Chemical Modulation of Gasdermin-Mediated Pyroptosis and Therapeutic Potential

Abstract

Pyroptosis, a lytic form of programmed cell death, both stimulates effective immune responses and causes tissue damage. Gasdermin (GSDM) proteins are a family of pore-forming executors of pyroptosis. While the most-studied member, GSDMD, exerts critical functions in inflammasome biology, emerging evidence demonstrates potential broad relevance for GSDM-mediated pyroptosis across diverse pathologies. In this review, we describe GSDM biology, outline conditions where inflammasomes and GSDM-mediated pyroptosis represent rational therapeutic targets, and delineate strategies to manipulate these central immunologic processes for the treatment of human disease.

Keywords: anti-tumor immunity; immunogenic cell death; inflammasome; inflammasomopathy; pore-forming protein.

Figure 1:. Schematic overview of critical residues and targetable sites of the gasdermins.

GSDMA-E contain an NT pore forming domain (light blue) and a CT inhibitory domain (dark blue). PJVK contains a putative pore forming domain (light blue) and a truncated CT domain (light purple). Activating (green) and inhibitory (black) cleavage sites are indicated by scissors. Residues annotated in black indicate those with known function. Residues annotated in purple italics indicate those with putative function due to conservation with other GSDM family members. Residues in red indicate sites modified by current GSDMD targeting drugs. Human GSDMD is activated by caspases 1,4,5,8 at D275 and by neutrophil elastase (ELANE) at C268 [,,,,–54]. GSDMD is inactivated by caspase-3 at D87 and by enterovirus 71 protease 3C at Q193 [113,121]. Among the other gasdermin family members, the D87 residue is conserved in GSDMB (D91), GSDMC (D85), and PJVK (D85), although proteolytic cleavage has not been shown at this site for GSDMC or PJVK. Important residues for GSDMD pore formation include C191 (oligomerization) and F240 (dimerization) [–45]. The F240 residue is conserved in all GSDM family members, whereas C191 is not. Mutation of I104N results in a hypomorphic gasdermin D allele, and this site is conserved in GSDMB [49]. Four residues in GSDMD^CT (L304, V308, V364, L367) are responsible for GSDMD-inflammatory caspase exosite interactions [50,51]. GSDMA and GSDME harbor an inhibitory phosphorylation site (T8, T6, respectively) [17]. A conserved residue is found in GSDMB, GSDMC and PJVK. There are no described proteases for GSDMA or PJVK. GSDMB is cleaved at K229 and K244 by granzyme A; some splice variants may undergo caspase-1 cleavage at D236 [28,32]. GSDMC is cleaved at D365 by caspase-8 [35]. GSDME is cleaved at D270 by caspase-3 or granzyme B [62,63,119,120]. Direct GSDMD inhibitors all modify C191 [44,45,57]. DMF additionally succinates C56, C268, C309, and C467. DMF additionally modifies GSDME on C45, C156, C168, C180, C235, C371, C408, and C489. See text for further details. Created with BioRender.com.

Figure 2:. Understanding GSDMD as a bottleneck for inflammasome-driven effects.

Inflammasome-mediated GSDMD activation and the resulting downstream effects are divided into four general steps: (I) inflammasome activation, (II) GSDMD cleavage, (III) GSDMD oligomerization and membrane insertion and (IV) downstream processes/outcomes. (I) Numerous inflammasome activating stimuli are sensed by either a canonical or non-canonical inflammasome sensor protein. Depicted triggers for various canonical inflammasomes include bacterial components (type III secretion system, flagellin), dsRNA, viral proteases, and other stimuli that facilitate proteasomal activation of NLRP1 and CARD8. Depicted stimuli for NLRP3 canonical inflammasome activation include aspergillus, toxoplasma, monosodium urate (and other) crystals, amyloid β, ATP, heme released from lysed red blood cells, DNA:RNA hybrids, and K+ efflux. The non-canonical inflammasome is activated by LPS from gram-negative bacteria. (II) GSDMD^FL (full-length) can be cleaved by multiple caspases and other proteases to release the GSDMD^NT fragment. (III) The liberated GSDMD^NT associates with membranes, oligomerizes, and inserts into the membrane as a higher-order multimeric pore (~27mer) upon conformational change. (IV) Assembled GSDMD pores cause both cell intrinsic and extrinsic effects. Cell intrinsic effects include ion fluxes (both efflux and influx), NETosis, and pyroptosis. Some DAMPs additionally produce cell intrinsic effects. Other DAMPs and factors released via GSDMD pores or following GSDMD-mediated NETosis or pyroptosis cause cell extrinsic effects, including immune cell activation, clotting, and tissue repair or damage. To the right are agents that function at each step. Of note, GSDMD can be activated outside of the context of inflammasomes and a variety of stimuli trigger cleavage of other GSDMs to produce similar downstream effects (see text for details). PLC = phospholipase C. Created with BioRender.com.