Positive allosteric modulators (PAMs) of cystathionine gamma lyase (CSE) increase hydrogen sulfide (H2S) and inhibit NLRP3 activation
Driss Ehirchiou (1), Twinu Chirayath (2), Sonia Nasi (2), Veronique Chobaz (2), Rainer Riedl (3), Cindy Blatter (3), Stefan Germann (3), Alexandra Brandenberger (3), Nathalie Busso (2, Alexander So (1)
Affiliation(s):
1. Sulfiscon Sa, Epalinges, Switzerland
2. Lausanne University Hospital, Lausanne, Switzerland
3. Zurich University Of Applied Sciences, Wädenswil, Switzerland
Background: Hydrogen sulfide (H2S) is a gasotransmitter involved in various pathophysiological processes, including inflammation. Physiological and pharmacological levels of H2S can be reached both in vitro and in vivo using H2S donors. H2S is produced in cells by three enzymes, one of which is cystathionine gamma lyase (CSE). The NLRP3 inflammasome is a multiprotein complex that can be activated by monosodium urate (MSU) crystals, the etiological factor in gout, as well as by calcium pyrophosphate (CPP) crystals, which are implicated in calcium pyrophosphate deposition disease (CPPD). When the inflammasome is activated, ASC oligomerizes, leading to the activation of caspase-1, which cleaves pro-IL-1β into its active, secreted form. We previously demonstrated that both exogenous and endogenous H2S inhibit NLRP3 inflammasome activation in macrophages in vitro and in murine peritoneal fluids in vivo [1].
Objectives: To test whether positive allosteric activators of CSE (CSE-PAMs) could inhibit NLRP3 inflammasome activation by MSU and CPP crystals.
Methods: A CSE-PAM (SAN523) was identified and tested for its ability to enhance H2S production by recombinant or cellular CSE. H2S production was measured using the H2S-specific fluorescent probe AzMc or the lead acetate method. Supernatants from primed murine bone marrow-derived macrophages (BMDMs) and human macrophage-like THP1 cells, stimulated with 500 µg/ml MSU or CPP crystals for 6h, in the presence or absence of SAN523 (0.1-50 µM), were analyzed by IL-1β Western blot (WB) and ELISA. In vivo, peritonitis was induced in mice by intraperitoneal (i.p.) injection of 1 mg MSU or CPP crystals, with SAN523 treatment (5 mg/kg) or vehicle (DMSO) administered intravenously (i.v.) 15 minutes prior to crystal injection. THP1 cells, stimulated with 500 µg/ml MSU or CPP crystals for 1h, in the presence or absence of SAN523 (50 µM), were analyzed by the maleimide assay for detection of protein persulfidation.
Results: SAN523 stimulated H2S production by human recombinant CSE and THP1 cells in a dose-dependent manner. Additionally, in primed THP1 macrophages stimulated with MSU or CPP crystals, SAN523 reduced ASC oligomerization (% ASC speck-positive cells over total cells: MSU = 11.5; MSU+SAN523 = 3.5, p = 0.037; CPP = 36.7; CPP+SAN523 = 14.7, p = 0.0009;) and inhibited IL-1β secretion in a dose-dependent manner (IC50 for MSU = 1µM, Figure 1A). A similar decrease in IL-1β secretion was observed in primed BMDMs. Inhibition of IL-1β secretion by SAN523 in both human and murine cell supernatants was confirmed by WB.
In the MSU- and CPP-induced peritonitis models, pretreatment with SAN523 significantly reduced inflammatory cell recruitment (millions of cells: MSU = 5.3; MSU+SAN523 = 3.7, p = 0.012, Figure 1B; CPP = 5.7; CPP+SAN523 = 0.2, p < 0.0001) and IL-1β levels in peritoneal fluid (IL-1β in pg/ml: MSU = 30.27; MSU+SAN523 = 2.27, p = 0.009, Figure 1C; CPP = 86.14; CPP+SAN523 = 34.37, p = 0.02), along with reductions in other inflammatory cytokines and chemokines such as IL-6, IL-1α, and Cxcl1. Finally, we identified that SAN523 acts, at least partly, via induction of protein persulfidation (Figure 1).
Conclusion: SAN523 enhances H2S production, leading to reduced NLRP3 inflammasome activation in vitro and in the MSU- and CPP-induced peritonitis models in vivo. This new compound may be of interest as a novel drug for acute gout, CPPD, and other IL-1β-mediated inflammatory diseases.
References:
[1] M. Castelblanco, J. Lugrin, D. Ehirchiou, S. Nasi, I. Ishii, A. So, F. Martinon, N. Busso, Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation, J Biol Chem 293(7) (2018) 2546-2557.
