Next Phase Newsletter

Juan Carlos Lopez

Andy Marshall

It is now nearly a decade since Dublin-based startup ​Inflazome burst onto the scene with the description of MCC950, the first nanomolar selective inhibitor of the inflammasome. Inflammasome-mediated low-grade inflammation has been associated with cancers, numerous chronic complex diseases—including inflammatory bowel disease, arthritis, metabolic dysfunction-associated steatohepatitis (MASH), atherosclerosis, Alzheimer’s, Parkinson’s and ALS—as well as rare autoinflammatory diseases, such as cryopyrin-associated periodic syndromes (CAPS). There is a wealth of compelling human genetic evidence from Muckle-Wells syndrome and other autosomal dominant familial genetic diseases showing gain-of-function mutations in NLRP3 are causative of autoinflammatory disease.

Given the ‘pipeline in a product’ potential of drugs targeting this pathway, big pharma has shown considerable interest, with Genentech/Roche snapping up Jecure Therapeutics for an undisclosed amount, and both Novartis and Roche splashing out hundreds of millions of dollars for pioneer companies IFM Tre and Inflazome, respectively. In 2022, Novo Nordisk licensed Ventus Therapeutics’ peripherally restricted NLRP3 inhibitor in a deal worth up to $703 million, lending weight to pharmacological inhibition of NLRP3 as a complement to glucagon-like peptide-1 agonists (GLP-1s) in cardiometabolic disease. And with several programs now entering the clinic, investment activity in the area has continued, with Enveda’s announcement last week of a $150 million series D round to fund a phase 1 trial for ENV-6946, an orally delivered gut-restricted small molecule targeting the NLRP3/tumor necrosis factor-like cytokine 1A (TL1A) pathway in inflammatory bowel disease.

Today, Haystack counts at least 17 independent companies pursuing inflammasome therapeutics (AC Immune, Azome Therapeutics, BioAge Labs, Cardiol Therapeutics, EpicentRx, Enveda, Halia Therapeutics, InflammX Therapeutics, Insilico Medicine, Neumora Therapeutics, NodThera, Olatec Therapeutics, Shaperon, Ventus Therapeutics, Ventyx Biosciences, ZyVersa Therapeutics and Zydus LifeSciences) and 8 programs now in clinical testing specifically targeting the key inflammasome component NLRP3.

While drugmakers have traditionally targeted downstream extracellular mediators of the inflammasome pathway (canakinumab or rilonacept against IL-1β or anakinra to block IL-1 receptor), NLRP3 represents a key upstream intracellular signaling hub, activated by innate immune pattern-recognition receptor (Toll like receptors 2/4) signaling via MyD88 and NFkappaB. Once activated, NLRP3 monomers unfold and associate into a massive 1.2 MDa oligomeric supracomplex with three other proteins: ASC, NEK7 and caspase 1. The mature complex then cleaves and activates proinflammatory cytokines interleukin (IL)-1β and IL-18 and primes gasdermin D to instigate cell pore formation and cell death via pyroptosis.

Discovering effective drugs against NLRP3 has proven challenging. The first NMR structure was obtained in 2016, but the structural basis for how NLRP3 ring-like oligomers associate with intracellular membranes and how its pyrin domains associate with ASC to orchestrate speck formation and caspase activation have only recently been elucidated. Thus far, the majority of small-molecule inhibitors (e.g., Inflazyme’s archetypal MCC950 and inzolemid; Zydus’s (ZYIL1), Olatec’s OLT117 and Jecure Therapeutics’ GDC-2394) form hydrogen bonds via a sulfonylurea group to NLRP3’s NACHT domain nucleotide-binding motifs, thereby obstructing ATP hydrolysis. Other companies are taking a different tack: thus, Halia Therapeutic’s small-molecule inhibitor orniflast and Monte Rosa Therapeutics’ MRT-8102 molecular glue target NEK7 rather than NLRP3.

But it has been less than straightforward to identify compounds with sufficient potency to target this pivotal innate immune signaling pathway without debilitating off-target effects. Indeed, several of the first wave of compounds entering the clinic have been dogged by serious toxicities, including liver problems (MCC950 and GDC-2394) and hypoglycemia (glyburide). Now, a team led by Rebecca Coll (Queen’s University Belfast) and Kevin Wilhelmsen (of BioAge Labs) reports in The Journal of Experimental Medicine the discovery and characterization of BAL-0028, a novel and selective small-molecule inhibitor of the human NLRP3 inflammasome.

Unlike previously studied inhibitors, BAL-0028 acts through a unique mechanism of action; it binds NLRP3’s NACHT domain at a site distinct from other inhibitors that act by directly interfering with ATPase activity. BAL-0028 has nanomolar potency against human and primate NLRP3 but, remarkably, has weak activity against the mouse target, highlighting species-specific differences.

As BAL-0028 showed very high plasma protein binding in mice, limiting its use in vivo, the team developed a derivative, BAL-0598, with improved pharmacokinetic properties. In a humanized NLRP3 mouse peritonitis model, BAL-0598 effectively reduced IL-1β and IL-6 production, confirming its anti-inflammatory activity in vivo. Importantly, both BAL-0028 and BAL-0598 inhibited hyperactive NLRP3 mutants associated with autoinflammatory diseases, in some cases more effectively than Vertex’s VX-765, a caspase 1 inhibitor, and compounds like MCC950, one of the best characterized NLRP3 inhibitors available.

The novel mechanism of action of BAL-0028 and BAL-0598 would suggest their off-target effects may be different from those associated with other NLRP3 inhibitors blocking ATP hydrolysis. The concern that such compounds might also bind other members of the NOD/NLR family (e.g., NLRP1, NLRP4 or AIM2 inflammasomes) is mitigated by most published studies indicating that NLRP3’s unique fold around the ATP binding site makes small-molecule binders selective for this family member alone. The most likely explanation from trials published to date is that the observed toxicities are associated with small molecule chemotype rather than any NLRP3 class-specific problem. In any case, the findings from this study support further investigation of these compounds as candidates for treating inflammatory and age-related diseases where NLRP3 plays a role. The race to develop a safe and effective NLRP3 inhibitor is on, with big pharma billion-dollar bets and startups jostling to create best-in-class assets across cancer, cardiovascular, neurodegenerative and metabolic disease.

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