development involves two waves of hematopoiesis: the primitive one during early embryonic development, and the definitive one, which occurs in later stages (Gore et al., 2018). Definitive hematopoiesis employs multipotent hematopoietic stem cells (HSCs), which ultimately migrate to the bone marrow, or kidney marrow in zebrafish, and give rise to all blood lineages (Birbrair and Frenette, 2016; Cumano and Godin, 2007). . Maturation of HSC involves diversification of lymphoid cell lineages (T, B, and NK cells) and myeloid / erythroid cells (megakaryocytes, erythrocytes, granulocytes, and macrophages) (Kondo, 2010; Kondo et al., 2003; Weissman, 2000). The decision of the destinations of erythroid and myeloid cells mainly depends on two factors of the GATA1 and SPI1 transcription (also known as PU.1) that show a cross-inhibitory relationship that results in physical interaction and direct competition between them for the target genes ( Nerlov et al., 2000; Rekhtman et al., 1999). However, there are many controversies about factors responsible for differentiation of terminal erythroid and myeloid cells and many unknown routes that are probably involved in their regulation (Cantor and Orkin, 2002; Hoppe et al., 2016). These unidentified routes may have important clinical implications, since hematopoietic lineage bias is associated with an increased incidence of diseases with prominent inflammatory components including atherosclerosis, autoimmunity, neurodegenerative disease, and carcinogenesis (Elias et ai, 2017). Inflammasomes are part of the innate immune system and as intracellular receptors and sensors regulate the activation of inflammatory caspases, specifically caspase-1 and caspase-1 (caspase-4 and caspase-5 in humans), which induce inflammation in response to infectious microbes. and endogenous warning signs (Latz et al., 2013; Martinon et al., 2009). Normally, the inflammasome multiprotein complexes contain sensing proteins (NOD-like receptors, NLR), adapter proteins (apoptosis-related stain-like protein containing a CARD, ASC), and effector caspases in a form of zymogen, all being capable to interact with each other through homotypic interactions (Broz and Monack, 2011; Sharma and Kanneganti, 2016). Recently, the GBP protein family has also been shown to be part of these multiprotein complexes (Pilla et al., 2014; Santos et al., 2018; Tyrkalska et al., 2016; Wallet et al., 2017; Zwack et al., 2017). The oligomerization of pro-caspases and their autoproteolytic maturation lead to the processing and secretion of proinflammatory cytokines interleukin-ΐ b (IL-1 b) and IL-18, and the induction of a form of programmed cell death called piroptosis (Lamkanfi and Dixit , 2014). Lately, it turned out that inflammasomes play crucial roles not only in sterile infection and inflammation but also in the maintenance of basic cellular functions and the control of cellular homeostasis (Rathinam & Fitzgerald, 2016). Thus, recently discovered regulatory roles for inflammasomes in cell metabolism, proliferation, gene transcription, and oncogenesis have been demonstrated (Rathinam & Fitzgerald, 2016; Sharma & Kanneganti, 2016). Although little is known to date about the impact of inflammasomes on hematopoiesis in general, it has been shown that the master erythroid transcription factor GATA1 could be cleaved in vitro by many caspases and in vivo by caspase-3 (De Maña et al ., 1999).

Zebrafish has recently emerged as a powerful and useful model for studying hematopoiesis (Berman et al., 2012; Ellett and Lieschke, 2010). Furthermore, genetic programs that control hematopoiesis in zebrafish are conserved with mammals, including humans, making them clinically relevant model systems (Jagannathan-Bogdan & Zon, 2013). This document shows for the first time the critical role played by the inflammasome in the regulation of erythroid / myeloid cell fate decision and terminal erythroid differentiation using zebrafish, mouse and human models. Furthermore, the results also have important clinical implications, since pharmacological inhibition of the inflammasome rescues models of zebrafish and mouse disease from neutrophilic inflammation and anemia.

Brief description of the figures

Figure 1. Inhibition of inflammasomes decreases the number of neutrophils in zebrafish. Single-celled Tg zebrafish embryos (mpx.eGFP) were injected with standard control (Std), Ase or MO of Gbp4 (a, b, g, h), and / or with antisense mRNA (As), Gbp4WT, Gbp4KS / AA , Gbp4ACARD, Gbp4DM, Ase or Caspa (eh). Alternatively, the Tg embryos (mpx.eGFP) that were not injected had the chorion manually removed at 24 or 48 hpf and were treated by immersion with DMSO or the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH) (c, d, i, j). Each point represents the number of neutrophils of a single larva, while the mean ± SEM for each group is also shown (a, c, e, g, i). The sample size (n) is indicated for each treatment. Representative images of complete larval green carcasses for different treatments are also shown. Scale bars, 500 pm. Caspase-1 activity in complete larvae was determined for each treatment at 72 hpf (n = 30) (b, d, f, h, j). * p <0.05; ** p <0.01; *** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test.

Figure 2. Inhibition of inflammasomes increases the number of erythrocytes in zebrafish. The Tg zebrafish embryos (lcr.eGFP) had their chorion manually removed at 24 hpf and were treated by immersion with DMSO or the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH) for 48 h (a ). Alternatively, Tg unicellular embryos (lcr.eGFP) were injected with standard control (Std) or Ase MO (b). Each point represents the percentage of GFP + cells from each group of 50 larvae, while the mean ± SEM for each group is also shown. Representative dot diagrams of green and blue channels of control morphants (b, e) treated with caspase-1 inhibitor (c) and Ase (f) are shown. *** p <0.001 according to the Student's t test. Figure 3. The inflammasome is intrinsically required for HSC differentiation but is dispensable for its appearance in zebrafish, (ah) Tg zebrafish embryos (runx1: GAL4 \ UAS.nfsb-mCherry) had their chorion manually removed at 24 or 48 hpf and were treated by immersion with DMSO or the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH) for 24 or 48 h (af). Alternatively, Tg unicellular embryos (runx1: GAL4 \ UAS.nfsb-mCherry) were injected with standard control (Std) or Ase MO (gh). Each point represents the HSC number of a single larva, while the mean ± SEM for each group is also shown. The sample size (n) is indicated for each treatment. Representative images of complete larval red carcasses for the different treatments (a, c, e, g) are also shown. Scale bars, 500 pm. Caspase-1 activity was determined for each treatment of 48 or 72 hpf larvae (n = 30) (b, d, f, h). (il) Tg larvae were fixed (runx1: gal4; UAS: Gbp4KS / AA) (i), Tg (mpx: gal4; UAS: Gbp4KS / AA) (j), Tg (runx1: gal4; UAS: AscACARD) (k ), Tg (mpx: gal4; UAS: AscACARD) (I) at 72 hpf and stained with Sudan black for neutrophil detection. Each point represents the number of neutrophils of a single larva, while the mean ± SEM for each group is also shown. The sample size (n) is indicated for each ns treatment, not significant; * p <0.05; ** p <0.01; *** p <0.001 according to the Student's t test.

Figure 4. Inflammasome activity is essential for zebrafish myelopoiesis. The Tg zebrafish larvae (mpx: GAL4; UAS.nsfb-mCherry) had their chorion removed manually at 48 hpf and were treated by immersion with metronidazole (Mtz) for 24 h and then with DMSO or the irreversible caspase-1 inhibitor. Ac- YVAD-CMK (C1 INH) for the next 4 days. Control groups were treated for 5 days with Mtz (all the time) (a) Each point represents the number of neutrophils from a single larva, while the mean ± SEM for each group is also shown (n = 30). (b) Representative images of complete larval red channels for different treatments and time points are also shown. Scale bars, 500 pm. *** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test. Figure 5. The infection cannot circumvent the inflammasome requirement for the production of neutrophils in zebrafish (ah). Single-celled Tg zebrafish embryos (mpx: eGFP) were injected with conventional control (Std), Gbp4, or Ase MO in combination with antisense mRNA (As), Gesta, Ase, Dandruff (c, d, g, h, i, j) or were not injected, the chorion was manually removed at 48 hpf and treated by immersion with DMSO or the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH) (a, b, e, f). Larvae at 48 hpf were then infected with S. typhimurium (Sl) in the otic vesicle (a, b) or yolk sac (g, h) and the number of neutrophils throughout the body was counted at 24 hpi (a, b) or 72 hpf (cf) and survival was determined for 5 days after infection (g, h). Each point represents the number of neutrophils of a single larva, while the mean ± SEM for each group is also shown. The sample size (n) is indicated for each treatment. Representative images of complete larval green carcasses for the different treatments (af) are shown. Scale bars, 500 pm. Caspase-1 activity in complete larvae was determined for each treatment at 72 hpf (n = 30) (b, d, f). (i- j). The levels of sp¡1 b, gatal a, mesf and gesf mRNAs in larval tails were measured by RT-qPCR at 24 hpf (i), whereas the levels of Gatala and histone H3 protein were determined using Western blotting in larval tails at 24 hpf (j). A densitometry analysis was performed to check the differences between ns treatments, not significant; * p <0.05; ** p <0.01; *** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test (af, i, j) or logarithmic range test with Bonferroni correction (g, h).

Figure 6. Pharmacological inhibition of caspase-1 in human CD34 + HSCs promotes erythroid differentiation. CD34 + cells were incubated with EPO for 5 days in the presence of DMSO or the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH, 50mM). The mRNA levels of the genes coding for the inflammasome components CASP1, PYCARD, NLRP3 and NLRC4 (a) and the differentiation markers GATA1, GYPA, TFRC and SLC4A1 (c) were measured by RT-qPCR, while the activity Caspase-1 was determined using the YVAD-AFC fluorogenic substrate (b). * p <0.05; ** p <0.01; *** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test. Figure 7. Pharmacological inhibition of caspase-1 impairs erythroid differentiation of K562 cells. K562 cells were incubated with 50 mM hemin for the indicated time in the presence or absence of the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH, 50 mM) and images of cell pellets were obtained (a, e, g) were lysed and resolved by SDS-PAGE and immunoblotted with anti-GATA1 and anti-ACTB (a, e, f) antibodies, processed for the quantification of caspase-1 activity using the fluorogenic substrate YVAD-AFC ( b, g) and for immunofluorescence using anti-CASP1 and anti-GATA1 (c, d) antibodies. Cell extracts of HEK293T transfected with GATA1 -FLAG and empty FLAG were included as mobility controls in a. Immunofluorescence overlap of caspase-1 and K562 DAPI stained nuclei differentiated for 48 h with hemin in d are shown. Scale bars, 5 pm. *** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test.

Figure 8. Pharmacological inhibition of caspase-1 releases zebrafish models of neutrophilic inflammation and anemia (ae). Wild-type larvae and spintl mutants were manually removed from the chorion and treated from 1-3 dpf with the inhibitor of Irreversible caspase-1 Ac-YVAD-CMK (C1 INH, 100 mM). The activity of caspase-1 (a), the gene expression ratio sp¡1 b / gata1 a (b), the dispersion of neutrophils (c) and the number of neutrophils (d, e) were then determined. Each point represents the number of neutrophils of a single larva, while the mean ± SEM for each group is also shown. Representative images of complete larval green carcasses for the different treatments are shown (e). Scale bar, 500 pm. (fh) Zebrafish unicellular embryos were injected with conventional control (Std) or Gatal MO, the chorion was manually removed at 24 hpf and they were treated by immersion with DMSO or the reversible caspase-1 inhibitor Ac-YVAD- CFIO (C1 INH) for 24-48 hpf. The inhibitor was then removed by washing and the larvae were incubated up to 72 hpf. Representative images of Gatal a deficient larvae with mild, moderate and severe anemia (f), quantification of the phenotype of larvae treated with DMSO or C1 INH (g) and immunoblotting of larval extracts with anti-Gata1 a, anti-Sp¡1 antibodies by anti-Actb. (a, b) n = 4; (c) n = 35, 35, 27 and 22. (d) n = 29, 28, 19 and 17. (g) n = 116 and 96. ns, not significant; * p <0.05; ** p <0.01; *** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test (ad) and Fisher's exact test

(g).

Figure 9. Pharmacological inhibition of caspase-1 frees mice treated with 5-FU from anemia. (a) Experimental design. Mice were injected i.p. with 5-FU on day 0 and then with 10 mg / kg of the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH) in PBS with 10% DMSO or vehicle only on days 6, 7, 10 and 12. Blood was collected at -1, 6, 10 and 14 d after injection of 5-FU (source in red) and analyzed on a ProCyte Dx hematology analyzer. (b-f). Erythrocyte counts (b), hemoglobin (c), hematocrit (d), platelets (e), and white blood cells (f) are shown as the mean ± SEM (n = 13). * p <0.05 according to 2-factor ANOVA followed by Bonferroni's multiple amplitude test.

Figure 10. Proposed model illustrating the regulation of erythroid / myeloid decision and terminal erythroid differentiation by the inflammasome. (a) Under homeostasis conditions, the activation of the inflammasome favors the myeloid differentiation of CMP promoting the cleavage of GATA! However, the inflammasome is also activated during terminal erythroid differentiation to inactivate GATA! (b) In chronic inflammatory diseases, excessive activation of inflammasomes in CMP results in disproportionate degradation of GATA1, resulting in myeloid bias; that is, neutrophilia and anemia (ACD). (c) The pharmacological inhibition of caspase-1 in chronic inflammation restores a normal myeloid / erythroid differentiation, reducing neutrophilia and improving anemia. CMP, common myeloid progenitors; GMP, granulocytic-monocytic parents; MEP, megakaryocyte-erythrocyte progenitors; N, neutrophils, M, monocytes / macrophages; E, erythrocytes.

Figure 11. Inhibition of inflammasomes decreased the number of macrophages in zebrafish larvae. Single-celled Tg zebrafish embryos (mpeg: eGFP) were injected with conventional control (Std), Ase or Gbp4 MO (a, b), or with antisense mRNA (As), Ase or / and Dandruff (ef). Alternatively, Tg (mpeg: eGFP) embryos were manually removed from the chorion at 48 hpf and treated by immersion with DMSO or the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH) (c, d). Each dot represents the number of macrophages from a single larva, while the mean ± SEM is also shown for each group (a, c, e). The sample size (n) is indicated for each treatment. Representative images of complete larval green carcasses for different treatments are also shown. Scale bars, 500 pm. Caspase-1 activity in complete larvae was determined for each treatment at 72 hpf (n = 30) (b, d, f). * p <0.05; ** p <0.01; *** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test.

Figure 12. Inhibition of inflammasomes decreases the number of neutrophils in zebrafish larvae. Single-celled Tg zebrafish embryos (lyz: dsRED) were injected with standard control (Std), Ase or MO of Gbp4 (a, b). Alternatively, Tg (lyz: dsRED) larvae were manually removed from the chorion at 48 hpf and treated by immersion with DMSO or the irreversible caspase-1 inhibitor Ac-YVAD-CMK (C1 INH) (c, d). Each point represents the number of neutrophils of a single larva, while the mean ± SEM for each group is also shown. The sample size (n) is indicated for each treatment. Representative images of complete larval red carcasses for the different treatments (a, b) are also shown. Scale bars, 500 pm. Caspase-1 activity in complete larvae was determined for each treatment at 72 hpf (n = 30). (b, d). * p <0.05; *** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test.

Figure 13. Inflammasome activity regulates levels of gatal expression in zebrafish larvae. Casper zebrafish unicellular embryos were injected with conventional control (Std), Ase or MO of Gbp4. At the indicated times, full assembly in situ hybridization (WISH) was performed using antisense probes to the gatal a, sp¡1 b, gesfr, cmyb, runxl and rag1 genes. The numbers in the drawings represent animals with the phenotype shown by total analyzed animals. Scale bar: 500 pm.

Figure 14. Expression of genes encoding for key inflammasome components are tightly regulated in human progenitor and hematopoietic cells. Relative levels of expression of GATA1, CASP1, PYCARD, NLRC4, NLRP3, NLRP1, GBP5, and IL1 B in human hematopoietic stem cells (HSC), lymphoid-primed multipotent progenitor (LMPP), common myeloid progenitors (CMP), megakaryocytic-erythroid progenitors (MEP) and granulocytic-monocytic progenitors (GMP) according to the GSE63270 data exposed from the GEO database. Each point represents the gene expression of a donor, while the mean ± SEM for each group is also shown (n = 7). ^* p <0.05; ^** p <0.01; ^*** p <0.001; according to ANOVA followed by Tukey's multiple amplitude test.

Figure 15. Expression of genes encoding inflammasome components is regulated during erythroid differentiation of K562 cells. K562 cells were incubated with hemin for 48 h and then mRNA levels of the NLRC4, NLRP3, PYCARD and CASP1 genes were determined by RT-qPCR (n = 3). ^* p <0.05; ^** p <0.01; ^*** p <0.001 according to ANOVA followed by Tukey's multiple amplitude test.

Figure 16. Caspase-1 cleaves human GATA1 in vitro at residue D300. (a) Schematic of human GATA1 showing the zinc finger domains and residues D276 and D300. (bd) HEK293T cells were transfected with FLAG-vacuum or FLAG-GATA1 (b, c) and vacuum-FLAG, wild-type GATA1-FLAG (W27), GATA1-FLAG (D276A), GATA1-FLAG (D300A) expression plasmids. ) or GATA1 -FLAG (D276A / D300A) (DM) (d). Twenty-four hours after transfection, GATA1 was removed from cell extracts with M2 anti-FLAG affinity gel and treated or not for 2 hr at 37 ° C with 10 Ul of recombinant human caspase-1. Full-length GATA1 and proteolytic fragments generated on SDS-PAGE were resolved and immunoblotted with anti-FLAG to visualize full-length and N-terminal anti-GATA1 (b, d) and (C-terminal).

Detailed description of the invention

Definitions

To facilitate review of the various examples of this disclosure, the following explanations of specific terms are provided:

The term "acyl" refers to the group of formula RC (O) - wherein R is an organic group. "Administration of" and "administering" a compound should be understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. The compound or composition can be administered to the subject by another person (eg, intravenously) or can be self-administered by the subject (eg, tablets).

The term "alkoxy" refers to a group of the formula —OR, in which R is an organic group such as an alkyl group, optionally substituted with an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group. Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxycyclopropoxy, cyclohexyloxy, and the like.

The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl , heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. A "lower alkyl" group is a saturated branched or unbranched hydrocarbon having from 1 to 10 carbon atoms. Alkyl groups can be substituted alkyls in which one or more hydrogen atoms are substituted with a substituent such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl or carboxyl.

The term "alkylamino" refers to alkyl groups as defined above in which at least one hydrogen atom is replaced with an amino group.

The term "alkenyl" refers to a hydrocarbon group of 2 to 24 carbon atoms and a structural formula that contains at least one carbon-carbon double bond.

The term "alkynyl" refers to a hydrocarbon group of 2 to 24 carbon atoms and a structural formula containing at least one carbon-carbon triple bond.

The term "aliphatic" is defined as including alkyl, alkenyl, alkynyl, halogenated alkyl, and cycloalkyl groups as described above. A "lower aliphatic" group is a branched or unbranched aliphatic group having from 1 to 10 carbon atoms. The term "amine" or "amino" refers to a group of the formula— NRR ', where R and R' can independently be hydrogen or an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl group. or heterocycloalkyl described herein.

The term "amide group" or "amido group" is represented by the formula— C (0) NRR ', where R and R' can independently be a hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl or heterocycloalkyl described herein.

An "animal" refers to living multicellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (such as cats and dogs), livestock (such as pigs, sheep, cows ), as well as non-domesticated animals, such as big cats. The term subject is applied regardless of the phase in the life cycle of the organism. Thus, the term subject is applied to an organism in utero or ovo, depending on the organism (i.e., whether the organism is a mammal or a bird, such as a domesticated or wild poultry).

The term "aryl" refers to any carbon-based aromatic group that includes, but is not limited to, benzene, naphthalene, etc. The term "aromatic" also includes "heteroaryl group", which is defined as an aromatic group having at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorous. The aryl group may optionally be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxyl, carboxylic acid, or alkoxy, or the aryl group can be unsubstituted.

"Carbonyl" refers to a radical of formula— C (O) -. Carbonyl-containing groups include any substituent containing a carbon-oxygen double bond (C = 0), including acyl groups, amides, carboxyl groups, esters, ureas, carbamates, carbonates, and ketones and aldehydes, such as substituents based on - COR or -RCHO where R is an alkyl, heteroalkyl, aliphatic, heteroaliphatic hydroxyl, or a secondary, tertiary or quaternary amine.

A "carboxyl residue" refers to any residue or group that includes— C (0) 0—. Illustrative carboxyl residues include carboxylic acid (-C (O) OH); a carboxylate (-C (O) OR) ester where R is an aliphatic or heteroaliphatic group); a carboxylate salt (- C (O) OM) where M is a cation such as L, Na or K.

The term "co-administered" or "to be co-administered" refers to the administration of the compound disclosed herein with at least one other therapeutic agent within the same general time period, and does not require administration at the same point in time ( although co-administration includes administering at the same moment of time). Therefore, co-administration can be on the same day or on different days, or in the same week or in different weeks.

A "covalent bond" refers to an interatomic bond between two atoms, characterized by the sharing of one or more pairs of electrons by the atoms. The terms "covalently linked" or "covalently linked" refer to converting two differentiated molecules into one contiguous molecule.

The term "cycloalkyl" refers to a non-aromatic carbon-based ring that is made up of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heterocycloalkyl group" is a cycloalkyl group as defined above in which at least one of the ring carbon atoms is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorous.

The terms "halogenated alkyl" or "haloalkyl group" refer to an alkyl group as defined above with one or more hydrogen atoms present in these groups substituted with a halogen (F, Cl, Br, I).

The term "heteroaryl" refers to a condensed or non-condensed mono or polycyclic (eg, b or tricyclic, or more) ring or radical system having at least one aromatic ring, having from five to ten atoms ring of the which one ring atom is selected from S, O and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon. A heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoimyl, benzoimyl, benzoimyl, benzoimyl, benzoimyl.

The term "heteroaralkyl" refers to an alkyl residue attached to a heteroaryl ring. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, and the like.

The term "heterocycloalkyl" refers to a non-aromatic 3-, 4-, 5-, 6-, or 7-membered ring or a fused or non-fused group b or tricyclic system, where (i) each ring contains between one and three selected heteroatoms regardless of oxygen, sulfur and nitrogen, (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms can be optionally oxidized , (iv) the nitrogen heteroatom can optionally be quaternized, and (iv) any of the above rings can be fused to a benzene ring. Representative heterocycloalkyl groups include, but are not limited to, [1, 3] dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl and tetrahydrofuryl.

The term "hydroxyl" is represented by the formula— OH.

The term "hydroxyalkyl" refers to an alkyl group having at least one hydrogen atom substituted with a hydroxyl group. The term "alkoxyalkyl group" is defined as an alkyl group having at least one hydrogen atom substituted with an alkoxy group described above.

"Inhibit" refers to inhibiting the complete development of a disease or condition. "Inhibit" also refers to any quantitative or qualitative reduction in binding or biological activity, relative to a control. A "mimetic" refers to a chemical entity that contains structural elements that can mimic the biochemical or biological action of another chemical entity. For example, in a peptidomimetic the three-dimensional arrangement of the chemical constituents of such a peptidomimetic mimics the three-dimensional arrangement of the peptide backbone and component amino acid side chains of another peptide resulting in an agent that is specific and / or selective for inhibition. of caspasa diana.

A "peptide" refers to amino acid residues that are linked together through amide linkages. When the amino acids are alpha-amino acids, either the optical L-isomer or the optical D-isomer can be used. The term "peptide" is specifically intended to cover amino acids that occur naturally, as well as those that are recombinantly produced or synthetic. The term "residue" or "amino acid residue" includes the reference to a natural, recombinant, or synthetic amino acid that can be incorporated into a protein, polypeptide, or peptide. Peptides can be modified by a variety of chemical techniques to produce peptidomimetics that have essentially the same activity as unmodified peptides, and optionally have other desirable properties. For example, carboxylic acid groups of the peptide may be provided, either side chain or carboxyl end, in the form of a pharmaceutically acceptable cation salt or esterified to form a C ₁ -C ₁₆ ester, or converted to an amide of formula NR ₁ R ₂ where R ₁ and R ₂ are each independently H or C ₁ -C ₁₆ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. The amino groups of the peptide, whether side-chain or amino-end, can be in the form of a pharmaceutically acceptable acid addition salt, such as the salts of HCI acid, HBr acid, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid and other organic salts, or they can be modified to dialkylamino or C ₁ -C ₁₆ -alkyl or further converted into an amide. Hydroxyl groups of the peptide side chains can become alkoxyl C ₁ -C ₁₆ ester _or a C ₁ -C ₁₆ using techniques well recognized. The phenyl rings and phenolic side chain peptide can be substituted with one or more halogen atoms such as fluorine, chlorine, bromine or iodine, or with C ₁ -C _16, alkoxyl C ₁ -C ₁₆ carboxylic acids and esters thereof, or amides of such carboxylic acids. The methylene groups in the peptide side chains can be extended to C ₂ -C ₄ alkylene homologues. The thiols can be protected with any one of several well recognized protecting groups, such as acetamide groups. Other peptide modifications include addition and / or deletion and / or substitution of one or more amino acid residues in the peptide chain, and / or replacement of one or more of the amide bonds with a non-amide bond, and / or replacement of one or plus amino acid side chains by a different chemical moiety, and / or protection of the N-terminus, the C-terminus, or one or more of the side chains by a protecting group, and / or introduction of double bonds and / or cyclization and / or stereospecificity in the amino acid chain to increase stiffness, and / or binding affinity and / or enhance resistance to enzymatic degradation of the peptides.

A "polypeptide" is a polymer in which the monomers are amino acid residues that are linked together through amide linkages.

The term "pharmaceutically acceptable salt or ester" refers to salts or esters prepared by conventional means including basic salts of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid , ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. "Pharmaceutically acceptable salts" of the compounds disclosed herein also include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N- methyl-glutamine, sina, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, ths (hydroxymethyl) aminomethane and tetrabutylammonium hydroxide. These salts can be prepared by conventional procedures, for example, by reacting the free acid with a suitable organic or inorganic base. Any chemical compound indicated in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts" also include the free acid, base and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). When the compounds disclosed herein include an acidic function such as a carboxyl group, then pairs of pharmaceutically acceptable cations suitable for the carboxyl group are well known to those skilled in the art and include alkali, alkaline earth, ammonium cations, quaternary ammonium and the like. Such salts are known to those of skill in the art. For additional examples of "pharmacologically acceptable salts," see Berge et al., J. Pharm. Sci. 66: 1 (1977). "Pharmaceutically acceptable esters" include those derivatives of compounds described herein that are modified to include a hydroxyl or carboxyl group. An in vivo hydrolyzable ester is an ester, which is hydrolyzed in the human or animal body to produce the original acid or alcohol. Pharmaceutically acceptable esters suitable for carboxyl include C1-6 alkoxymethyl esters for example methoxy-methyl, C1-6 alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C1-6 alkylcycloalkoxycarbonyloxy esters for example 1 -cyclohexylcarbonyl- oxyethyl; 1,3-dioxolen-2-ynylmethyl esters, for example 5-methyl-1,3-dioxolen-2-ynylmethyl; and C1-6 alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyl-oxyethyl which can be formed at any carboxyl group in the compounds.

An in vivo hydrolyzable ester containing a hydroxyl group includes inorganic esters such as phosphate esters and α-acyloxyalkyl ethers and related compounds which as a result of in vivo hydrolysis of the ester decomposition give the original hydroxyl group. Examples of α-acyloxyalkyl ethers include acetoxy-methoxy and 2,2-dimethylpropionyloxy-methoxy. A selection of in vivo hydrolyzable ester forming groups for hydroxyl include alkanoyl, benzoyl, phenylacetyl, and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl, and N- (dialkylaminoethyl) -N-alk Lcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl. Examples of benzoyl substituents include morpholino and piperazine attached from a ring nitrogen atom via a methylene group to the 3- or 4-position of the benzoyl ring.

For therapeutic use, salts of the compounds are those in which the counterion is pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable, for example, in the preparation or purification of a pharmaceutically acceptable compound.

The pharmaceutically acceptable acid and base addition salts as mentioned herein above are intended to comprise the therapeutically active non-toxic base and acid addition salt forms that the compounds can form. Pharmaceutically acceptable acid addition salts can be conveniently obtained by treating the base form with such an appropriate acid. Appropriate acids include, for example, inorganic acids such as hydroacids, for example hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid ), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like. Instead, such salt forms can be converted by treatment with an appropriate base to give the free base form.

Compounds containing an acidic proton can also be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Suitable base salt forms comprise, for example, ammonium salts, alkali and alkaline earth metal salts, for example, lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, for example benzathine salts, N-methyl-D-glucamine, hydrabamine, and salts with amino acids such as, for example, arginine, sina and the like.

The term "addition salt" as used herein above also encompasses the solvates that the compounds described herein can form. Such solvates are, for example, hydrates, alcoholates, and the like.

The term "quaternary amine" as used herein above defines the quaternary ammonium salts that compounds can form by reaction between a basic nitrogen of a compound and an appropriate quaternizing agent, such as, for example, an alkylhalide , arylhalide or arylalkylhalide optionally substituted, for example methyliodide or benzyliodide. Other reagents with good leaving groups can also be used, such as alkyl trifluoromethanesulfonates, alkyl methanesulfonates, and alkyl p-toluenesulfonates. A quaternary amine has a positively charged nitrogen. Pharmaceutically acceptable counterions include chlorine, bromine, iodine, trifluoroacetate, and acetate. The counterion of choice can be introduced using ion exchange resins.

It will be appreciated that the compounds described herein may have complexing, chelating, metal-binding properties and therefore may exist as metal complexes or metal chelates.

The term "prodrug" is also intended to include any covalently linked carrier that releases a disclosed compound or an original compound thereof in vivo when the prodrug is administered to a subject. Since prodrugs often have enhanced properties relative to the active pharmaceutical agent, such as solubility and bioavailability, the compounds disclosed herein can be administered as a prodrug. Thus, prodrugs of the compounds disclosed herein, methods of administering prodrugs, and compositions containing such prodrugs are also contemplated. Prodrugs of the disclosed compounds are normally prepared by modifying one or more functional groups present in the compound such that the modifications are cleaved, either by routine manipulation or in vivo, to give the original compound. In particular, ester prodrugs are specifically contemplated herein. Similarly, prodrugs include compounds having an amino or sulfhydryl group functionalized with any group that cleaves to give the corresponding free amino or free sulfhydryl group. Examples of prodrugs include, without limitation, compounds having an acylated hydroxyl, amino and / or sulfhydryl group with an acetate, formate or benzoate group.

Protected derivatives of the disclosed compounds are also contemplated. The term "protecting group" or "blocking group" refers to any group that when attached to a functional group avoids or reduces the group's susceptibility to the reaction. "Protecting group" generally refers to groups well known in the art. technique used to prevent selected reactive groups, such as carboxyl, amino, hydroxyl, mercapto, and the like, from undergoing undesired reactions, such as oxidation, reduction, nucleophilic, electrophilic, and the like. The terms "deprotecting,""deprotected," or "deprotecting," as used herein, are intended to refer to the process of removing a protecting group from a compound.

A "therapeutically effective amount" or "diagnostically effective amount" refers to an amount of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Ideally, a therapeutically effective amount or a diagnostically effective amount of an agent is an amount sufficient to inhibit or treat the disease without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount or diagnostically effective amount of an agent will depend on the subject being treated, the severity of the condition, and the manner of administration of the therapeutic composition.

"Treatment" refers to a therapeutic intervention that improves a sign or symptom of a disease or condition after it has begun to develop. As used herein, the term "improve", with reference to a disease or condition, refers to any observable beneficial effect of the treatment. The beneficial effect may be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all of the clinical symptoms of the disease, a slower progression of the disease, a improvement of the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. The term "treating a disease" refers to inhibiting the complete development of a disease or condition, for example, in a subject who is at risk of suffering from a disease such as cancer, particularly metastatic cancer. A "prophylactic" treatment is a treatment administered to a subject who does not show signs of a disease or shows only early signs in order to reduce the risk of developing a pathology. Particular examples of the agents disclosed herein include one or more asymmetric centers; therefore these compounds can exist in different stereoisometric forms. Accordingly, compounds and compositions can be provided as single pure enantiomers or as stereoisomeric mixtures, including racemic mixtures. In certain embodiments, the compounds disclosed herein are synthesized in or purified to be in a substantially enantiopure form, such as in an enantiometric excess of 90%, an enantiometric excess of 95%, or an enantiometric excess of 97%, or even in more than 99% enantiometric excess, such as in enantiopura form.

Some of the compounds described herein may also exist in their tautometric form.

In the context of the present invention, the term "anemia associated with chronic diseases" (ADC) is understood as a form of anemia observed in chronic infection, chronic immune activation and cancer.

In the context of the present invention, the term "chemotherapy-induced anemia" is understood as anemia of cancer patients receiving chemotherapy. In the context of the present invention, the term "Diamond-Blackfan anemia" is understood as a genetic disorder characterized by reduced levels of the GATA1 protein due to a damaged translation in the GATA1 mRNA bosbosome.

Detailed description of the invention

As used herein, the singular terms "a", "one" and "he / she" include plural referents unless the context clearly indicates otherwise. Furthermore, as used herein, the term "comprise" means "includes." It is further understood that all nucleotide sizes or amino acid sizes, and all values of molecular mass or molecular weight, given for nucleic acids or polypeptides or other compounds are approximate, and are provided for the description. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

An evolutionary conserved signaling pathway is disclosed herein that binds the inflammasome with HSC differentiation for the first time. During periods of hematopoietic stress induced by chemotherapy or viral infection, activation of NLRPI a prolongs cytopenia, bone marrow hypoplasia, and immunosuppression. Interestingly, this effect is mediated by caspase-1-dependent but ASC-independent pyroptosis of hematopoietic progenitor cells. Furthermore, the NLRP3 inflammasome has been found to lead to clonic expansion and pyroptopotic cell death in myelodysplastic syndromes (Basiorka et al., 2016). The results show that although the inflammasome is dispensable for the appearance of HSC in zebrafish, it intrinsically regulates the differentiation of HSC under homeostasis conditions at two different levels: decision of the fate of erythroid / myeloid cells and terminal erythroid differentiation (figure 10). Although CASP1 can target several proteins to regulate both processes, one possible scenario is the cleavage of GATA1 at residue D300 by CASP1, resulting in the rapid degradation of GATA1, since processed GATA1 could not be detected in zebrafish larvae. or K562 cells. Decreased GATA1 levels upon inflammation activation result in increased SPI1 levels concomitantly leading to enhanced erythropoiesis and reduced myelopoiesis, as lineage choice is initiated, or at least executed and enhanced, by these two cross-antagonistic factors of the transcript. Similarly, but without the involvement of SP 11, terminal erythroid differentiation requires cleavage of GATA1 by CASP1. Therefore, it was observed that pharmacological inhibition of CASP1 leads to GATA1 accumulation and altered erythroid differentiation of both HSC CD34 ⁺ and K562 cells (Figure 10), since GATA1 inhibits terminal erythroid differentiation in vitro. Although it remains to be elucidated the signals responsible for the activation of the inflammasome in the decision of the fate of erythroid / myeloid cells and terminal erythroid differentiation as well as the components Inflammasome involved, zebrafish genetics studies show that Gbp4 and Ase are both intrinsically required in vivo by HSC to regulate their differentiation. Mild CASP1 activation is anticipated to prevent pyrophotic cell death from hematopoietic cells. This can be achieved by the assembly of small ASC spots and / or the low abundance of caspase-1 in hematopoietic progenitor cells and erythroid precursors, as it occurs in neutrophils that have sustained release of IL-1b without pyroptosis compared to macrophages ( Boucher et al., 2018; Chen et al., 2014).

Hematopoietic lineage bias is associated to increase the incidence of diseases with prominent inflammatory components including atherosclerosis, autoimmunity, neurodegenerative disease, and carcinogenesis (Elias et al., 2017). In particular, neutrophilic dermatosis is characterized by the accumulation of neutrophils on the skin and skin lesions (Marzano et al., 2018). It was observed that the robust neutrophilia of a zebrafish model of skin inflammation is reversed by pharmacological inhibition of Dandruff, despite the fact that skin lesions and neutrophil infiltration are largely unaffected. To the best of our knowledge, this is the first evidence showing that inflammasome activation alters granulopoiesis through Sp¡1 / Gata1 imbalance and, more importantly, that its pharmacological inhibition restores Sp¡1 / Gata1 balance and count neutrophils (figure 10). Furthermore, the critical role of the inflammasome in the regulation of Sp¡1 / Gata1 was also highlighted by the pharmacological inhibition ability of Caspa to restore Erythroid and Gatal hemoglobin levels, and to reduce Spi1 levels, in a zebrafish model de Gatal reduced, as occurs in Diamond-Blackfan anemia (Danilova and Gazda, 2015). Similarly, pharmacological inhibition of CASP1 accelerates recovery from anemia in 5-FU-treated mice without affecting leukocyte and platelet counts. Taken together, all of these results point towards the ability to inhibit inflammasomes as a therapeutic approach to treat human diseases with associated hematopoietic lineage bias, such as neutrophilic inflammation, anemia associated with chronic diseases, chemotherapy-induced anemia and Diamond-Blackfan anemia. . The availability of an orally active CASP1 inhibitor, VX-765, with high specificity, excellent pharmacokinetic properties, and efficacy in mouse models of rheumatoid arthritis and skin inflammation (Wannamaker et al., 2007) further supports clinical trials of CASP1 inhibitors in disorders with hematopoietic lineage bias.

Therefore, a first aspect of the invention relates to a composition comprising at least one caspase-1 inhibitor for use in a method of treatment of a disease selected from the list consisting of anemia associated with chronic diseases, induced anemia from chemotherapy and Diamond-Blackfan anemia.

The present invention is limited to the aforementioned anemia and not other anemia such as autoimmune hemolytic anemia (AHA) or aplastic anemia (AA) due to the following reasons. AHA is caused by the generation of antibodies against red blood cells that shortened your life. This disorder can present as primary (idiopathic) or secondary to autoimmune disorders, malignant tumors or infections. Aplastic anemia (AA) is characterized by pancytopenia and hypocellular bone marrow caused by decreased hematopoietic stem cells. The combination of various genetic alterations with low penetrance, together with environmental factors, contributes to the development of AA.

Treatment of patients with AHA and AA with caspase-1 inhibitors is highly unlikely to result in beneficial effects, since such treatment although will increase GATA1 levels by forcing erythropoiesis, such newly formed red blood cells will be destroyed by autoantibodies in HA, and in AA, deficiency in hematopoietic stem cells will still result in damaged erythropoiesis despite increased GATA1 levels.

In contrast, the treatment of anemia associated with chronic diseases (ACD) will cure it satisfactorily by pharmacological inhibition of caspase-1 as already made possible from the experimental evidence provided in the examples, since ACD is associated with hyperactivation of the inflammasome and caspase-1. Therefore, treatment with caspase-1 inhibitors will restore GATA1 levels. Similarly, Diamond-Blackfan anemia is a genetic disorder characterized by reduced levels of GATA1 due to damaged translation into the ribosome of GATA1 mRNA. Therefore, treatment with caspase-1 inhibitors will restore GATA1 levels; this is demonstrated in the examples provided in the present specification. Finally, chemotherapy-induced anemia is caused by transient depletion of the hematopoietic progenitor cell compartment and increased levels of GATA1 through inhibition of caspase-1 will force erythropoiesis of the remaining and newly formed progenitor cell, leading to the cure of anemia , as demonstrated in the examples provided herein.

Caspase-1 inhibitors useful for the above treatment method have the motif: X or X-W, where X is a selective caspase-1 structure relative to other cysteine proteases. The specificity and / or selectivity of a substrate for a caspase can be determined by biochemical and cell-based assays on related enzymes.

In certain embodiments, X has a structure comprising: Ar-A2-A1 -, wherein Ar is an optionally substituted aryl or optionally substituted heteroaryl; and A1 and A2 are each individually an amino acid residue, or A1 and A2 together form a peptidomimetic. The caspase-1 X selective structure can include at least one additional amino acid in addition to A1 and A2. Such additional amino acid (s) may be the same or different compared to the amino acids described below for A1 and A2. However, in certain embodiments X consists only of A1 and A2. Amino acids for A1 and A2 can be natural or unnatural amino acids (eg, recombinant or synthetic). A1 and A2 can be the same or different amino acids.

Illustrative amino acids for A1 and A2 can be represented by— N (R1) - C (R2) (R3) - C (O) - where R1 is H; R2 and R3 are each individually selected from H, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, or an optionally substituted heteroaryl, or R2 and R3 together form a cycloalkyl structure; or R1 and R2 together form an azacyclic structure. Several specific amino acids for A2 are:

Ar can be an optionally substituted aryl or heteroaryl. The optionally substituted aryl can be a single 5-, 6- or 7-membered ring such as phenyl or a fused ring such as naphthyl or quinolinyl. The optionally substituted heteroaryl can include a heteroatom selected from N, O, or S. Illustrative heteroaryl groups include furanyl, pyranyl, pyrroyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, isoindolyl, indoyl, quinolinyl, isothiazolyl, and isoxazolyl. A heteroaryl preferred is pyrindyl. Illustrative substituents include halogen, amino, aminoalkyl (eg, NMe2), aminoacyl (eg, AcHN), halogenated alkyl, alkoxy, and tetrazolyl. The Ar group can include a carbonyl radical (- C (O) -) that binds A ² . In selected embodiments, Ar is optionally substituted benzoyl which means that X has the structure: Ph (optionally substituted) - C (0) -A ² -A ¹ , where Ph is phenyl.

Several specific examples for Ar are:

W represents a "warhead" comprising - NH - CH (Y) (Z). The electrophilic warhead reversibly modifies caspase so that caspase cannot interact with or cleave a caspase substrate. Although not limited by any theory, the novel structure of the warhead disclosed herein is believed to allow covalent binding with an active site thiol in caspase by optimizing hydrophobic and hydrophilic interactions between its inhibitory compound and caspase. , specific intermolecular hydrogen binding between the inhibitory compound and caspase, and proper alignment of the enzyme nucleophilic thiol and the covalent modifier in the inhibitory compound.

Y is a structure that allows the inhibitor compound to form a reversible covalent bond with a caspase 1. In particular, Y allows the formation of a reversible bond with a nucleophilic amino acid residue of caspase 1. This covalent bond is considered reversible in fact. that the newly formed enzyme-inhibitor bond of thioimidate or intermediate thioboronate can be broken through hydrolysis or simple inversion of the reaction to generate both free inhibitor and free enzyme. Illustrative Y groups include cyano (- CN), cyano-substituted alkyl (eg, -CH2CN), boronic acid (-B (OH) 2), or boronic acid-substituted alkyl (eg, -CH2B (OH) 2 ). Z is a carboxyl residue or a carboxylic acid mimetic. Illustrative Z groups include cyano (-CN), cyano-substituted alkyl (eg, -CH2CN), acid boronic (- B (OH) ₂ ), alkyl substituted by boronic acid (for example, - CH ₂ B (OH) ₂ ), carboxylic acid (- CO ₂ H), alkyl substituted by carboxylic acid (for example, - CH ₂ CO ₂ H), carboxylate ester (for example, -C0 ₂ (alkyl), or -CH ₂ C0 ₂ (alkyl)), tetrazolyl, tetrazolyl-substituted alkyl (for example, -CH ₂ -tetrazoyl), or an amido (eg, - CONH, - CH ₂ CONH (OH), - CH ₂ CONH (OMe), or - CH ₂ CONH (CN)). Carboxylic acid mimetics have a proton with a pKA in the range of 4 to 9, which is close to that of carboxylic acid as shown below:

In accordance with certain embodiments disclosed herein, A ² , A ¹ and Ar are selected from the specific structures disclosed above; Y is selected from cyano or boronic acid; and Z is selected from— CH ₂ B (OH) ₂ or— CH ₂ C (0) - O-lower alkyl. According to certain embodiments disclosed herein, A ² is selected from:

A¹ se selecciona de:

A ¹ is selected from:

Y is selected from cyano or boronic acid; and Z is selected from— CH2B (OH) 2 or— CH2C (0) - O-lower alkyl. According to certain embodiments disclosed herein, caspase 1 inhibiting agents include a 3-cyanopropanil residue incorporated in caspase 1 inhibitor supports.

en la que According to particular embodiments, the compounds disclosed herein have the structure of formula II:

in which

R ¹ is H, - C (0) R ⁸ , - C (0) C (0) R ⁸ , - S (0) ₂ R ⁸ , - S (0) R ⁸ , - C (0) 0R ⁸ , - C (0) N (H) R ⁸ , - S (0) ₂ N (H) - R ⁸ , - S (0) N (H) - R ⁸ , - C (0) C (0) N ( H) R ⁸ , -

C (0) CH = CHR ⁸ , - C (0) CH ₂ 0R ⁸ , - C (0) CH ₂ N (H) R ⁸ , - C (0) N (R ⁸ ) _2I - S (0) ₂ N (R ⁸ ) _2I - S (0) N (R ⁸ ) _2I - C (0) C (0) N (R ⁸ ) _2I - C (0) CH ₂ N (R ⁸ ) _2I - CH ₂ R ⁸ , - CH ₂ -alkenyl-R ⁸ , or - CH ₂ -alkynyl-R ⁸ ;

R ² is H and each R ⁶ is independently— H, an amino acid side chain, or - R ⁸ ; or R ² and R ⁶ together with the atoms to which they are attached form a 3- to 7-membered cyclic or heterocyclic ring system;

R ²² is— C (R ⁶ ) 2— O— N (R ⁶ ) -;

R ³ is H and each R ⁴ is independently— H, an amino acid side chain, or - R ⁸ ; or R ³ and R ⁴ together with the atoms to which they are attached form a 3- to 7-membered cyclic or heterocyclic ring system;

R ⁵ is— H;

R ²¹ is— CN or— C (0) 0R ⁹ ;

R ²⁰ is— C (0) 0R ⁹ , or a heteroaryl;

R ⁹ is— H, alkyl, or— CN; and m is 0 or 1; provided that at least one of R ²⁰ or R ²¹ includes— CN.

In more specific examples, the compounds have a formula III:

In certain embodiments disclosed herein, the Caspase inhibition Clso of the disclosed compounds is less than 100 nM. The compounds may have an aqueous solubility of greater than 10 pg / ml, a LogD of less than 5, and a molecular weight of less than 650 Dalton.

Illustrative examples of specific compounds are listed below:

Ejemplos ilustrativos adicionales de compuestos específicos son: ac-YVAD-CMK,

Additional illustrative examples of specific compounds are: ac-YVAD-CMK,

z-YVAD-FMK

z-WEHD-FMK

Ac-YVAD-FMK

Ac-YVAD-CHO

Ac-YVAD-FMK

Los compuestos dados a conocer en el presente documento pueden sintetizarse generalmente tal como se ¡lustra en el documento US9365612B2, que se incorpora en el presente documento como referencia. ZD-CH2-DCB,

The compounds disclosed herein can generally be synthesized as illustrated in US9365612B2, which is incorporated herein by reference.

A second aspect of the disclosure includes pharmaceutical compositions prepared for administration to a subject, for use as reflected in the first aspect of the invention, and which includes a therapeutically effective amount of one or more of the compounds disclosed in This document. The therapeutically effective amount of a disclosed compound will depend on the route of administration, the species of the subject and the physical characteristics of the subject being treated. Specific factors that can be considered include severity and phase of the disease, weight, diet, and concurrent medications. The relationship of these factors to determining a therapeutically effective amount of the disclosed compounds is understood by those skilled in the art.

Pharmaceutical compositions for administration to a subject may include at least one additional pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like in addition to the molecule of choice. The pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional. Remington's Pharmaceutical Sciences by EW Martin, Mack Publishing Co., Easton, Pa., 19th edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the compounds disclosed herein.

In general, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually contain injectable liquids that include pharmaceutically and physiologically acceptable liquids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (eg, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. .

The pharmaceutical compositions disclosed herein include those formed from pharmaceutically acceptable salts and / or solvates of the disclosed compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable organic or inorganic acids and bases. The particular disclosed compounds possess at least one basic group which can form acid-base salts with acids. Examples of basic groups include, but are not limited to, amino and amino groups. Examples of inorganic acids that can form salts with such basic groups include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid. The basic groups can also form salts with organic carboxylic acids, sulfonic acids, sulfo acids or phospho acids or N-substituted sulfamic acid, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, embonic acid , nicotinic acid or isonicotinic acid and, in addition, with amino acids, for example with a-amino acids, and also with methanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonic acid, ethane-1,2-disulfonic acid, benzenedisulfonic acid, 4-methylbenzenesulfonic acid , naphthalene-2-sulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid (with formation of cyclamates) or with other compounds organic acid coughs, such as ascorbic acid. In particular, suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art.

Certain compounds include at least one acidic group that can form an acid-base salt with an inorganic or organic base. Examples of salts formed from inorganic bases include salts of the compounds disclosed herein with alkali metals such as potassium and sodium, alkaline earth metals, including calcium and magnesium, and the like. Similarly, salts of acidic compounds with an organic base, such as an amine, are contemplated (as used herein, terms referring to amines are to be understood to include their conjugated acids unless the context clearly indicates free amine is provided), including salts formed with basic amino acids, aliphatic amines, heterocyclic amines, aromatic amines, pyridines, guanidines and amidines. Of the aliphatic amines, the acyclic aliphatic amines, and cyclic and acyclic di and trialkyl amines are particularly suitable for use in the disclosed compounds. In addition, quaternary ammonium counterions can also be used.

Particular examples of suitable amine bases (and their corresponding ammonium ions) for use in the present compounds include, without limitation, pyridine, N, N-dimethylaminopihdine, diazabicyclononane, diazabicycloundecene, N-methyl-N-ethylamine, diethylamine, triethylamine, diisopropylethylamine, mono-, bis- or tris- (2- hydroxyethyl) amine, 2-hydroxy-tert-butylamine, tris (hydroxymethyl) methylamine, N, N-dimethyl- N- (2-hydroxyethyl) amine, tri- (2-hydroxyethyl) amine, and N-methyl-D-glucamine. For additional examples of "pharmacologically acceptable salts," see Berge et al., J. Pharm. Sci. 66: 1 (1977).

The compounds disclosed herein can be crystallized and can be provided in a single crystalline form or as a combination of different crystalline polymorphs. As such, the compounds can be provided in one or more physical forms, such as different crystalline forms, crystalline forms, liquid crystalline, or non-crystalline (amorphous). Such different physical forms of the compounds can be prepared using, for example, different solvents or different solvent mixtures for recrystallization. Alternatively or additionally, different polymorphs can be prepared, for example, by performing recrystallizations at different temperatures and / or by altering the cooling rates during recrystallization. The presence of polymorphs can be determined by X-ray crystallography, or in some cases by another spectroscopy technique, such as solid phase NMR spectroscopy, IR spectroscopy, or by differential scanning calorimetry.

The pharmaceutical compositions can be administered to subjects by a variety of modes of administration to the mucosa, including by oral, rectal, intranasal, intrapulmonary, or transdermal administration, or by topical administration to other surfaces. Optionally, the compositions can be administered by non-mucosal routes, including intramuscular, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intrathecal, intracerebroventricular or parenteral. In other alternative embodiments, the compound can be administered ex vivo by direct exposure to cells, tissues, or organs originating from a subject.

To formulate the pharmaceutical compositions, the compound can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the compound. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, local anesthetics (eg, benzyl alcohol), isotonic agents (eg, sodium chloride, mannitol, sorbitol), absorption inhibitors (eg, Tween 80 or Miglyol 812), enhancement agents may be included. solubility (eg, cyclodextrins and derivatives thereof), stabilizers (eg, serum albumin), and reducing agents (eg, glutathione). May include adjuvants such as aluminum hydroxide (eg Amphogel, Wyet Laboratories, Madison, NJ), Freund's adjuvant, MPL ™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.) And IL-12 (Genetics Institute, Cambridge, Mass.), Among many other suitable adjuvants well known in the art. When the composition is a liquid, the tonicity of the formulation, as measured with reference to the 0.9% (w / v) saline physiological solution tonicity taken as a unit, is normally adjusted to a value where no Substantial and irreversible tissue damage will be induced at the site of administration. Generally, the tonicity of the solution is adjusted to a value of from about 0.3 to about 3.0, such as from about 0.5 to about 2.0, or from about 0.8 to about 1.7.

The compound can be dispersed in a base or vehicle, which can include a hydrophilic compound that has an ability to disperse the compound, and any desired additives. The base can be selected from a wide variety of suitable compounds, including but not limited to, copolymers of carboxylic polyacids or salts thereof, carboxylic anhydrides (eg, maleic anhydride) with other monomers (eg, methyl (meth) acrylate , acrylic acid and the like), hydrophilic vinyl polymers, such as poly (vinyl acetate), poly (vinyl alcohol), polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose, and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and non-toxic metal salts thereof. Often, a biodegradable polymer is selected as the base or vehicle, for example, poly (lactic acid), poly (lactic acid-glycolic acid) copolymer, poly (hydroxybutyric acid), poly (hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerol fatty acid esters, sucrose fatty acid esters and the like can be employed as carriers. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, crosslinking, and the like. The vehicle can be provided in a variety of forms, including viscous or fluid solutions, gels, pastes, powders, microspheres, and films for direct application to a mucous surface.

The compound can be combined with the base or vehicle according to a variety of methods, and the compound can be released by diffusion, vehicle disintegration, or associated formation of water channels. Under some circumstances, the compound is dispersed into microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, for example isobutyl 2-cyanoacrylate (see, for example, Michael et al., J. Pharmacy Pharmacol 43: 1-5, 1991), and disperses in a biocompatible dispersion medium, which produces sustained administration and biological activity over a prolonged period of time.

The compositions of the disclosure may alternatively contain as pharmaceutically acceptable carriers, substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, eg, sodium acetate , sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.

Pharmaceutical compositions for administering the compound can also be formulated as a solution, microemulsion, or other ordered structure suitable for a high concentration of active ingredients. The vehicle may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Adequate fluidity can be maintained for the solutions, for example, by using a coating such as lecithin, by maintaining a desired particle size in the case of dispersible formulations, and by using surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the compound can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the compound can be administered in a sustained release formulation, for example in a composition that includes a delayed release polymer. These compositions can be prepared with carriers that will protect against rapid release, for example a controlled release carrier such as a polymer, microencapsulated delivery system, or bioadhesive gel. Prolonged administration in various compositions of the disclosure can be accomplished by including absorption retarding agents in the composition, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use according to the disclosure include any biocompatible controlled release materials that are inert to the active agent and that can incorporate the compound and / or other biologically active agent. Vapors of such materials are known in the art. Useful controlled release binders are materials that are metabolized slowly under physiological conditions after administration (eg, to a mucous surface, or in the presence of body fluids). Appropriate binders include, but are not limited to, biocompatible polymers and well copolymers. known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to the surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.

The pharmaceutical compositions of the disclosure are normally sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients listed herein, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the compound and / or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed herein. In the case of sterile powders, the methods of preparation include vacuum drying and lyophilization which produces a powder of the compound plus any additional desired ingredients of a previously filtered sterile solution thereof. Prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

According to the various methods of treatment of the disclosure, the compound can be administered to a subject in a manner consistent with conventional methodologies associated with the management of the disorder for which treatment or prevention is sought. According to the disclosure herein, a prophylactic or therapeutically effective amount of the compound and / or other biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate disease. or selected state or one or more symptoms thereof.

The actual dosage of the compound will vary according to factors such as the indication of the disease and the particular condition of the subject (for example, age, size, physical state, degree of symptoms, susceptibility factors, and the like, of the subject), time and route of administration, other drugs or treatments that are Being administered simultaneously, as well as the specific pharmacology of the compound to provoke the desired biological activity or response in the subject. Dosage regimens can be adjusted to provide an optimal prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or deleterious side effects of the compound and / or other biologically active agent are clinically overcome by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of a compound and / or other biologically active agent within the methods and formulations of the disclosure is from about 0.01 mg / kg of body weight to about 20 mg / kg of body weight, such as from about 0.05 mg / kg to about 5 mg / kg of body weight, or from about 0.2 mg / kg to about 2 mg / kg of body weight.

The dosage may be varied by the attending physician to maintain a desired concentration at a target site (eg, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of administration, eg, transepidermal, rectal, oral, pulmonary, or intranasal administration versus intravenous or subcutaneous administration. The dosage can also be adjusted based on the rate of release of the administered formulation, eg, from an intrapulmonary aerosol versus powder, sustained oral release versus injection or transdermal particle delivery formulations, and so on.

The present disclosure also includes kits, packages and multi-container units containing the pharmaceutical compositions, active ingredients and / or means described herein for administering the same for use in the prevention and treatment of diseases and other conditions in subjects. mammals. Kits for diagnostic use are also provided. In one embodiment, these kits include a container or formulation containing one or more of the compounds described herein. In one example, this component is formulated into a pharmaceutical preparation for administration to a subject. The compound is optionally contained in a bulk dispersion container or unit or multiple unit dosage form. Can be provided optional dispersion media, for example a pulmonary or intranasal aerosol applicator. The packaging materials optionally include a label or instruction indicating for what purposes of treatment and / or in what manner the pharmaceutical agent packaged therewith can be used.

Examples

Materials and methods

Animals

Zebrafish (Danio rerio H.) were obtained from the Zebrafish International Resource Center and mated, their phase evaluated, bred, and processed as described (Westerfield, 2000). The roy lines ³⁹⁷³⁹ ; nacre ^{w2 / w2} (casper) (White et al., 2008), Tg (mpx: eGFP) ' ¹¹⁴ (Renshaw et al., 2006), Tg (mpeg1: eGFP) ⁹ ' ²² , Tg (mpeg1: GAL4) ⁹¹²⁵ (Ellett et al., 2011), Tg (lyz: dsRED) ^nz50 (Hall et al., 2007), Tg (mpx: Gal4. VP16) ' ²²² (Davison et al., 2007), Tg (lcr: eGFP) ^cz3325 (Ganis et al., 2012), Tg (runx1: GAL4) ^utn6 (Tamplin et al., 2015), Tg (UAS: nfsB-mCherry) ^c264 (Davison et al., 2007) and Tg (spint1a) ^hl2217 ( Carney et al., 2007; Mathias et al., 2007) have been previously described. The experiments carried out comply with the guidelines of the Council of the European Union (Directive 2010/63 / EU) and the Spanish Royal Decree RD 53/2013. Experiments and procedures were carried out as approved by the Bioethics Committees of the University of Murcia (approval numbers # 75/2014, # 216/2014 and 395/2017).

C57BL / 6 mice were purchased from Janvier Laboratory. All experiments were carried out in accordance with the French guidelines for animal handling and approved by the Inserm Ethics Committee.

DNA construct and transgenics generation

The uas: AscACARD-GFP construct was generated by MultiSite Gateway assemblies using LR Clonase II Plus (Life Technologies) according to conventional protocols and using Tol2kit vectors previously described (Kwan et al., 2007). Expression constructs Gbp4, Gbp4KS AA, Gbp4ACARD, were previously described. Gbp4KS AA / ACARD (double mutant, DM) and uas: gbp4KS / AA (Tyrkalska et al., 2016); Asc-Myc and Caspa (Masumoto et al., 2003); and Gcsfa (Liongue et al., 2009).

The Tg line (UAS: gbp4KS / AA) ^{ums3 was} previously ^described (Tyrkalska et al., 2016). Tg (UAS: ascACARD-GFP) ^{ums4 was generated by} microinjecting 0.5-1 ni into the yolk sac of single- ^cell embryos a solution containing 100 ng / pl of uas: ascACARD-GFP and uas: gbp4KS AA constructs, respectively, and 50 ng / mI of Tol2 RNA in microinjection buffer (Tango ^c 0.5 buffer and 0.05% phenol red solution) using a microinjector (Narishige).

Morpholino, RNA injection and chemical treatments for zebrafish larvae

Specific morpholinos (Gene Tools) were resuspended in 1mM nuclease free water (Table S1). In vitro transcribed RNA was obtained following the manufacturer's instructions (mMESSAGE mMACHINE kit, Ambion). Morpholinos and RNA were mixed in microinjection buffer and microinjected into the yolk sac of single-cell embryos using a microinjector (Narishige) (0.5-1 or per embryo). The same amount of MO and / or RNA was used in all experimental groups.

In some experiments, 1 -2 dpf embryos had their chorion removed and treated for 24 h up to 7 dpf at 28 ° C by bath immersion with caspase-1 inhibitors Ac-YVAD-CMK (irreversible) or Ac- YVAD-CHO (reversible) (100mM, Peptanova) diluted in water with egg supplemented with 1% DMSO or with metronidazole (Mtz, 5mM, Sigma-Aldrich).

Live imaging, Sudan black staining of neutrophils, neutrophil ablation, and erythrocyte determination in zebrafish larvae

At 48 and 72 hpf, the larvae were anesthetized in tricaine and mounted in 1% (w / v) low melting point agarose (Sigma-Aldrich) dissolved in egg water (de Oliveira et al., 2013). Images were captured with a Lumar V12 epifluorescence stereomicroscope equipped with green and red fluorescent filters while the animals were kept on their agar matrices at 28.5 ° C. All images were acquired with the camera integrated into the stereomicroscope and used for subsequent counting of the total number of neutrophils, macrophages or HSC in complete larvae. In order to reduce pigmentation and improve Sudan black staining signal, 24 hpf larvae were incubated in 200 mM 1-phenyl 2-thiourea (PTU) up to 72 hpf, when anesthetized in buffered tricaine and fixed for overnight at 4 ° C in 4% methanol-free formaldehyde. The next day, all larvae were rinsed with PBS three times, incubated for 15 min with Sudan black (# 380B-1 KT, Sigma-Aldrich) and washed extensively in 70% EtOH in water. After that, progressive rehydration was performed: 50% EtOH in PBS and 0.1% Tween 20 (PBT) (Sigma-Aldrich), 25% EtOH in PBT and PBT alone. Finally, the larvae were immediately visualized using a Scope.AI stereomicroscope equipped with a digital camera (AxioCam ICc 3, Zeiss) (Le Guyader et al., 2008).

For neutrophil ablation, Tg larvae (mpx: Gal4. VP16; UAS.nsfb-mCherry) were treated at 2 dpf with 5 mM Mtz and kept in the dark. At 72 hpf, the drug was withdrawn and the larvae were treated up to 7 dpf with 1% DMSO alone or containing Ac-YVAD-CMK (100 mM). The inhibitor was refreshed every 24 h and images of the larvae were obtained once a day up to 7 dpf and the number of neutrophils was determined (Davison et al., 2007; Halpern et al., 2008).

Erythrocyte counts were determined by flow cytometry. At 3 dpf, groups of 50 Tg larvae (lcr.eGFP) were anesthetized in tricaine, ground with a blade and incubated at 28 ° C for 30 min with Liberase 0.077 mg / ml (# 05401119001, Roche). After that, 10% FBS was added to inactivate Liberase and the resulting cell suspension was passed through a 40 m 40ti cell filter. Sytox Blue (Life Technologies) was used as a vital dye to exclude dead cells. Flow cytometric acquisitions were performed on a FACSCALIBUR (BD). Analyzes were performed using FlowJo software (Treestar).

Zebrafish Larvae Infection Assays

For infection experiments, the S. typhimurium 12023 strain (wild type) provided by Prof. Holden was used. Overnight, cultures were diluted in Luria-Bertani (LB) medium 1/5 in LB with 0.3M NaCI, incubated at 37 ° C until an optical density of 1.5 to 600 nm was reached, and finally they were diluted in Sterile PBS. 2 dpf larvae were anesthetized in embryonic medium with 0.16 mg / ml tricaine and 10 bacteria were injected into the yolk sac or otic vesicle. Larvae were allowed to recover in egg water at 28-29 ° C, and were monitored for clinical signs of disease or mortality over 5 days and neutrophil recruitment up to 24 hpi (Tyrkalska et al., 2016) .

Full Assembly In Situ Hybridization (WISH) in Zebrafish Larvae

Transparent Casper embryos were used for WISH (Thisse et al., 1993). Gatala, spilb, gcsfr, cmyb, runxl and rag1 sense and antisense RNA probes were generated using the DIG RNA label kit (Roche Applied Science) from Idealized plasmids. Embryo images were obtained using a Scope.AI stereomicroscope equipped with a digital camera (AxioCam ICc 3, Zeiss).

Cell culture and erythroid differentiation assays

Peripheral blood CD34 ⁺ cells were collected from a single donor (R003272, 08/25/2016). Cells were thawed rapidly in a 37 ° C water bath, then serially diluted with thawing buffer (1% FBS in PBS) to 32 ml total volume, and finally centrifuged for 10 min at room temperature ( 250xg). After discarding the supernatant, cells were resuspended in 20 ml of expansion media containing: serum-free medium for expansion and culture of hematopoietic cells - SFEM (# 09650, Stem Cell), 1% cytokine cocktail 100 - CC100 (# 02690, Stem Span CC100) and 2% Penicillin-Streptomycin Mix (P / S, # 15140122, Thermo Fischer Scientific), and grown in standing flasks at 37 ° C. At 72 h after thawing, 30 ml of new expansion media were added. After six days of expansion, all cells were centrifuged again for 10 min at room temperature and resuspended in erythroid differentiation medium (EDM) containing: 98% filtered SFEM, 2% P / S, 1 U / ml EPO, 5 ng / ml IL-3, 20 ng / ml SCF (stem cell factor), 2 mM dexamethasone and 1 mM b-estradiol, and divided into 2 T75 flasks, of which one was treated with DMSO and the other with AcYVAD-CMK (10, 50 and 100 mM, Peptanova). On the third day of differentiation, new inhibitor was added to the EDM and the old medium was replaced. Cells were collected at different time points (0, 2, 3 and 5 days of differentiation), centrifuged, they were washed twice with PBS, instantly frozen in liquid nitrogen and stored at -80 ° C.

K562 cells (CRL-3343; American Type Culture Collection) were maintained in RPMI supplemented with 10% FCS, 2mM glutamine and 1% penicillin-streptomycin (Life Technologies). Cells were maintained and divided before confluence every 72 h. For differentiation, cells were treated with 50mM hemin (# 16009-13-5, Sigma-Aldrich), prepared as previously described (Smith et al., 2000), in the presence of 0.1 DMSO % alone or containing 100 mM Ac-YVAD-CMK. Cells were harvested at different time points (0, 6, 12, 24, 48 hours after the addition of hemin), centrifuged, washed with PBS, and stored at -80C.

Caspase-1 assay activity

Caspase-1 activity was determined with the Z-YVAD-AFC fluorometric substrate (Caspase-1 substrate VI, Calbiochem) as previously described (Angosto et al., 2012; Lopez-Castejon et al., 2008; Tyrkalska et al., 2016). Briefly, zebrafish larvae, and CD34 ⁺ and K562 cells were lysed in hypotonic cell lysis buffer [4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) 25mM, Ethylene glycol-bis (2-aminoether ether) -N, N, N ', N'-tetraacetic acid (EGTA) 5 mM, dithiothreitol (DTT) 5 mM, protease inhibitor cocktail 1:20 ( Sigma-Aldrich), pH 7.5] on ice for 10 min. For each reaction, 80 pg of protein was incubated for 90 min at 23 ° C with 50 mM Z-YVAD-AFC and 50 mI of reaction buffer [3 - [(3- colamidopropyl) dimethylammonium] -1-propanesulfonate (CHAPS) 0.2%, HEPES 0.2M, 20% sucrose, 29mM DTT, pH 7.5] After incubation, the fluorescence of the AFC released from the Z-YVAD-AFC substrate was measured with a FLUOstart spectrofluorimeter ( BGM, LabTechnologies) at an excitation wavelength of 405 nm and an emission wavelength of 492 nm. A representative caspase-1 assay activity of the three carried out is shown accompanying each cell count.

Focal laser microscopy

Cellware cells were seeded in 12-cell Poly-L-Lys (Corning), 50,000 cells in 100 ml were allowed to bind to the cover for 10 min at room temperature, then medium and treatment were added. After hemin treatment, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 10 min, incubated 20 min at room temperature with 20 mM glycine, permeabilized with 0.5% NP40, and blocked for 1 hr. with 2% BSA. Cells were then labeled with corresponding primary antibody, followed by Alexa 568-conjugated secondary antibody (Thermo Fisher Scientific). Samples were mounted using Dako mounting medium and examined with an AOBS confocal laser scanning microscope and Leica software (Leica Microsystems). Images were acquired in a 1,024 ^c 1,024 pixel format in sequential scan mode between frames to avoid crosstalk. The objective used was HCX PL APO CS ^c 63 and the pin value was 1, corresponding to 114.73 pm.

Immunoblotting

The lysis buffer for mammalian cell lysis contained 50mM Tris-HCI (pH 7.5), 150mM NaCI, 1mM EDTA, 1mM EGTA, 1% (w / v) NP-40 and protease inhibitor. new (1/20, P8340, Sigma-Aldrich), whereas for lysis of zebrafish larvae it contained 1% SDS. Protein quantification was performed with the BCA kit using BSA as standard. Cell lysates (40 pg) in SDS sample buffer were subjected to electrophoresis on a polyacrylamide gel and transferred to PVDF membranes. The membranes were incubated for 1 hr with TTBS containing 5% (w / v) skimmed milk powder or 2% (w / v) BSA. The membranes were immunoblotted in the same buffer 16 h at 4 ° C with the indicated primary antibodies. The blots were then washed with TTBS and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies diluted 2,500 times in 5% (w / v) skim milk in TTBS. After repeated washes, the signal was detected with the enhanced chemiluminescence reagent and ChemiDoc XRS Biorad. The primary antibodies used are: rabbit polyclonal antibody against human GATA1 (1/200, # sc1234, Santa Cruz Biotechnology) for confocal assay, rabbit mAb against human GATA1 (1/200, # 3535, Cell Signaling) for immunoblotting, rabbit polyclonal antibody against CASP1 (1/200, No. sc56036 Santa Cruz Biotechnology) for confocal assay, rabbit polyclonal antibody against Gatal a and Sp¡1 b of zebrafish (1/2000, No. GTX128333 and GTX128266, GeneTex), antibody rabbit polyclonal against histone H3 (1/200, # ab1791, Abcam) and mouse-produced ANTI-FLAG® M2-Peroxidase (HRP) monoclonal antibody (A8592 Sigma-Aldrich). Densitometry analysis has been performed using Fiji Image J software (Schindelin et al., 2012).

Immunoprecipitation and Recombinant Caspase-1 Assay

Co-immunoprecipitation assays were also performed as previously described (Tyrkalska et al., 2017), with minor modifications. Cells were washed twice with PBS, solubilized in lysis buffer (50mM Tñs-HCI, pH 7.7, 150mM NaCl, 1% NP-40 and protease inhibitor cocktail) for 30 min with stirring and they centrifuged (13.000 ^c g, 10 min). The cell lysate (1 mg) was incubated for 2 h at 4 ° C with gentle shaking with 40 ml of ANTIFLAG® M2 suspension (A2220 Sigma-Aldrich). Immunoprecipitates were washed four times with lysis buffer containing 0.15 M NaCI, washed twice with PBS, and incubated with 10 ul of recombinant caspase-1 (# GTX65025, GeneTex) in reaction buffer (Hepes 50 mM, pH 7.2, 50mM NaCl, 0.1% Chaps, 10mM EDTA, 5% glycerol and 10mM DTT) for 2h at 37 ° C. The resin was boiled in SDS sample buffer 5 min at 95 ° C and bound proteins resolved on 4-15% SDS-PAGE (BioRad TGX # 456-1084) and transferred to PVDF membranes for 1 at 300 mA. Immunoblotting was probed with antibodies to FLAG and GATA1 (see above).

Gene expression analysis

Total RNA was extracted from 10 ⁶ CD34 ⁺ or K562 cells, complete embryos / larvae (60) or larval tails (100) with TRIzol reagent (Thermo Fisher Scientific) following the manufacturer's instructions and treated with DNase I, quality for amplification ( 1 U / Dg of RNA; Invitrogen). SuperScript III RNase FI ^D reverse transcriptase (Invitrogen) was used to synthesize the first strand cDNA with 1 Dg oligo (dT) 18 primer of total RNA at 50 ° C for 50 min. Real-time PCR was performed with an ABI PRISM 7500 instrument (Applied Biosystems) using SYBR Green PCR Core reagents (Applied Biosystems). Reaction mixtures were incubated for 10 min at 95 ° C, followed by 40 cycles of 15 s at 95 ° C, 1 min at 60 ° C, and finally 15 s at 95 ° C, 1 min at 60 ° C and 15 s at 95 ° C. For each mRNA, expression was normalized gene to the ribosomal protein S11 (rps11) for zebrafish or b-actin (ACTB) for the content of human cells in each sample following the Pfaffl method (Pfaffl, 2001). The primers used are shown in (Table S2). In all cases, each PCR was performed with samples in triplicate and repeated with at least two independent samples.

GEO data analysis

Microalignment gene expression profiles were extracted from Gene Expression Omnibus (GEO) data set GSE63270 using the geo2r code in the R Studio software. The expression levels of GATA1, IL1B, CASP1, PYCARD, NLRP1, NLRP3, NLRC4 and GBP5 were analyzed at different stages of erythroid differentiation from seven healthy human donors, including HSC, CMP, MEP and GMP.

Mouse treatment and blood sampling

5-FU (120 mg / kg in PBS on day 0) and Ac-YVAD-CMK (10 mg / kg in PBS containing 10% DMSO on days 6, 7, and 12) were injected intraperitoneally. . 50 ml of blood samples from orbital mice were collected at -1, 6, 10, and 14 days, and red blood cells, hemoglobin, hematocrit, platelets, and white blood cells were determined using a ProCyte Dx hematology analyzer following the manufacturer's instructions.

Statistic analysis

Data are shown as mean ± SEM and analyzed by vacancy analysis (ANOVA) and a multiple Tukey or Bonferroni amplitude test to determine differences between groups. Differences between two samples were analyzed using the Student's t-test. Fisher's exact test was used for the analysis of contingency tables. A logarithmic range test with the Bonferroni correction for multiple comparisons was used to calculate the statistical differences in survival of the different experimental groups.

Results Inflammasome inhibition decreases the number of neutrophils and macrophages in zebrafish larvae

Using transgenic zebrafish lines with Tg green fluorescent neutrophils (mpx.eGFP) ¹¹¹⁴ or Tg macrophages (mpeg1: eGFP) ^gl22 , the total number of both cell populations in complete larvae was quantified at 72 hpf. Genetic inhibition of various components of the inflammasome, specifically Gbp4, Ase and Caspa, the functional homolog of mammalian CASP1 (Kuri et al., 2017; Masumoto et al., 2003; Tyrkalska et al., 2016), resulted in numbers significant reductions of both neutrophils (figure 1 a, 1 b) and macrophages (figure 1 1 a, 11 b). Similarly, pharmacological inhibition of caspase-1 with the irreversible inhibitor Ac-YVAD-CMK (Tyrkalska et al., 2016) also resulted in reduced numbers of myeloid cells (Figure 1c, 1d, 11c, 11d). . These results were confirmed using a Tg {lyz: dsRED) ^nz50 independent transgenic line with labeled neutrophils (Figure 12a-12d). Similarly, the forced expression of the Gbp4 GTPase-deficient mutant (KS / AA) as well as its double mutant (DM: KS / AA; ACARD), both of which behave as dominant negatives (DN) and inhibit activation of inflammasome-dependent caspase-1 (Tyrkalska et al., 2016), resulted in a reduced number of neutrophils (Figure 1e, 1f). Furthermore, although the activation of the inflammasome by the forced expression of either Gbp4, or Ase or Caspa could not increase the numbers of neutrophils (Figure 1e-1h) or macrophages (Figure 11 e-11f), it was able to rescue the myeloid cell number and caspase-1 activity in Gbp4 and Ase deficient fish (Figure 1g-1j). Remarkably, however, the simultaneous expression of Ase and Caspa significantly increased the number of neutrophils (Figure 1 i, 1j) and macrophages (Figure 11 e, 11 f).

The inflammasome regulates the differentiation of HSC but it is dispensed for its appearance

Differentiation of HSC and progenitor cells into various types of blood cells is controlled by multiple extrinsic and intrinsic factors, and dysregulation in hematopoiesis can result in various hematologic abnormalities (Morrison et al., 1997; Yang et al., 2007). Chronic inflammatory disorders are commonly associated with neutrophilia and anemia, the so-called chronic disease anemia (ACD). Therefore, it was next examined whether the The inflammasome also regulated erythropoiesis using a transgenic Tg zebrafish line (lcreGFP), which has specific erythroid GFP expression (Ganis et al., 2012). The results showed that the inflammasome activity had the opposite effect on erythrocytes than on myeloid cells; that is, the abundance of erythrocytes increased after inhibition of the pharmacological and genetic inflammasome, as tested by flow cytometry (Figure 2a-2f). However, the expression of cmyb and runxl, which begins at 36 hpf and marks emerging definitive hematopoietic stem and progenitor cells (Burns et al., 2005), was not affected in Gbp4 and Ase deficient larvae at 48 hpf, as tested by complete assembly in situ hybridization (WISH) (Figure 13a). Similarly, the expression of rag1, which is expressed in differentiated thymic T cells, was apparently not affected by 5 dpf in inflammasome-deficient larvae (Figure 13a). Taken together, these results suggest a specific role for the inflammasome in regulating the balance between myelopoiesis and erythropoiesis.

To further confirm the role of the inflammasome in HSC differentiation, the number of HSC in the transgenic Tg line (runx1: GAL4; UAS.nfsB-mCherry) that has marked HSC (Tamplin et al., 2015), after inhibition, was quantified. pharmacological or genetic of the inflammasome at different stages of development (24 and 48 hpf). Caspase-1 inhibition did not result in changes in HSC number at any point in the treatment, confirming the result in Ase deficient larvae (Figure 3a-h). Furthermore, genetic inhibition of the inflammasome in neutrophils and HSC by forced expression of DN forms of Ase (AscACARD) or Gbp4 (Gbp4KS / AA) (Tyrkalska et al., 2016) using the specific promoters mpx and runxl, respectively, showed that the number of neutrophils decreased in HSC, but not in neutrophil inflammasome-deficient larvae (Figure 3 -3). Taken together, these results confirm the dispersibility of the inflammasome for the appearance and renewal of HSC, but that it is intrinsically required for HSC differentiation.

Zebrafish is an elegant model for cell ablation using specific transgenic lines that express bacterial nitroreductase, encoded by the nfsB gene, under the control of specific promoters (Davison et al., 2007). The enzyme nitroreductase converts the drug metronidazole (Mtz) into a cytotoxic product, which induces cell death in expressing cells to achieve tissue-specific ablation that has no effect on other cell populations (Curado et al., 2007; Curado et al., 2008; Prajsnar et al., 2012). Using this approach, neutrophils were excised in Tg (mpx: Gal4; UAS.nfsB-mCherry) applying Mtz for 24 h and then neutrophil recovery was analyzed in the presence or absence of the caspase-1 inhibitor for 6 days (Figure 4a). Mtz robustly reduced the number of neutrophils, which began to recover 4 days after ablation in control larvae (Figure 5B, 5C). However, pharmacological inhibition of the inflammasome affected neutrophil recovery after ablation and strongly decreased neutrophil abundance in unresected larvae (Figure 4b, 4c). As expected, continuous Mtz treatment resulted in drastic neutrophil decrease but did not show any toxic effect on control larvae that did not express nitroreductase (Figure 4b, 4c). These results indicate that the inflammasome is essential for the myeloid differentiation of HSC.

Inhibition of inflammasomes impairs demand-driven myelopoiesis. In response to infection, hematopoietic tissue enhances the production and mobilization of neutrophils, which are short-lived and are needed in large quantities to fight infection. This process is called demand-driven or emergency hematopoiesis (Hall et al., 2012). To check whether only steady-state hematopoiesis or demand-driven hematopoiesis were regulated by the inflammasome, 48 hpf was infected with Salmonella typhimurium in the otic vesicle of larvae and the number of total neutrophils was counted at 24 hpi in the presence or absence of the irreversible caspase-1 inhibitor Ac-YVAD-CMK. It was observed that pharmacological inhibition of the inflammasome could cancel out infection-driven myelopoiesis, which resulted in an increased number of neutrophils in infected larvae (Figure 5a, 5b). Notably, the forced expression of granulocyte colony-stimulating factor (Gcsf), which stimulates both steady-state and demand-driven granulopoiesis in zebrafish (Hall et al., 2012; Stachura et al., 2013), The number of neutrophils was dramatically increased to similar levels in wild-type, Ase-deficient larvae, as well as in larvae treated with the caspase-1 inhibitor, without affecting caspase-1 activity (Figure 5c-5f). However, Gcsf was unable to rescue the increased susceptibility to S. typhimurium infection of Ase-deficient larvae and larvae. treated with caspase-1 inhibitor (figure 5g, 5h), confirming previous results in Gbp4-deficient larvae (Tyrkalska et al., 2016). All these results also suggest that the inflammasome regulates the decision of myeloid / erythroid fate in addition to the function of mature myeloid cells.

The inflammasome displaces the Spi1 / Gata1 balance favoring myeloid differentiation

Regulation of Sp1 and Gatal has been shown to be critical for differentiation of myeloid and erythroid cells, respectively, in all vertebrates. Since inhibition of the inflammasome resulted in hematopoietic lineage bias, i.e., reduced myeloid blood cells and increased erythroid blood cells, then spi1 and GATA1 levels were analyzed by RT-qPCR and WISH. Increased transcript levels of gatal at 24 hpf were observed in Gbp4 and Ase deficient larvae, while levels of genes encoding spi1 and pivotal downstream macrophage and neutrophil growth factors, specifically macrophage colony stimulating factors and Granulocytes (mesf and gesf genes) were not greatly affected (Figures 5¡). Importantly, Gatal protein levels were also adjusted by the inflammasome, since genetic inhibition of either Ase or Gbp4 was able to increase Gatal, while the forced expression of Ase and Dandruff, which resulted in an increased number of neutrophils and macrophages (Figure 1 i, 1 j, 11 e, 11 f), Gatal robustly decreased (Figure 5j). Thus, the inflammasome regulates myeloid / erythroid cell fate decision by adjusting GATA1 levels.

The regulation of HSC differentiation by the inflammasome is evolutionarily conserved

Next, we sought to determine if the inflammasome also regulates human hematopoiesis. Bioinformatic analysis of gene expression profiles coding for inflammasome components in normal hematopoietic stem and progenitor cells (GEO dataset GSE63270) (Jung et al., 2015) revealed the expected increased levels of GATA1 in parent common myeloid (CMP) and megakaryocytic-erythroid progenitors (MEP). In contrast, CASP1 transcript levels decreased in both CMP and MEP, while PYCARD, which encodes ASC, NLRP3, NLRP1 and GBP5, was only reduced in MEP but not in CMP. However, IL1B levels did not show a clear pattern associated with erythropoiesis or myelopoiesis.

Tight regulation of CASP1, PYCARD, NLRP3 and NLRC4 was then confirmed during erythropoietin (EPO) -induced erythroid differentiation of CD34 ⁺ HSC in vitro. Therefore, the transcript levels of all of them were reduced on day 3 or 5 after the addition of EPO (Figure 6a). More interestingly, caspase-1 activity was reduced after the addition of EPO and remained at undetectable levels until 5 days of differentiation, the longest time analyzed (Figure 6b). These data led to analyze the impact of pharmacological inhibition of CASP1 on erythroid differentiation of HSC CD34 ⁺ by EPO. Surprisingly, inhibition of CASP1 resulted in increased GATA1 transcript levels on 3 days of erythroid differentiation and those of the gene encoding the erythroid differentiation markers glycophorin A (GYPA), transferrin receptor (TFRC), and transport protein band 3 anion (SLC4A1) 3 or 5 days after the addition of EPO (Figure 6c).

To further explore the relevance of the inflammasome in erythroid differentiation, the human erythroleukemic cell line K562, which can differentiate into erythrocytes in the presence of hemin, was then used (Andersson et al., 1979; Koeffler and Golde, 1980). GATA1 levels and activity were found to increase in the early stages of erythropoiesis, while they decreased in the later stage to allow for terminal erythroid differentiation (Ferreira et al., 2005; Whyatt et al., 2000). As expected, hemin was observed to promote a gradual accumulation of hemoglobin and reduced GATA1 protein levels from 0 to 48 h (Figure 7a, 7c). Notably, the NLRC4, NLRP3, and CASP1 transcript levels gradually increased, while those of PYCARD peaked at 12 hr and then decreased to baseline levels. Furthermore, CASP1 activity (Figure 7b) and protein levels (Figure 7c) progressively increased during erythroid differentiation. Furthermore, CASP1 was evenly distributed both in the cytosol and in the nucleus (Figure 7d). Surprisingly, pharmacological inhibition of CASP1 in K562 cells impaired hemin-induced erythroid differentiation, assessed as hemoglobin accumulation, and inhibited GATA1 reduction at both 24 (Figure 7e) and 48h (Figure 7f, 7g).

CASP1 can target various proteins to regulate HSC differentiation. One possibility is that CASP1 directly cleaves GATA1, as reported for CASP3, which down-regulates erythropoiesis by cleavage of GATA1 (De María et al., 1999). Therefore, it was studied whether recombinant human CASP1 could cleave human GATA1 in vitro. The results showed that recombinant CASP1 cleaved GATA1 generating N and C-terminal proteolytic fragments of approximately 30 and 15 kDa, respectively. Cleavage of CASP1 from GATA1 at residues D276 and / or D300 can generate the fragments obtained, thereby generating single and double CASP1 mutants (D276A and D300A) and it was found that CASP1 could only cleave GATA1 at residue D300. Taken together, all these results reveal a novel, conserved evolutionary role of the inflammasome in the regulation of the decision of the erythroid / myeloid fate and terminal erythroid differentiation by means of GATA1 excision.

Pharmacological inhibition of the inflammasome rescues zebrafish and mouse models from neutrophilic inflammation and anemia

Hematopoietic lineage bias is associated with chronic inflammatory diseases, cancer, and aging (Elias et al., 2017; Marzano et al., 2018; Wu et al., 2014). Neutrophilic dermatosis is a group of diseases characterized by the accumulation of neutrophils on the skin (Marzano et al., 2018). A Spintl a zebrafish mutant was used as a model for neutrophilic dermatosis, as it is characterized by strong infiltration of neutrophils into the skin (Carney et al., 2007; Mathias et al., 2007). Larvae deficient in Spintl a were found to have increased caspase-1 activity (Figure 8a) and altered spiUgatal ratio (Figure 8b). Notably, although pharmacological inhibition of caspase-1 was unable to rescue cutaneous infiltration of neutrophils from Spintl a-deficient animals (Figure 8c, 8e), it was able to rescue their robust neutrophilia (Figure 8d, 8e). The Diamond-Blackfan anemia model, a ribosomopathy caused by ineffective translation of GATA1 (Danilova and Gazda, 2015), was then performed in zebrafish larvae by reducing GATA1 levels using a specific morpholino. Morpholino was first assessed and it was found that 1.7 ng / egg resulted in larvae with mild, moderate and severe anemia (figure 8f), while 0.85 ng / egg and 3.4 ng / egg had few effects or drastic, respectively. Therefore, it was examined whether pharmacological inhibition of caspase-1 could rescue hemoglobin alterations from Gatal-deficient larvae. The results show that treatment of larvae for 24 h with the reversible caspase-1 inhibitor Ac-YVAD-CHO partially rescued the hemoglobin defects in Gatal-deficient larvae and Sp¡1 / Gatal protein levels (Figure 8g, 8h). Together these results demonstrate that pharmacological inhibition of caspase-1 rescues hematopoietic lineage bias in vivo.

To further confirm the above results in another preclinical model, HSC was partially excised in mice with 5-fluorouracil (5-FU) (Coppin et al., 2016) and then the effects of pharmacological inhibition of caspase-1 on the recovery of blood cells (figure 9a). Two single injections at 6 and 7 days after injection of irreversible caspase-1 inhibitor Ac-YVAD-CMK 5-FU (10 mg / Kg) were able to rescue the 5-FU-induced anemia at 10 days, assessed as red blood cell count and hemoglobin and hematocrit levels (Figure 9b-d). However, vehicle-treated mice recovered from anemia much later (14 d after 5-FU treatment) (Figure 9b-d). Notably, platelet counts (Figure 9e) and total white blood cells (Figure 9f) were unaffected by CASP1 inhibition. These results demonstrate that pharmacological inhibition of caspase-1 rescues chemotherapy-induced anemia in mouse models.

Claims

Claims one . Composition comprising at least one caspase-1 inhibitor for use in a method of treatment of a disease selected from the list consisting of anemia associated with chronic diseases, chemotherapy-induced anemia and Diamond-Blackfan anemia. 2. Composition for use according to claim 1, in which the caspase 1 inhibitor has the motif: X or X-W, where X is a selective structure of caspase-1 in relation to other cysteine proteases, in which The selectivity of a substrate for a caspase is determined by biochemical and cell-based assays on related enzymes, and where W represents a chemical moiety comprising— NH— CH (Y) (Z), where the electrophilic warhead modifies from caspase is reversibly so that caspase cannot interact with or cleave a caspase substrate, where Y is a structure that allows the inhibitor compound to form a reversible covalent bond with caspase-1, and where Z is a moiety carboxyl or a carboxylic acid mimetic. 3. Composition for use according to claim 1, wherein the caspase-1 inhibitor is ac-YVAD-CMK, comprising the following chemical structure:

any salt thereof or any stereoisomeric form. 4. Composition for use according to claim 1, wherein the caspase-1 inhibitor is z-YVAD-FMK, which comprises the following chemical structure:

any salt thereof or any stereoisomeric form. 5. Composition for use according to claim 1, wherein the caspase-1 inhibitor is z-WEHD-FMK, which comprises the following chemical structure:

any salt thereof or any stereoisomeric form. 6. Composition for use according to claim 1, wherein the caspase-1 inhibitor is z-WEHD-CHO, which comprises the following chemical structure:

any salt thereof or any stereoisomeric form. 7. Composition for use according to claim 1, wherein the caspase-1 inhibitor is Ac-YVAD-CHO, which comprises the following chemical structure:

any salt thereof or any stereoisomeric form. 8. Composition for use according to claim 1, wherein the caspase-1 inhibitor is ac-YVKD-CHO, which comprises the following chemical structure:

any salt thereof or any stereoisomeric form. 9. Composition for use according to claim 1, wherein the caspase-1 inhibitor is Ac-YVAD-FMK, which comprises the following chemical structure:

any salt thereof or any stereoisomeric form. 10. Composition for use according to claim 1, wherein the caspase-1 inhibitor is Ac-YVAD-AOM, comprising the following chemical structure:

any salt thereof or any stereoisomeric form. 11. Composition for use according to claim 1, wherein the caspase-1 inhibitor is Z-D-CH2-DCB, comprising the following chemical structure:

any salt thereof or any stereoisomeric form. 12. Composition for use according to claim 1, wherein the caspase-1 inhibitor is VX-765, comprising the following chemical structure:

any salt thereof or any stereoisomeric form. 13. Composition for use according to claim 1, wherein said composition is a pharmaceutical composition prepared for administration to a subject, comprising a therapeutically effective amount of one or more of the compounds as defined in any one of claims 1 to 12.