Covalent allosteric inhibitors of NLRP3

NLRP3 has received considerable attention as an inflammatory disease target. The protein normally exists in an auto-inhibited conformation, but in response to various stimuli it changes shape to help form a multimeric complex called the inflammasome. This in turn activates various inflammatory pathways, including cell death. Several drugs targeting NLRP3 have gone into the clinic, but none have been approved. In a new open-access Br. J. Pharmacol. paper, Brian Cook, Gabriel Simon, and colleagues at Vividion describe a covalent inhibitor that binds to a previously unknown site.

 

As we wrote nearly a decade ago, Vividion pioneered the use of chemoproteomics to identify covalent binders in cells and cell lysates. While exploring the mechanism of the previously reported NLRP3 inhibitor MCC950, they found that cysteine 280 became resistant to modification by electrophiles in the presence of the ligand. MCC950 does not contain an electrophile, arguing against direct interaction with Cys280 and suggesting instead that binding causes a conformational change to the protein which makes Cys280 less accessible. In other words, protein inhibition correlated with Cys280 being less exposed.

 

A screen of Vividion’s library yielded no hits against Cys280 but more than 300 against cysteine 463. Interestingly, modification of C463 also caused a decrease in reactivity of Cys280, suggesting a conformational change similar to the one caused by MCC950. Thus, the researchers pursued Cys463 binders.

 

Two parameters were used to guide compound optimization, target engagement and promiscuity. The first number, TE₅₀, measures the extent of covalent engagement of a given cysteine after 1 hour in cell lysates. The second, P₅₀₀, measures the percentage of all measured cysteines in the sample labeled by at least 40% after incubation with 500 µM compound for 1 hour. One of the initial hits, VVD-124476, gave a low micromolar value for TE₅₀ but labeled some 20% of the measured proteome. Indeed, it was previously reported as a covalent ligand for a completely different protein. To be useful as a NLRP3 inhibitor, the researchers needed to reduce promiscuity, and the most straightforward approach is to reduce general reactivity.

 

To decrease reactivity, the researchers swapped the acrylamide warhead with a butynamide (VVD-142397). Butynamides are reported to be less reactive than acrylamides, a result corroborated at Vividion with chemoproteomic data on 400 matched molecular pairs. The acrylamide to butynamide swap improved selectivity some 20-fold, though it also decreased potency against NLRP3. Increasing the core ring size regained activity, and rigidifying the molecule led to further improvements. Further optimization ultimately led to VVD-338213 and several related molecules.

 

Although the researchers do not report k_inact/K_I values, a back of the envelope calculation puts it around 1200 M^-1s^-1 for VVD-338213, a potency where cell activity might be expected. Happily this turned out to be the case: the molecule blocked IL-1β secretion from human cells at lower concentrations than MCC950. Importantly, in cells where Cys463 had been mutated to alanine, MCC950 was still active while the covalent molecules were inactive.

 

In addition to promising cell activity, many of the covalent molecules had reasonable stability in whole blood, good permeability, and low efflux. (I do wish the ligand efficiency reactivities of the molecules were reported; after all Vividion, introduced this metric.) Some of the molecules also showed good brain penetration. In vivo pharmacodynamic studies are complicated by the fact that Cys463 is not conserved in mice, but fortunately the researchers could run studies on transgenic mice in which human NLRP3 had been introduced. In these animals, IL-1β was decreased by both VVD-338213 and by MCC950.

 

The researchers were also able to obtain a cryo-EM structure of one of their molecules bound to NLRP3, which confirmed covalent binding to Cys463. More importantly, it revealed that the binding site is in a cryptic pocket some 20 Å from where MCC950 binds. (One wonders if the computational technique we discussed last week would have been able to predict this pocket.) Similarly to the WRN inhibitor Vividion has taken into the clinic, the molecule appears to make only hydrophobic contacts with the protein, with no polar interactions. Mechanistically, the “Cys463 ligands act as molecular doorstops, stabilizing the inactive conformation and preventing the structural rearrangements necessary for transition to the active inflammasome disc.”

 

This is a lovely paper and a case study in transforming a highly reactive molecule into a selective molecule with in vivo activity. It is interesting that the starting molecule, with 28 non-hydrogen atoms, flagrantly violates the rule of three. Last year we asked whether covalent fragments need to be larger than their non-covalent counterparts, and this paper provides another example where the answer is yes. Whether or not this chemical series ultimately advances, it is nice to have another chemical probe for this target.