Covalent modalities represent an important component of the modern medicinal chemist’s toolbox for pursuing challenging targets in drug discovery. By taking a “covalent-first” approach to identifying druggable pockets on challenging-to-drug targets, we and others have expanded accessible target space and driven fragment-like hits to clinical-stage molecules. The field has long recognized intrinsic warhead reactivity as a key parameter to monitor, typically addressed by determining k inact and K I values, which are impractically time-intensive and can be misleading regarding reactivity. Here we present an alternative way to normalize potency for electrophilicity utilizing glutathione (GSH) consumption data, which enables us to extract target-specific improvements in potency, a metric we term ligand reactivity efficiency (LRE). Our hope is that the details of our approach and this metric will simplify the rational design of covalent drugs for fellow practitioners in the field.

Shows VVD-065 increases the KEAP1-CUL3 protein-protein interaction without affecting the KEAP1 protein half-life

Shows cysteine-directed selectivity analysis of VVD-065 and related compounds

Shows chemoproteomic determination of KEAP1 Cys151 engagement and subsequent downstream signaling effects of VVD-065 related compounds

Shows additional analysis of the tumor growth inhibition study in the KLN205 syngeneic model, and evaluation of the tolerability of VVD-065 in combination with chemotherapy in patient-derived xenograft models

Shows differential cellular response to VVD-065, showing activity in wild-type cells but inactivity or reduce activity in cells with certain KEAP1 or NFE2L2 mutations

Shows VVD-065 enhances NRF2 ubiquitination by increasing the KEAP1- CUL3 protein-protein interaction

Shows immunoblots revealing that the activity of VVD-065 is dependent on KEAP1/ CUL3 but independent of cellular redox status

Shows further evaluation of VVD-065, including assessment of the acetamide analog's lack of activity, determination of VVD-065 reaction kinetics, and additional structural analysis of the compound

Shows distribution of physicochemical properties for the covalent fragment library and initial chemoproteomic screening results

Shows the anti-proliferative activity of VVD-065, observed across various cell lines and potentiated in 3D culture, is fundamentally linked to the presence of KEAP1 Cys151

Shows TGI effects of VVD-065 and NRF2 genetic ablation in a variety of cell line derived xenograft models

BACKGROUND AND PURPOSE:The NLRP3 inflammasome is an attractive therapeutic target for multiple inflammatory conditions. Although inhibitors have been developed, their chemical diversity is limited, and their properties are not ideal for brain penetrance, which is desirable for treating neuroinflammatory disorders. EXPERIMENTAL APPROACH:We applied our chemoproteomics platform to survey our electrophilic fragment collection to identify inhibitors of NLRP3. We focused our attention on compounds that bind Cys463, as this residue was identified as an allosteric sensor of NLRP3 function. KEY RESULTS:A novel inhibitor series was identified bearing a butynamide electrophile and a unique spirocyclic lactam core. Compounds from this series displayed mid-nanomolar potency and were found to inhibit IL-1β secretion in a Cys463-dependent manner. Cryo-EM structures revealed that ligand binding to Cys463 stabilizes an inactive conformation, thereby preventing structural rearrangements required for inflammasome activation. These compounds displayed attractive pharmacokinetic properties and, notably, Kp,uu values >0.5, suggesting the potential to address neuroinflammatory disorders. Administration of a representative compound to humanized mice resulted in clear NLRP3 Cys463 target-engagement and profound suppression of LPS- and ATP-induced IL-1β secretion, demonstrating clear proof-of-concept in vivo. CONCLUSION AND IMPLICATIONS:Chemoproteomics-based ligand discovery is intrinsically function-agnostic and has the potential to identify novel pockets on even well-characterized protein targets. Here, optimization of ligands targeting Cys463 of NLRP3 within a previously uncharacterized allosteric pocket led to a unique and potent inhibitor series with attractive physicochemical and pharmacokinetic properties for the potential treatment of diseases involving aberrant innate immune activation in both central and peripheral tissues.

Shows synthesis routes of compounds

Shows global proteomic analysis of VVD-065 treated cells

Shows VVD-065 activity on NRF2 targets

Shows pharmacodynamic effects and tolerability of VVD-065 evaluated in patient-derived xenograft (PDX) models

Shows buffer composition in HTRF assay