Rescue from a fiery death: A therapeutic endeavor

Cells die in many ways. Apoptosis is an immunologically silent cell death program that is important for embryonic development. By contrast, pyroptosis is a proinflammatory mode of cell death that is triggered by diverse infectious and sterile insults. Many cell types are pyroptosis-competent, including hematopoietic, epithelial, and endothelial cells. Inhibiting pyroptosis ameliorates disease, including septic shock and autoinflammation, in multiple preclinical mouse models. Thus, attenuating aberrant or excessive pyroptosis may be clinically beneficial, despite a role of pyroptosis in pathogen defense. Pyroptosis is driven by the pore-forming fragment of the gasdermin D (GSDMD) protein, which is proteolytically generated by the cysteine protease caspases 1, 4, 5, and 11 in response to activation of the inflammasome (a multiprotein complex that detects infectious and sterile insults). Recent clinical findings emphasize the critical role of pyroptosis in human inflammatory disease and cancer, and this has galvanized focus on GSDMD as a therapeutic target.

The term pyroptosis was coined in 2001 (1) from the Greek word “pyro,” meaning fire (or fever), to describe the caspase-1–dependent proinflammatory programmed cell death of bacteria-infected macrophages. Subsequently, pyroptosis was invoked whenever cell death was prevented by caspase-1 deficiency, whereas apoptosis and necrosis (accidental cell death) were defined by their morphological characteristics. Pyroptotic cells can exhibit features of both necrosis (plasma membrane rupture) and apoptosis (DNA damage). Damage to the plasma membrane during pyroptosis is regarded as a combustive event because it releases powerful mediators of inflammation, including interleukin-1β (IL-1β), a potent endogenous pyrogen that propagates an inflammatory immune response in neighboring cells. Indeed, caspase-1 was initially named IL-1β–converting enzyme because of its crucial role in cleaving latent pro–IL-1β into active IL-1β.

In 2015, GSDMD was identified as the dominant mediator of pyroptotic cell rupture and IL-1β release (2, 3). Inflammasome-activated caspases 1, 4, 5, and 11 cleave GSDMD to generate an exceptionally potent pore-forming fragment that rapidly induces membrane rupture (2–5) (see the figure). Hence, the original definition of pyroptosis as caspase-1–dependent cell death (1) has evolved because GSDMD can be cleaved by caspases 4, 5, and 11 independently of caspase-1 (2, 3). One option is to redefine pyroptosis as gasdermin-dependent cell death (2), although this fails to incorporate apoptotic-like features, which are prominent in certain cells and arise from caspase-1 activation of the executioners of apoptosis, caspases 3 and 7.

Caspase-1 and caspase-4 (caspase-11 in mice) are activated within canonical and noncanonical inflammasomes, respectively. Canonical inflammasome complexes are nucleated by intracellular sensors such as NLRP3 (nucleotide-binding domain leucine-rich repeat–containing protein 3) that respond to diverse infectious or sterile insults. Many sterile inflammatory diseases (that develop without pathogens), including gout and atherosclerosis, have been linked to NLRP3 activation. Indeed, there are already numerous reports of compounds targeting NLRP3. For example, the sulfonylurea MCC950 inhibits NLRP3 directly by engaging its nucleotide-binding domain. Although it is a useful tool compound, clinical use of MCC950 is hampered by off-target liver toxicity. The quest for more specific inhibitors will be facilitated by the recently elucidated high-resolution structure of NLRP3 (6). The noncanonical inflammasome that activates caspases 4, 5, and 11 is triggered by cytoplasmic lipopolysaccharide (LPS, also known as endotoxin) derived from Gram-negative bacteria. Notably, deficiency of the Gsdmd gene in mice largely abrogates cell rupture and IL-1β release in response to both canonical and noncanonical inflammasome activation (2, 3).

GSDMD primarily exists as an inactive precursor in the cytosol. A conserved amino-terminal pore-forming domain (PFD) and a self-inhibitory carboxyl-terminal domain (CTD) are separated by a flexible linker region. Crystal structures of full-length human or mouse GSDMD or mouse GSDMA3 (a family member and ortholog of human GSDMA) indicate that the globular CTD binds to the PFD through two interfaces and keeps the PFD in check (4). These features—intrinsic pore-forming activity within the PFD and autoinhibition by the CTD—appear to be shared by other gasdermin family members (GSDMA, GSDMB, GSDMC, GSDMD, and GSDME), with DFNB59 being the exception. Little is known about the physiological roles of GSDMA, -B, and -C, but GSDME can undergo caspase-3–mediated proteolytic activation and rupture the plasma membrane in response to chemotherapy drugs.

GSDMD is cleaved by caspases 1, 4, 5, and 11 at a specific residue within the linker region (Asp²⁷⁵ in humans) (2, 3). Liberated from the GSDMD-CTD, the GSDMD-PFD directly binds to negatively charged lipids in the plasma membrane (4, 5). A structure of the GSDMA3 pore in synthetic liposomes (7) indicates that cleaved GSDMA3-PFD undergoes a dramatic conformational change and forms a 27-fold oligomeric pore with distinct membrane-spanning β strands. The symmetrical ring has an inner diameter of ∼18 nm, large enough to permit passive and nonselective ion flow that would collapse the intracellular electrochemical gradient and promote plasma membrane rupture. Precisely when cells die during pyroptosis is unclear. Although cell rupture is a hallmark of pyroptosis, it is conceivable that cell death (as measured by loss of mitochondrial membrane potential) precedes cell rupture. Whether cleaved GSDMD forms pores in cells that are similar in size to GSDMA3 pores observed in synthetic liposomes remains to be confirmed.

The inflammatory cytokines pro–IL-1β and pro–IL-18 must be proteolytically activated by caspase-1 and released into the extracellular milieu to signal the alarm to neighboring cells. In the noncanonical inflammasome pathway, this is achieved by caspases 4, 5, and 11 initially generating GSDMD-PFD. Membrane perturbation then activates the NLRP3 inflammasome and caspase-1 (3). In theory, the 18-nm inner diameter of the gasdermin pore (7) is large enough for passive release of IL-1β (4.5-nm diameter) as well as other intracellular proinflammatory molecules [collectively called damage-associated molecular patterns (DAMPs)], including adenosine triphosphate (ATP), IL-1α, and IL-18. Subsequent cell rupture releases larger molecules such as lactic acid dehydrogenase (a commonly used marker of cell rupture), DNA-bound histones, and possibly even organelles. Following pyroptosis, intracellular pathogens are acutely exposed to a sterilizing environment composed of the complement pathway and neutrophils. Additionally, pathogens trapped inside pyroptotic corpses can be engulfed by phagocytes, thereby limiting infection. Accordingly, mice lacking the genes encoding caspase-1 (Casp1), caspase-11 (Casp11), or both, as well as mice in which the Gsdmd gene is ablated, are unable to efficiently clear intracellular bacteria, including Francisella novicida and Salmonella Typhimurium (8).

The plasma membrane may not be the sole target of GSDMD. The GSDMD-PFD also binds to cardiolipin in vitro (4, 5), but, in cells, this phospholipid is found almost exclusively within the inner mitochondrial membrane and may not be accessible. Therefore, whether GSDMD-induced mitochondrial damage contributes to cellular demise is presently unknown. In neutrophils, GSDMD mediates the coordinated rupture of nuclear and plasma membranes (9), promoting the release of weblike chromatin structures called neutrophil extracellular traps (NETs). This process, called NETosis, enhances host defense by capturing extracellular pathogens and limiting microbial dissemination. However, when NETosis is excessive, as in sepsis, it can contribute to organ failure owing to endothelial damage and subsequent capillary blockage. Cultured neutrophils that lack Casp11 or Gsdmd are protected from NETosis in response to LPS and Gram-negative bacteria (9, 10), underscoring the potential benefit of GSDMD inhibitors in treating sepsis and other NETosis-driven diseases.

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Caspases 1, 4, 5, and 11 are not the only proteases that are able to cleave GSDMD. In macrophages infected with strains of the Yersinia bacteria, caspase-8, an initiator of apoptosis, cleaves GSDMD at the same site as caspase-1 to induce cell rupture (11). It remains uncertain, however, if this membrane rupture is a primary determinant of cell death or a secondary event following apoptosis. The serine protease neutrophil elastase can cleave GSDMD at distinct residues within the linker to generate a cytolytic GSDMD-PFD, although the physiological importance of this neutrophil elastase–mediated GSDMD cleavage remains ambiguous.

GSDMD pore formation may not be the point of no return. For example, GSDMD releases IL-1β without causing macrophage death when exposed to particular host-derived oxidized lipids (12). The IL-1β–releasing GSDMD pore may be patched rapidly by machinery such as the ESCRT (endosomal sorting complex required for transport) system, which can repair punctured plasma membranes (13).

Pyroptosis contributes to pathology in numerous mouse models of inflammatory disease. For example, loss of Casp11 or Gsdmd protects mice from LPS-induced septic shock (3). IL-1β blocking therapies, such as the U.S. Food and Drug Administration–approved IL-1β monoclonal antibody canakinumab, have proven successful in treating periodic fever syndromes, including familial cryopyrin-associated periodic syndromes, which arise from activating mutations in the NLRP3 gene. Additionally, canakinumab treatment significantly reduced cardiovascular events in a large phase 3 study of more than 10,000 patients with a prior history of myocardial infarction. This confirms a role for sterile inflammation, and IL-1β in particular, in atherosclerotic heart disease. Unexpectedly, canakinumab treatment correlated with an extraordinary reduction in lung cancer–associated mortality (14). This suggests that targeting IL-1β may be beneficial in treating certain human cancers. Currently, however, it is unclear if IL-1β contributes to tumor initiation, progression, or both. Therefore, clinical trials with cancer patients are required.

Given the major role of GSDMD in IL-1β release and pyroptosis (2, 3), targeting GSDMD with a small-molecule therapeutic is especially attractive. Such inhibition should have more potent anti-inflammatory properties than targeting IL-1β because GSDMD induces pyroptosis and releases proinflammatory DAMPs besides IL-1β. Currently, there are several small molecules reported to inhibit the function of GSDMD. Necrosulfonamide (15), an inhibitor of human mixed-lineage kinase domain–like protein (MLKL), the pseudokinase required for necroptosis (a form of programmed necrosis), covalently modifies a critical residue (Cys¹⁹¹) in human GSDMD. Another GSDMD inhibitor is a pyrazolo benzoxazepine compound (10) that can bind to both uncleaved and cleaved GSDMD. Although the therapeutic potential of these GSDMD inhibitors is unknown, preclinical data provide proof of concept for GSDMD as a druggable target. The contribution of GSDMD to human pathology remains an open question. This can only be addressed by testing GSDMD inhibitors in relevant indications in the clinic. The future of pyroptosis biology holds much promise, as it may provide a new dimension to the treatment of inflammatory pathologies, including some cancers.