Beta-hydroxy-beta-methylbutyric acid as an inhibitor of gsdmd and its anti-cell pyroptosis applications

Abstract

The present invention relates to the use of beta-hydroxy-beta-methylbutyric acid (HMB) or a pharmaceutically acceptable salt thereof as an inhibitor of Gasdermin D (GSDMD) in the manufacture of a medicament for the treatment of pyroptosis. HMB modulates pyroptosis in macrophages by blocking palmitoylation of GSDMD and has the potential to be a novel therapeutic agent for the treatment of inflammatory diseases driven by pyroptosis disorders.

Description

Technical Field

[0001] This invention relates to the field of chemical biology. More specifically, this invention relates to the application of β-hydroxy-β-methylbutyric acid as an inhibitor of Gasdermin D (GSDMD) and its anti-pyroptosis effect. Background Technology

[0002] Pyroptosis is an inflammatory type of programmed cell death characterized by cell swelling, membrane perforation, and release of inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-18 (IL-18) [1] ([1] refers to reference 1, hereinafter the same). In response to pathogen challenges, pyroptosis can be triggered by classical or non-classical inflammasome pathways [2-4]. Gasdermin (GSDM)D plays a key role in the execution of pyroptosis, with the activated N-terminal (NT) fragment of GSDMD forming transmembrane pores, mediating cytokine release, and disrupting ion and water homeostasis [5-8]. In the GSDM protein family, GSDMD is widely distributed in most organs and immune cells and is closely involved in innate immune responses to pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMP) [9,10]. It has been reported that excessive pyroptosis can lead to a variety of inflammatory diseases, and GSDMD has been significantly upregulated in these diseases, including inflammatory bowel disease (IBD)

[11] , ischemia-reperfusion injury

[12] , sepsis

[13] , cancer[14,15], making GSDMD an attractive target for the treatment of various inflammatory diseases.

[0003] Inhibiting GSDMD-mediated pyroptosis is becoming a promising strategy for treating inflammatory diseases, and efforts have recently been made to develop chemical inhibitors targeting GSDMD. Researchers have identified several GSDMD inhibitors, such as necrosulfonamide (NSA)

[16] , disulfiram (DSF)

[17] , and dimethylfumarate (DMF)

[18] . Most of these GSDMD inhibitors have shown significant alleviating effects in preclinical disease models of sepsis. CN119745860A discloses that the small molecule compound SFII, as a GSDMD inhibitor, can resist pyroptosis and exert anti-inflammatory effects to prevent or treat atherosclerosis. However, recent studies have found that GSDMD has a pyroptosis-independent protective function in epithelial cells[19,20], which complicates the selection of GSDMD inhibitors as clinical drugs, especially for inflammatory diseases with epithelial damage. Therefore, it is crucial to develop and study novel GSDMD inhibitors as therapeutics for a range of diseases.

[0004] β-hydroxy-β-methylbutyrate (HMB) is a bioactive metabolite derived from the essential amino acid leucine. It has been used as a supplement to enhance athletic performance, promote muscle hypertrophy, and increase muscle strength

[21] . Multiple clinical studies have shown that HMB supplementation is beneficial to both healthy individuals and clinical populations, and its biosafety has been demonstrated in various populations, including athletes, the elderly, and clinical patients such as those with chronic obstructive pulmonary disease (COPD) or cancer[22-25]. Summary of the Invention

[0005] As mentioned above, pyroptosis is a lytic, inflammatory form of programmed cell death mediated by Gasdermin proteins, and is involved in the pathogenesis of various inflammatory diseases, such as inflammatory bowel disease and ischemia-reperfusion injury. Gasdermin D (GSDMD) is a key executor of pyroptosis and a promising therapeutic target, but strategies for safely modulating its activity remain to be explored. Here, we discovered that β-hydroxy-β-methylbutyrate (HMB), a leucine-derived metabolite with proven clinical biosafety, is a potent inhibitor of GSDMD-dependent pyroptosis. - / - Metabolomics analysis of wild-type macrophages revealed significant accumulation of HMB during pyroptosis. Mechanistically, HMB attenuates Akt signaling and inhibits palmitoyltransferase ZDHHC7, thereby blocking palmitoylation of GSDMD cysteine ​​residues. This modification is crucial for GSDMD membrane localization, and HMB inhibition disrupts pore formation, cytokine release, and pyroptosis. In mouse models, HMB administration significantly reduced DSS-induced colitis and pulmonary ischemia-reperfusion injury, similar to the protective effect of GSDMD inhibition. Our findings not only depict a metabolite-GSDMD regulatory axis via palmitoylation but also highlight the potential of HMB (a clinically safe nutritional supplement) as a novel therapeutic agent for pyroptosis-driven inflammatory diseases.

[0006] Therefore, according to one aspect of the invention, the present invention provides the use of β-hydroxy-β-methylbutyric acid or a pharmaceutical salt thereof in the preparation of an inhibitor of GSDMD.

[0007] According to another aspect of the present invention, the present invention provides the use of β-hydroxy-β-methylbutyric acid or a pharmaceutical salt thereof as an inhibitor of GSDMD in the preparation of medicaments for anti-pyroptosis (i.e., inhibition of GSDMD-mediated pyroptosis).

[0008] In a preferred embodiment, the cells are immune cells, including macrophages and neutrophils, preferably macrophages, more preferably bone marrow-derived macrophages (BMDMs).

[0009] In another preferred embodiment, the drug is used to treat or prevent inflammatory diseases, particularly those mediated or caused by pyroptosis of immune cells (such as macrophages).

[0010] In another preferred embodiment, the inflammatory disease is an epithelial inflammatory disease, particularly an inflammatory disease with epithelial damage.

[0011] In another preferred embodiment, the drug is used to treat or prevent inflammatory bowel disease (such as colitis), (lung) ischemia-reperfusion injury, sepsis, atherosclerosis, or cancer.

[0012] In another preferred embodiment, the drug further includes an additional GSDMD inhibitor. This additional GSDMD inhibitor may include necrosulfonamide (NSA) disulfiram and dimethyl fumarate, small molecule compounds such as SFII, as mentioned above.

[0013] In another preferred embodiment, β-hydroxy-β-methylbutyric acid or a pharmaceutically acceptable salt thereof can inhibit the increase in pro-inflammatory cytokine levels. Preferably, the pro-inflammatory cytokines include interleukin-1β (IL-1β) and interleukin-18 (IL-18).

[0014] In another preferred embodiment, β-hydroxy-β-methylbutyric acid or a pharmaceutically acceptable salt thereof can inhibit macrophage inflammation.

[0015] In another preferred embodiment, β-hydroxy-β-methylbutyric acid or a pharmaceutically acceptable salt thereof can inhibit inflammation-induced macrophage pyroptosis.

[0016] In another preferred embodiment, the pharmaceutical salt is selected from alkali metal salts and alkaline earth metal salts of β-hydroxy-β-methylbutyric acid, preferably calcium or sodium salts.

[0017] In a preferred embodiment, β-hydroxy-β-methylbutyric acid or a pharmaceutically acceptable salt thereof inhibits GSDMD-induced pyroptosis by blocking palmitoylation of GSDMD cysteine ​​residues.

[0018] In a preferred embodiment, β-hydroxy-β-methylbutyric acid or its pharmaceutical salt attenuates Akt signaling, inhibits palmitoyltransferase ZDHHC7, thereby blocking the palmitoylation of GSDMD cysteine ​​residues.

[0019] In a preferred embodiment, the drug further comprises pharmaceutical excipients. Pharmaceutical excipients may include fillers, binders, disintegrants, lubricants, preservatives, antioxidants, solubilizers, flavoring agents, emulsifiers, absorption enhancers, surfactants, etc. The drug may be in the form of tablets, suspensions, capsules, granules, ointments, creams, gels, injections, aerosols, patches, etc.

[0020] This invention reveals that HMB, a supplement that enhances motor function, is an inhibitor of GSDMD and pyroptosis. We elucidate that HMB primarily inhibits pyroptosis by suppressing ZDHHC7 through the Akt signaling pathway, which blocks palmitoylation of GSDMD cysteine ​​residues, thereby reducing its membrane binding and impairing pyroptosis. Furthermore, HMB demonstrated significant therapeutic effects in two mouse disease models, including colitis and pulmonary ischemia-reperfusion injury. Our findings provide a new option for GSDMD inhibitors as clinically viable drugs with good biocompatibility and offer mechanistic insights into the repurposing of HMB for the treatment of various inflammatory diseases. Attached Figure Description

[0021] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein:

[0022] Figure 1 β-Hydroxy-β-methylbutyrate (HMB) inhibits pyroptosis in bone marrow-derived macrophages (BMDMs). (A) Heatmap of the relative concentrations of the top 50 metabolites detected by metabolomics. (B) Seven metabolites showing significant changes among the top 50 metabolites. (C, D, E, and G) Bone marrow-derived macrophages (BMDMs) were treated as shown, and lactate dehydrogenase (LDH), interleukin-1β (IL-1β), or tumor necrosis factor-α were measured by enzyme-linked immunosorbent assay (ELISA). (F) Western blot analysis of proteins expressing GSDMD in bone marrow-derived macrophages (BMDMs). (H) Relative concentration of β-hydroxy-β-methylbutyric acid (HMB) detected by liquid chromatography-mass spectrometry (LC-MS); (I and J) Measurement of lactate dehydrogenase (LDH) and interleukin-1β (IL-1β) by enzyme-linked immunosorbent assay (ELISA); (K) Expression of GSDMD in bone marrow-derived macrophages (BMDMs) treated with endogenous β-hydroxy-β-methylbutyric acid (HMB) inhibitors by Western blotting. *P<0.05, **P<0.01; One-way ANOVA. Error bars represent mean ± standard error (SEM).

[0023] Figure 2β-hydroxy-β-methylbutyrate (HMB) inhibited pyroptosis in THP-1 cells. (A, B, and C) THP-1 cells were induced to differentiate with phorbol 12-myristate 13-acetate (PMA, 100 ng / mL) (Bio-Biobio (Guangzhou) Co., Ltd.) as indicated. Lactate dehydrogenase (LDH), interleukin-1β, or tumor necrosis factor-α were measured by enzyme-linked immunosorbent assay (ELISA). (D and E) GSDMD was detected by liquid chromatography-mass spectrometry (LC-MS). - / - Relative concentrations of β-hydroxy-β-methylbutyrate (HMB) and leucine in bone marrow-derived macrophages (BMDMs). *p<0.05; one-way ANOVA. Error bars represent mean ± standard error (SEM).

[0024] Figure 3 Palmitoylation of GSDMD inhibits pyroptosis. (A) Representative mass spectra of GSDMD palmitoylation immunoprecipitated from bone marrow-derived macrophages (BMDMs), and the relative abundance of cysteine ​​palmitoylation modification as shown by mass spectrometry. (B) Co-immunoprecipitation (CO-IP) (C and D) Measurement of cell death in THP-1 cells and bone marrow-derived macrophages (BMDMs) by enzyme-linked immunosorbent assay (ELISA). (E) Immunoblot of GSDMD in the membrane of THP-1 cells transfected as shown. *P<0.05, **P<0.01; One-way ANOVA. Error bars represent mean ± standard error (SEM).

[0025] Figure 4β-Hydroxy-β-methylbutyrate (HMB) inhibits the PI3K-Akt signaling pathway to reduce cysteine ​​palmitoylation of GSDMDs. (A) Analysis of ZDHHC2, 7, and 12 mRNA expression in bone marrow-derived macrophages (BMDMs) treated as indicated by real-time quantitative polymerase chain reaction (RT-qPCR) (PCR primers used for detection are shown in Table 1). (B and C) Measurement of cell death and interleukin-1β in bone marrow-derived macrophages (BMDMs) by enzyme-linked immunosorbent assay (ELISA). (D) KEGG analysis of the most significantly enriched signaling pathways in bone marrow-derived macrophages (BMDMs) treated with β-hydroxy-β-methylbutyrate (HMB) or phosphate-buffered saline (PBS). (E) Analysis of ZDHHC7 mRNA expression in bone marrow-derived macrophages (BMDMs) treated as indicated by real-time quantitative polymerase chain reaction (RT-qPCR) (PCR primers used for detection are shown in Table 1). (F) Immunoblot analysis of Akt in bone marrow-derived macrophages (BMDMs) processed as shown (P-AKT and AKT detection antibodies were purchased from MedChemExpress). *P<0.05, **P<0.01; one-way ANOVA. Error bars represent mean ± standard error (SEM).

[0026] Table 1

[0027]

[0028] Figure 5 Oral administration of β-hydroxy-β-methylbutyric acid (HMB) alleviated dextran sulfate sodium (DSS)-induced enteritis in mice. (A) Weekly monitoring of body weight changes and (B) Disease Activity Index (DAI), and (C) colon length in mice on day 36. (D) Analysis of mRNA expression of pro-inflammatory cytokines in mouse colons by reverse transcription quantitative polymerase chain reaction (RT-qPCR) (PCR primers used for detection are shown in Table 2). (E and F) Immunoblot analysis of GSDMD, Akt, and p-Akt in mouse colons. Error bars represent mean ± standard error (SEM). **P < 0.01, ***P < 0.001. Two-way ANOVA and Sidak's multiple comparison test were used to analyze body weight changes and DAI, and two-tailed unpaired Student's t-test was used to analyze colon length.

[0029] Figure 6Oral administration of β-hydroxy-β-methylbutyric acid (HMB) alleviated lung ischemia-reperfusion injury in rats. (A) Hematoxylin-eosin (H&E) staining and (B) rat lung injury score. (C and D) Detection of interleukin-1β (IL-1β) and interleukin-18 (IL-18) by enzyme-linked immunosorbent assay (ELISA). (E, F and G) Analysis of mRNA expression of IL-1β, IL-18 and GSDMD in rat lungs by RT-qPCR (PCR primers used for detection are shown in Table 2). (H) Immunoblotting analysis and (I) Relative expression of GSDMD in membrane protein samples. (J) Immunoblotting analysis and (K) Relative expression of GSDMD in cytoplasmic protein samples. Error bars represent mean ± standard error (SEM). **P<0.01, ***P<0.001, ****P<0.0001.

[0030] Table 2

[0031]

[0032] Detailed Implementation

[0033] Unless otherwise indicated, the terms used herein have their general technical meanings as understood by those skilled in the art.

[0034] The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. All materials and reagents used in the following reactions are commercially available products unless otherwise specified.

[0035] Experimental methods

[0036] animal experiments

[0037] This study was approved by the Experimental Animal Research Center and the Animal Experiment Ethics Committee of Tsinghua University. Mice and rats were housed in a 12-hour light / 12-hour dark cycle environment with free access to food and water. SPF-grade male C57BL / 6J mice (5 weeks old) and SD rats (7-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After one week of acclimatization, the animals were randomly assigned to different experimental groups. - / - The mice were provided by the National Institute of Biological Sciences (NIBS) of China.

[0038] DSS colitis model

[0039] Animals were treated with 2.5% DSS (MP Biomedicals) in drinking water for 5 days, and then the DSS was replaced with tap water for another 7 days, depending on the requirements of each experiment. Animal weight was monitored daily, and intestinal tissue was collected at the end of the experiment to assess colitis and tissue regeneration.

[0040] Lung ischemia-reperfusion injury

[0041] Prior to surgery, rats were anesthetized by intraperitoneal injection of 5% sodium pentobarbital (10 mL / kg), fixed in a supine position, and underwent tracheotomy. They were then connected to a ventilator for mechanical ventilation. The ventilator parameters were adjusted to a tidal volume of 20 mL / kg, a frequency of 60 breaths / min, and an inspiratory-to-expiratory ratio of 1:2.5. Thoracotomy was performed, and the left hilum was clamped with an arterial clamp for 30 minutes to induce ischemia. The arterial clamp was then removed to simulate reperfusion for 1 hour, after which tissue samples were collected.

[0042] Tissue collection and processing

[0043] Mice or rats were euthanized by cervical dislocation. Lung, ileum, colon, cecum, and ileum tissues were collected and immediately frozen at -80°C for RNA extraction. The lung, colon, or ileum was also immersed in different tissue fixatives for histological analysis.

[0044] Colon explant culture

[0045] The colon was removed from the mouse, briefly rinsed in phosphate-buffered saline (PBS), and then rinsed three times with cold PBS containing gentamicin (20 μg / ml), penicillin G (200 μg / ml), and streptomycin (200 μg / ml) to remove residual intestinal bacteria. It was then cultured in supplemented medium containing penicillin G (200 μg / ml) and streptomycin (200 μg / ml)

[40] . After incubation at 37°C for 24 hours, the medium was collected and the production of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA).

[0046] Cytotoxicity assay and interleukin-1β ELISA detection

[0047] The relevant cells were treated as instructed. Cell death was measured by lactate dehydrogenase (LDH) assay using the CytoTox 96 non-radioactive cytotoxicity assay kit (Promega). Cell viability was determined by the CellTiter-Glo luminescent cell viability assay (Promega). To measure interleukin-1β release, primary BMDM cells (extracted from mice, see "Preparation and culture of bone marrow-derived macrophages from mice for functional analysis", DOI:10.1016 / j.xpro.2020.100246) were pre-stimulated with lipopolysaccharide (LPS) (1 μg / mL) for 2 h, and the release of mature interleukin-1β was measured using an interleukin-1β enzyme-linked immunosorbent assay (ELISA) kit (Neobioscience Technology Company).

[0048] RNA sequencing analysis

[0049] On day 5 after treatment with sodium dextran sulfate (DSS), samples from wild-type (WT) and GSDMD were collected. - / - Macrophages were isolated from the colon of mice. RNA isolation, library construction, and sequencing were performed using the BGISEQ-500 sequencing platform (Beijing Genomics Institute). Readings were mapped to the mouse genome (GRCm38.p5) using HISAT2. KEGG pathway analysis was performed using R packages, identifying differentially expressed genes (P-values ​​were calculated using Dr. Tom, a custom data mining system developed by BGI, available at http: / / report.bgi.com).

[0050] Click Chemistry

[0051] Cells were labeled overnight with 50 μM Alk-14 (Cayman) or adec15-alkynyl acid (16C-BYA, homemade) in DMEM supplemented with 10% fetal bovine serum, centrifuged, washed with cold phosphate-buffered saline (PBS), and resuspended in 1% NP-40 lysis buffer (a mixture of 50 mM aminomethane hydrochloride (Tris-HCl) pH 7.5, 150 mM sodium chloride (NaCl), 10% glycerol, and 1% Nonidet P-40 supplemented with protease inhibitors). Cells were then lysed on ice using an ultrasonic cell disruptor for 2 min. The lysates were centrifuged at 4 °C and 12,000 g for 10 min, and the collected supernatant was added to anti-GSDMD antibody (for intravenous GSDMD) or anti-Flag magnetic beads (for Flag-tagged proteins) for GSDMD immunoprecipitation. After incubation, the cells were washed three times with lysis buffer for a copper (Cu(I))-assisted click reaction. In summary, magnetic beads were incubated for 1 h in D-PBS (Dulbecco's Phosphate Buffered Saline) containing 1 μM tetramethylrhodamine azide (Tamra-azide, Cayman) or biotin-azide (Cayman), 1 mM copper sulfate (CuSO4), 0.1 mM tris((1-benzyl-4-triazolyl)methyl)amine, and 1 mM tris(2-carboxyethyl)phosphine (pH 7.0). The beads were washed three times with D-PBS and resuspended in D-PBS. To label tetramethylrhodamine (TAMRA), immunoprecipitated samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and scanned using a Typhoon 7000 variable-mode imager (GE Healthcare LifeSciences) to record TAMRA fluorescence signals. Biotin-labeled immunoprecipitated samples were eluted with 6 M urea for streptavidin pull-down assays.

[0052] RNA extraction, reverse transcription, and quantitative polymerase chain reaction (qPCR)

[0053] Total RNA was isolated from homogenized tissue using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the expression of the target gene. For analysis, after normalization with a control, 2... -ΔΔCT The method calculates the fold change in expression of the target gene.

[0054] Cytotoxicity assay (LDH release)

[0055] Following macrophage stimulation, the supernatant was collected and processed using CytoTox according to the manufacturer's instructions. The non-radioactive cytotoxicity assay kit (Promega) quantifies LDH release. The percentage of cytotoxicity is calculated based on the maximum LDH release from unstimulated cells lysed with 1% Triton X-100.

[0056] Protein immunoblotting analysis

[0057] Proteins were separated by 10%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes, which were treated with blocking buffer at room temperature for 1 hour. The PVDF membranes were then treated with antibodies in the following manner: mouse anti-caspase-1 (Casp-1) monoclonal antibody (1:1000, Proteintech), rabbit anti-GSDMD polyclonal antibody (1:1000, Proteintech), and mouse anti-β-actin monoclonal antibody (1:1000, Proteintech), incubated overnight at 4°C. The membranes were then washed and treated with horseradish peroxidase (HRP)-conjugated secondary antibodies: HRP-conjugated affinity-purified goat anti-rabbit IgG (H+L) (1:5000, Proteintech) and HRP-conjugated affinity-purified goat anti-mouse IgG (H+L) (1:5000, Proteintech). Protein expression levels were assessed using an electrochemiluminescence (ECL) protein blot analysis system (Pierce Biotechnology) for protein expression analysis.

[0058] Statistical analysis

[0059] Numerical values ​​are expressed as mean ± standard error (SEM). Data were analyzed using two-tailed Student's t-test, one-way ANOVA, two-way ANOVA, or repeated measures ANOVA. Differences between groups were then assessed using the post-hoc Tukey-Kramer test. P < 0.05 was considered statistically significant. All statistical analyses were performed using Graphpad Prism (version 8.0).

[0060] Example 1

[0061] The close association between HMB and pyroptosis

[0062] Numerous studies have shown that cellular metabolism plays a crucial role in regulating inflammatory responses. Metabolites derived from the tricarboxylic acid cycle (Krebs cycle), such as succinate and itaconic acid, have been shown to regulate the expression of inflammatory genes in lipopolysaccharide (LPS)-activated macrophages [26-30]. GSDMD is a key gene involved in pyroptosis, and its knockout can alleviate pyroptosis

[11] . To investigate which metabolites are closely associated with pyroptosis, we examined the effects of GSDMD knockout on intracellular metabolites. Bone marrow-derived macrophages (BMDMs) were pretreated with LPS (100 ng / ml) for 2 h and then exposed to Nig (10 μM) for 1 h to induce pyroptosis. Metabolites were detected using liquid chromatography-mass spectrometry (LC-MS)

[31] . A total of 500 metabolites were detected in the cells, and 7 of the 50 metabolites with the highest concentrations showed significant changes ( Figure 1 (A and B). BMDMs were treated with these seven metabolites at concentrations mentioned in previous studies [32-38] 12 hours prior to LPS-nigrain induction. Of these seven metabolites, β-hydroxy-β-methylbutyric acid (HMB) (Shanghai Yuanye Biotechnology Co., Ltd.) effectively blocked LPS-nigrain-induced lactate dehydrogenase (LDH) release, a marker of pyroptosis. Figure 1 HMB's inhibitory effect on pyroptosis is concentration-dependent; 4 mM is the concentration that reduces LPS-Nigerin-induced BMDMs (…). Figure 1 (D and E) and THP-1 cells ( Figure 2 The optimal concentrations for LDH and IL-1β release in (A and B) were also observed. HMB also inhibited the 30 kDa cleavage fragment of GSDMD in BMDMs. Figure 1 F), but does not affect tumor necrosis factor-α (TNF-α), which is a component of BMDMs ( Figure 1 G) and THP-1 cells ( Figure 2 These results indicate that HMB specifically inhibits pyroptosis without suppressing TNF-α-related inflammation.

[0063] Example 2

[0064] Endogenous HMB regulates pyroptosis through leucine metabolism.

[0065] HMB is an active metabolite of leucine, one of the three essential branched-chain amino acids (BCAAs; leucine, valine, and isoleucine)

[24] . It can be obtained exogenously through diet or synthesized endogenously during leucine metabolism. Our results showed that the concentrations of HMB and leucine were reduced in LPS-Nigeriacin-activated wild-type BMDMs, but increased in GSDMDs. - / - Normal recovery in BMDMs ( Figure 2 This suggests that HMB and leucine may play important roles in macrophage pyroptosis. To assess the involvement of endogenous HMB, we used the branched-chain transaminase (BCAT) inhibitor BAY-252 (MedChemExpress) to block the production of endogenous HMB. This resulted in a reduction of 30 kDa cleavage fragments of LDH, IL-1β, and GSDMD in BMDMs, indicating that endogenous HMB is involved in regulating macrophage pyroptosis. Figure 3 AC).

[0066] Example 3

[0067] HMB blocks palmitoylation of GSDMD at key cysteine ​​residues.

[0068] In previous studies, fumarate reacted with key cysteine ​​residues in Gasdermin D (GSDMD) to form S-(2-succinyl)-cysteine, an irreversible post-translational modification (PTM) that prevents GSDMD from interacting with caspase, thereby limiting its processing, oligomerization, and induction of cell death [18,39]. To verify whether β-hydroxy-β-methylbutyrate (HMB) also affects GSDMD via post-translational modification in the context of pyroptosis, we performed liquid chromatography / mass spectrometry (LC / MS) peptide profiling. The results showed that HMB inhibited cysteine ​​residues C39 and C192 in mouse GSDMD (C38 and C191 in human GSDMD). Figure 3 Palmitoylation of GSDMD (A). We also used click chemistry to confirm the effect of HMB on inhibiting palmitoylation modification of GSDMD. Palmitoylated proteins were labeled with Alk-14, followed by pull-down of GSDMD with a specific antibody. Under click chemistry conditions, Alk-14 further bound to a fluorescent group, thereby visualizing palmitoylated GSDMD. To further confirm the inhibitory effect of HMB, we co-treated lipopolysaccharide (LPS)-nigericin (Nig)-induced THP-1 cells with HMB (Shanghai Yuanye Biotechnology Co., Ltd., 4 mM) and the palmitoylation inhibitor 2-bromopalmitic acid (2-BP) (Merck, 50 μM). Both HMB and 2-BP significantly reduced the palmitoylation level of GSDMD (A). Figure 3 B).

[0069] Palmitoylation regulates various cellular processes, such as membrane binding, which may affect the binding of GSDMD to the cell membrane

[40] . Upon cleavage, the N-terminal domain of GSDMD (GSDMD-N) oligomerizes in the plasma membrane (PM) to form pores, increasing membrane permeability and triggering the release of interleukin-1β (IL-1β) and pyroptosis. To investigate the importance of palmitoylation for GSDMD-N function in pyroptosis, we prepared recombinant human GSDMD-N (gene ID: 79792), GSDMD-N-C38A, and GSDMD-N-C191A (mouse GSDMD-N (gene ID: 69146), GSDMD-N-C39A, and GSDMD-N-C192A), in which the key palmitoylation site was mutated to alanine to block palmitoylation. We then tested whether palmitoylation was essential for GSDMD-N-induced pyroptosis. Figure 3 As shown, wild-type GSDMD-N induced greater release of lactate dehydrogenase (LDH) in THP-1 cells and bone marrow-derived macrophages (BMDMs) than GSDMD-N-C38A and GSDMD-N-C191A. Figure 3 (C and D). In membrane binding experiments, wild-type GSDMD-N showed significantly enhanced binding to the plasma membrane compared to the mutant. Figure 3 This indicates that palmitoylation of GSDMD is crucial for its membrane binding during pyroptosis.

[0070] Example 4

[0071] ZDHHC7-mediated inhibition of GSDMD palmitoylation by HMB

[0072] Cysteine ​​palmitoylation (S-palmitoylation) is a reversible post-translational modification catalyzed by palmitoylation enzymes of the DHHC family and can be reversed by acylprotein thioesterases [41, 42]. To investigate the effects of HMB on gene expression in BMDMs, we performed RNA sequencing, which showed that HMB suppressed the mRNA levels of zinc finger DHHC palmitoylation enzymes 2, 7, and 12 (ZDHHC2, ZDHHC7, and ZDHHC12). Figure 4A). When ZDHHC7 was knocked down with small interfering RNA (siRNA) (ZDHHC2 siRNA (Thermo Fisher Scientific, 134988), ZDHHC17 siRNA (Thermo Fisher Scientific, 140245), ZDHHC12 siRNA (Thermo Fisher Scientific, 131467)) instead of ZDHHC2 or ZDHHC12, the inhibitory effect of HMB on pyroptosis was eliminated. Figure 4 (B and C), indicating that HMB regulates the palmitoylation of GSDMD via ZDHHC7.

[0073] Example 5

[0074] The Akt signaling pathway links the regulation of HMB with ZDHHC7.

[0075] To further elucidate the mechanism by which HMB inhibits ZDHHC7, we performed pathway enrichment analysis on differentially expressed genes in BMDM after HMB treatment. Among the enriched pathways, the lysosomal signaling pathway and the PI3K-AKT signaling pathway were found to be associated with cysteine ​​palmitoylation. Figure 4 , D)[43,44]. PI3K-IN-1 (MedChemExpress, 20 μmol / L) is a potent inhibitor of PI3K / Akt signaling[45,46], while MG-132 (Z-Leu-Leu-Leu-al)(abcam, 5 μmol·L -1 ZDHHC7 is a proteasome inhibitor [47,48], and inhibiting the PI3K / Akt signaling pathway reduces the mRNA level of ZDHHC7. Figure 4 Furthermore, we observed increased phosphorylation of AKT in LPS- and Nig-treated BMDM, which was inhibited by HMB, indicating that the PI3K / AKT pathway plays a crucial role in mediating the protective effect of HMB in pyroptosis. Figure 4 F).

[0076] Example 6

[0077] HMB reduces GSDMD-driven inflammatory diseases in vivo

[0078] To confirm the effect of HMB on colitis, age-matched mice were given water containing 2.5% sodium dextran sulfate (DSS) for 5 days, followed by normal water for 7 days, repeated three times for a total of 36 days. During this period, the mice developed persistent symptoms of chronic enteritis. Mice were administered HMB (1.0 g / kg) or saline via gavage. Oral administration of HMB significantly alleviated DSS-induced enteritis, as evidenced by significantly reduced body weight loss, lower Disease Activity Index (DAI) scores, longer colons, and increased mRNA expression levels of pro-inflammatory cytokines in the HMB-treated group. Figure 5 In addition, the protein expression of phosphorylated AKT (p-Akt) and GSDMD in the colon of mice treated with HMB was significantly reduced. Figure 5 These results collectively indicate that HMB has a protective effect against DSS-induced colitis.

[0079] To further investigate the potential therapeutic effects of HMB in other inflammation-related diseases, we established a lung ischemia-reperfusion model in SD rats. Lung ischemia-reperfusion injury was induced by oral administration of 1 mL of HMB solution (4 mM) or saline for 21 consecutive days. Oral administration of HMB significantly alleviated lung ischemia-reperfusion injury, as evidenced by reduced lung pathological damage and a lower injury score. Figure 6 (A&B) and decreased levels of inflammatory cytokines ( Figure 6 Furthermore, HMB treatment reduced GSDMD expression in both membrane and cytoplasmic protein samples. Figure 6 These animal experimental results indicate that HMB not only has a significant protective effect against DSS-induced colitis, but also against pulmonary ischemia-reperfusion injury.

[0080] GSDMD-mediated pore-forming-driven pyroptosis is crucial for immune inflammatory responses, and its dysregulation exacerbates tissue damage in diseases such as inflammatory bowel disease (IBD) and ischemia-reperfusion injury [11,12]. While pharmacological inhibition of GSDMD has emerged as a promising therapeutic strategy, developing drugs that can inhibit GSDMD-driven pyroptosis while preserving its protective effects in epithelial cells remains a key challenge. Here, we find that HMB, a leucine-derived metabolite with proven safety in clinical populations

[22] , is a novel GSDMD inhibitor that selectively targets pyroptosis and protects epithelial integrity. Our findings fill a critical gap in the development of safe and cell-type compatible therapies for epithelial-associated inflammatory diseases.

[0081] While post-translational modifications such as phosphorylation and ubiquitination have been well studied in the regulation of pyroptosis

[49] , the role of S-palmitoylation (a reversible post-translational modification discovered decades ago that is now known to affect nearly 3,000 human proteins) remains largely unexplored

[50] . We demonstrate that HMB inhibits palmitoylation of GSDMD cysteine ​​residues by downregulating the palmitotransferase ZDHHC7, which is regulated by AKT signaling. This mechanism differs from existing GSDMD inhibitors in that it modulates enzymatic modification rather than a direct GSDMD-cysteine ​​interaction. Importantly, HMB inhibition of ZDHHC7 specifically disrupts GSDMD membrane localization and pore formation while preserving other epithelial protective pathways, which may explain its protective effect in vivo.

[0082] For many years, β-hydroxy-β-methylbutyric acid (HMB) has been safely used to enhance muscle anabolism in athletes and cachectic patients

[21] , laying a solid foundation for its clinical translation. In muscle tissue, HMB supplementation is known to increase muscle fiber size and reduce inflammatory signaling

[24] , consistent with our findings on the protective effect of HMB against inflammation

[51]

[52] . This dual ability to both improve tissue damage and suppress inflammation makes HMB a viable candidate for clinical repurposing in immune epithelial inflammatory diseases. Our study extends the therapeutic use of HMB to inflammatory diseases by leveraging its inherent biocompatibility. In a dextran sulfate sodium (DSS)-induced colitis and pulmonary ischemia-reperfusion model, HMB significantly reduced pathological damage, demonstrating its potential as an effective treatment for epithelial inflammatory diseases. However, drug research related to pyroptosis is still in its early stages, and precise therapeutics that can modify proteins or even amino acid residues will be the main direction of future research. As a metabolite-based inhibitor, HMB has a unique advantage in this field, and our study on palmitoylation modification of GSDMD cysteine ​​residues will provide a theoretical basis for such research.

[0083] Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

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Claims

1. The use of β-hydroxy-β-methylbutyric acid or a pharmaceutically acceptable salt thereof as an inhibitor of Gasdermin D (GSDMD) in the preparation of an anti-pyroptosis drug, wherein the drug is used to treat or prevent ischemia-reperfusion injury.

2. The application according to claim 1, wherein the medicinal salt is selected from alkali metal salts and alkaline earth metal salts.

3. The application according to claim 1, wherein the medicinal salt is a calcium salt or a sodium salt.

4. The application according to claim 1, wherein β-hydroxy-β-methylbutyric acid or a pharmaceutically acceptable salt thereof inhibits GSDMD-induced pyroptosis by blocking palmitoylation of GSDMD cysteine ​​residues.

5. The application according to any one of claims 1 to 4, wherein the drug further comprises a pharmaceutical excipient.

Images