Credit: C&EN/Protein Data Bank, PDB ID 3JBL (NLRP3); Protein Data Bank, PDB ID 6CB8 (gasdermin-D) | Cryogenic electron microscopy models of inflammasome rings (center, ring with hooks) and gasdermin pores (bottom right)
There are some things we are better off without. Our lungs would be happier if never exposed to cigarette smoke. Our livers would be healthier if never stuffed with fat. Our arteries would be content without being clogged with cholesterol. And, some scientists argue, our brains would stay sharper without the amyloid-β plaques and tau tangles that accumulate in old age. All these things, and more, have one thing in common: they incite inflammation in our bodies. And they do so by activating a hypersensitive protein called NLRP3.
A protein called NLRP3 has been linked to a suspiciously long list of conditions, including atherosclerosis, Alzheimer’s disease, inflammatory bowel disease, and nonalcoholic steatohepatitis. It’s part of a large protein complex called the inflammasome that, when activated, triggers a chain of events that causes cells to erupt and spill an inflammation-inducing soup. Biotech start-ups and pharma companies are now eagerly developing NLRP3 inhibitors that they think could address a wide swath of common diseases. But drug hunters face a daunting task: many features of the inflammasome, including its exact structure, remain a mystery.
For that reason, Kate Schroder sometimes wonders if we’d be better off without NLRP3.
Schroder, one of the world’s leading experts in inflammasome biology, has studied NLRP3 for over a decade, and today she directs a center at the University of Queensland dedicated to inflammasomes. NLRP3 is the most deeply researched, probably the most poorly understood, and undoubtedly the most perplexing member in a family of protein complexes called inflammasomes. These complexes are found in our immune cells, where they instigate inflammation—lots of it. The NLRP3 inflammasome in particular is a culprit in a suspiciously long list of diseases.
About 8 years ago, Schroder got roped into helping Matthew Cooper, a chemist at her university, and Luke O’Neill, an inflammation biologist from Trinity College Dublin, test hundreds of molecules designed to inhibit NLRP3. The output of that work was a high-profile paper describing how a molecule dubbed MCC950 was a selective NLRP3 inhibitor. It wasn’t the first compound to block the protein complex, but it was by far the best.
In 2016, Cooper moved to Ireland to start a company called Inflazome with O’Neill. Their goal was to turn compounds inspired by MCC950 into bona fide NLRP3-blocking drugs. Today, as CEO, Cooper doesn’t skip a beat when selling the potential. “I thought this was going to be a transformational medicine,” he says with complete conviction. “I thought this could cure, or at least abate, Parkinson’s, Alzheimer’s, cancer, ALS, depression, cardiovascular disease, arthritis, chronic kidney disease, and asthma. Those things don’t happen very often in life. And I didn’t want anybody else to screw it up.”
The discovery of MCC950 triggered an NLRP3 gold rush. Inflazome is one of about a dozen companies with programs dedicated to stopping NLRP3 or related inflammasome proteins. Three NLRP3 start-ups have already been acquired by big pharma firms. And by Cooper’s count, MCC950 has been tested in 80 animal models of more than 50 diseases.
Stephen Glover, CEO of ZyVersa Therapeutics, repeats an old drug industry adage for a single therapy that can potentially do so much: “It would be a pipeline within a product.”
If it sounds too good to be true, it might be. Although study after study suggests that the NLRP3 inflammasome hurts more than it helps, those data are all in animals, not humans. And finding drugs to selectively stop NLRP3 will be difficult. Scientists say that they don’t even have a detailed image of the protein—information that typically provides a launching point for designing molecules that disrupt its activity. And NLRP3 is implicated in so many diseases that drug companies are flummoxed with where to begin.
H. Martin Seidel, CEO of IFM Therapeutics and former head of business development at the Novartis Institutes for BioMedical Research, says NLRP3 inhibitors have the potential to become the new statins—the pills taken daily by 40 million people in the US to lower cholesterol. But in addition to heart disease, NLRP3 inhibitors might help ward off Alzheimer’s disease, cancer, fatty liver diseases, and a constellation of other common ailments linked to chronic inflammation.
“We’ve joked here that we are all going to be taking one of these at some point,” Seidel says. “And we’ve thought long and hard about this, but there is no other target that readily comes to mind that would have the potential breadth that NLRP3 inhibitors would have.”
When Fabio Martinon discovered the inflammasome as a graduate student around 2001, he had no idea how important it would turn out to be. In fact, he argued with his mentor, University of Lausanne’s Jürg Tschopp, against calling it the inflammasome—a name that suggested it played a central role in the immune system. But they didn’t know that yet, because Martinon had studied it only in a test tube.
Martinon showed that three different proteins clump together into a large complex and activate a cytokine called IL-1β. Well known to immunologists, IL-1β promotes inflammation and was the target of several drug programs in the early 2000s. Tschopp named this IL-1β-activating complex the inflammasome, even though they had little evidence it actually existed in humans. The lab had a hard time getting the paper published.
Over the next decade, Tschopp and others would describe the general features of inflammasomes, which are security systems that prompt cells to self-destruct. All inflammasomes have an adapter protein called ASC and an enzyme called caspase-1, but the third component is a rotating cast of sensor proteins, which detect viral or bacterial intruders. Among the sensor proteins, NLRP3 is unusual because it detects what scientists call sterile danger signals—toxins that build up in our bodies from bad diets or old age. That’s why Schroder calls NLRP3 “the grumpy old man” of inflammation.
When a sensor protein is triggered, it kick-starts a complex chain of events that causes the cell to explode. The cell’s spilled guts include IL-1β, which incites the immune system, and a tangled mass of inflammasomes, which other immune cells can engulf, prompting the self-destruction sequence to start all over again.
It’s easy to imagine how this process can quickly run out of control—and why stopping it might treat so many diseases.
In the late 1990s, Chris Gabel was unwittingly trying to do just that. Gabel was a scientist at Pfizer looking for compounds that stopped immune cells from secreting IL-1β. He discovered that glyburide, a diabetes drug, blocked IL-1β release, but only at high doses. Drug companies hoped that IL-1β blockers would become big-selling drugs for conditions like rheumatoid arthritis, and Pfizer mounted a 3-year medicinal chemistry campaign that used glyburide as a starting point to find more-potent IL-1β inhibitors.
The result of that work was a series of diarylsulfonylurea compounds dubbed cytokine release inhibitory drugs (CRIDs). Pfizer tested one called CRID3 in a small clinical trial, but it had a shorter half-life and weaker potency than expected. Worse, when given in higher doses, CRID3 caused liver damage. With no clue how the molecule worked, “they put the concept on the shelf and shuttered the program,” Gabel says. “That was a very sad day in my life.”
The CRID3 study was largely forgotten until 2007, when Mohamed Lamkanfi, a postdoc at Genentech, came upon it while looking for compounds to inhibit IL-1β release. The year before, Martinon and Tschopp had shown that inflammation in gout is caused by monosodium urate and calcium pyrophosphate dihydratecrystals that activate the NLRP3 inflammasome. And in 2008, several studies indicated that asbestos, silica, and aluminum salt crystals trigger the NLRP3 inflammasome and subsequent lung fibrosis.
NLRP3 was starting to look like a promising drug target. “But at that time people were skeptical whether molecules like NLRP3 could be targeted at all,” Lamkanfi recalls. In 2009, the Genentech team showed that glyburide blocked IL-1β release because it was an NLRP3 inhibitor, albeit a weak one.
Around that time, Trinity’s O’Neill, a longtime IL-1β expert, was starting to develop an interest in the inflammasome. Together with Cooper, he followed up on Genentech’s glyburide study by testing CRID3’s ability to inhibit NLRP3. By 2012, his team found that CRID3, which was renamed MCC950, for “Matt Cooper compound 950,” was a highly specific NLRP3 inhibitor with nanomolar potency.
But MCC950 clearly had liabilities. Cooper and O’Neill discussed whether they should launch an effort to make better, more potent, and safer NLRP3 inhibitors, using MCC950 as a starting point. “Oh, we’ll never beat Pfizer,” O’Neill told his collaborator. “They must have loads of compounds in their warehouse.”
“Of course we can beat Pfizer,” Cooper retorted. “Let’s take it on.”
Designing selective NLRP3 inhibitors turned out to be hard work. For starters, scientists can’t find the protein’s natural ligand and at this point are doubtful that one even exists. The shrimp-shaped NLRP3 lacks an obvious binding pocket for a drug to latch on to, and worst of all, no one has published a full structure of the NLRP3 inflammasome. In the lab, the proteins clump together and form ginormous complexes, making it nearly impossible for scientists to snap a clean picture of NLRP3’s structure.
While NLRP3 is a hot target for drug designers, companies are taking a wide range of strategies to tackle the inflammasome.
Olatec Therapeutics: Small-molecule NLRP3 inhibitors
Bristol-Myers Squibb: NLRP3 activators for cancer immunotherapy acquired from IFM Therapeutics
Inflazome: Small-molecule NLRP3 inhibitors
Novartis: Small-molecule NLRP3 inhibitors acquired from IFM Therapeutics and developed in-house
Ardan ImmunoPharma: Small-molecule activators and inhibitors of the TMEM176B ion channel, which is an inhibitor of the inflammasome
Genentech: NLRP3 inhibitors acquired from Jecure Therapeutics
IFM Therapeutics: Small-molecule inhibitors of the NLRP1, NLRP6, NLRP10, and NLRC4 inflammasomes; small-molecule activators of the AIM2 inflammasome for cancer immunotherapy
Inflammasome Therapeutics: Small-molecule inflammasome inhibitors
NodThera: Small-molecule NLRP3 inhibitors expected to begin clinical studies this year
Quench Bio: Small-molecule gasdermin-D inhibitors
Ventus Therapeutics: Small-molecule inhibitors of innate immune system proteins, including inflammasomes
ZyVersa Therapeutics: Antibody inhibitors of the inflammasome protein ASC
After Cooper and O’Neill identified MCC950 as a selective NLRP3 inhibitor, Cooper wanted to hold off on publishing for a few years to give Avril Robertson, the head chemist on his team, time to create hundreds of analogs to MCC950. Schroder designed assays to study the new compounds’ effects on NLRP3, and O’Neill’s lab tested them. Those years of quiet work formed the basis of two patents filed in February 2015—the same day they published their MCC950 study in Nature Medicine.
Their paper inspired an inflammasome biotech boom. Cooper and O’Neill founded Inflazome in 2016, the same year that Gabel, whom a venture capital firm had tracked down, helped launch NodThera.
Versant Ventures funded its own NLRP3 inhibitor company, called Jecure Therapeutics, with $20 million in early 2017. Less than 2 years later, it was acquired by Genentech for an undisclosed sum. And IFM Therapeutics, a firm focused on innate immunity at large, has sold two NLRP3 subsidiaries—one to BMS and one to Novartis—in deals potentially worth more than $1 billion each.
Novartis is particularly attuned to the potential of NLRP3 inhibitors thanks to its IL-1β blocker, canakinumab. It’s one of three IL-1β inhibitors approved to treat rare inflammatory conditions, including ones caused by mutations in NLRP3.
But start-ups are keen to note the advantages that NLRP3 inhibitors could have over the IL-1β drugs, all of which must be injected. NLRP3 blockers could be taken as pills and could have a better chance of getting into the brain. Scientists also hope that NLRP3 inhibitors could be safer than the IL-1β blockers, which can cause infections.
Chemists from most NLRP3 companies used MCC950 as a starting point to design more potent and, they hope, safer molecules. But how, exactly, the compound inhibited NLRP3 was a mystery until last year, when two separate groups—one led by Robertson’s and Schroder’s labs and another led by Pablo Pelegrin at the Biomedical Research Institute of Murcia in Spain—simultaneously published papers outlining its potential mechanism of action.
Both studies indicated that MCC950 binds near a region of NLRP3 that is important for adenosine triphosphate (ATP) hydrolysis, the universal energy currency of cells. NLRP3 appears to require ATP for activation, Schroder says. But it’s still unclear whether that ATP is necessary for helping the shrimp-shaped NLRP3 proteins flex from their inactive to active forms or whether the ATP is required to hold the NLRP3 ring in its shrimp cocktail conformation.
A few companies emphasize that their programs are unrelated to MCC950. Olatec Therapeutics, for instance, has already tested a β-sulfonyl nitrile NLRP3 inhibitor in Phase II clinical studies. Inflammasome Therapeutics is designing chemically modified antivirals that inhibit multiple inflammasomes. And Joerg Eder, head of innate immunity at Novartis, says the company was working on its own NLRP3 inhibitors, designed to enter the brain, before it acquired the sulfonylurea-based compounds from IFM.
Academic chemists are keen to get in on the action, too, but are finding it difficult without knowing NLRP3’s structure. Tsan Sam Xiao from Case Western Reserve University says that screening chemical libraries for NLRP3 inhibitors has been disappointing so far because the hits have already been discovered or are known to have multiple targets. “It turns out that screening for inflammasome inhibitors is a highly complex and highly competitive business.”
In 2001, Hal Hoffman, an allergist and immunologist at the University of California San Diego, discovered that mutations in a protein that he named cryopyrin were responsible for a rare condition that caused some people to break out in hives when exposed to the cold. Cryopyrin was later renamed NLRP3, and the cold allergy is one of three rare genetic diseases caused by mutations that make NLRP3 hyperactive. Collectively, these diseases are called cryopyrin-associated periodic syndromes (CAPS).
People with CAPS are prescribed IL-1β inhibitors, but several inflammasome companies think the disease provides a perfect proving ground for their compounds too. “If it doesn’t work in CAPS, we’ll fall off our seats,” O’Neill says.
But after CAPS, prioritizing which diseases to tackle gets harder. Gout is a popular pick, since it should be easy to see whether NLRP3 inhibitors reduce inflammation. Companies are also pursuing—often in parallel—more complicated conditions like inflammatory bowel disease, the liver disease nonalcoholic steatohepatitis (NASH), and Parkinson’s disease.
The abundance of options has created an unusual situation: companies are actually relieved to discover their NLRP3 inhibitors don’t help in every disease model. “It didn’t work in epilepsy,” Cooper says. “And we were like, ‘Great! At least there’s one indication where it doesn’t work!’ ”
Of all the diseases that NLRP3 is implicated in, its link to Alzheimer’s may be the most tantalizing. In 2008, researchers from the University of Massachusetts Medical School discovered that amyloid-β, the protein that accumulates in the brains of people with Alzheimer’s disease, activates NLRP3 inflammasomes. In 2012, Michael Heneka, a neuroscientist at the University of Bonn, and the UMass team demonstrated that knocking out the genes that encode NLRP3 or caspase-1 in a mouse model of Alzheimer’s prevents the memory loss that normally afflicts the mice in old age.
Even more provocatively, in 2017 the groups showed that when the brain’s resident immune cells, microglia, explode after inflammasome activation, a mangled mess of inflammasomes spill out into the space between brain cells and catalyze the formation of amyloid-β oligomers and aggregates. Because these amyloid-β aggregates spread slowly from one brain region to the next in people with Alzheimer’s, Heneka wondered if stopping the inflammasomes would stop the spread of amyloid-β in the mouse brains.
Astonishingly, blocking the inflammasome protein ASC—with an antibody or by genetic knockout—halted the march of amyloid-β. The study suggests that inflammation “is not just a responsive phenomenon” in neurodegeneration, Heneka says. “It is actively contributing to the disease progression, which makes it a pharmacological target.”
Heneka thinks that amyloid-β triggers NLRP3 because amyloid-β is similar to structures found on bacteria. It’s as if the inflammasomes are trying to protect the brain from an infection that doesn’t exist. Inflammasome activation then facilitates the spread of amyloid-β, which triggers more inflammasome activation, creating a vicious cycle.
But Hoffman points out there’s a “chink in the armor” of this theory. Despite having hyperactive inflammasomes, his patients with CAPS don’t have higher rates of neurodegenerative diseases or other conditions linked to NLRP3. “These companies are very excited about all the diseases they think they are going to cure, and I am not sure it will be 100% as effective as they think,” Hoffman says.
It is unlikely that NLRP3 inhibitors will treat every single disease that NLRP3 is implicated in, according to Adam Keeney, CEO of NodThera. For complex conditions like NASH, in which inflammation is just one component of the disease, Keeney envisions NLRP3 inhibitors working best alongside other drugs. “But NLRP3 is as good a target that you will find,” he adds. Now it’s just a matter of seeing if NLRP3 inhibitors can live up to their promise in the first round of clinical trials, which for many companies have just begun or will commence soon. “The next 3 years will make or break the field.”
So far, most companies are keeping their clinical strategies, and the structures and mechanisms of their molecules, secret. Since most NLRP3 inhibitor programs are based on MCC950, companies are eager to differentiate themselves. One way they are doing that is by designing NLRP3 inhibitors that are targeted toward a particular area in the body, such as the brain, gastrointestinal tract, or skin.
But as far as new chemistry goes, most inflammasome companies think they’ve got the space covered. “Starting another NLRP3 inhibitor company would be a challenge at this point,” Keeney says. Unless, he adds, someone has a “completely new way of thinking about the chemistry.”
Some companies think they do. “The NLRP3 field is crowded with molecules that look similar and absolutely empty of things that are different,” says Marcelo Bigal, president and CEO of a stealth start-up called Ventus Therapeutics, which is backed by Versant Ventures. Bigal says that Ventus scientists have developed a technique for snapping pictures of inflammasome structures by a method he calls “paralyzing the monomers”—the individual shrimplike NLRP3 proteins, for instance. Those images will allow the firm to rationally design small molecules that inhibit the formation of oligomers, like the NLRP3 ring, he adds. Ventus is working on multiple innate immune proteins and inflammasomes, not just NLRP3, Bigal adds.
IFM’s Seidel says his company is exploring inflammasomes beyond NLRP3, as well as proteins that work upstream or downstream of NLRP3. One attractive target is gasdermin-D, a downstream protein that prompts that dramatic cellular explosion after inflammasome activation.
Many inflammasome experts think that gasdermin-D will be the next big target in innate immune system drug discovery. Xiao, from Case Western, gave up trying to find new NLRP3 inhibitors and instead focuses on gasdermin-D. And Martinon, the scientist who discovered the inflammasome, says the discovery linking gasdermin-D to pyroptosis—cell death after inflammasome activation—in 2015 was “the biggest breakthrough we’ve had in this field in decades, maybe ever.”
Last month, a start-up called Quench Bio raised $50 million to develop small-molecule inhibitors of gasdermin-D. CEO Samantha Truex says that Quench will focus on identifying inflammatory diseases in which an NLRP3 inhibitor won’t be enough, such as lupus and rheumatoid arthritis.
In other words, the inflammasome gold rush is just getting started.
And while it will be years before we know whether NLRP3 inhibitors can do a fraction of what companies hope for, they could still make a big dent in disease.
Schroder, meanwhile, is still trying to work out the biological purpose of NLRP3, a protein that so often seems to do more bad than good. “A bunch of the top killers in the Western world are all associated with NLRP3 activation,” Schroder says. “So if you can take some kind of once-a-day pill and help prevent yourself from getting Alzheimer’s, Parkinson’s, cardiovascular disease, atherosclerosis, and chronic liver disease, and if there are no side effects, then why wouldn’t you?”