Identification and Validation of a Novel WNK2 Inhibitor: A New Genetically Informed Target for Osteoarthritis Drug...

Osteoarthritis (OA) is a disease characterized by loss of joint space, degeneration of cartilage at articular surfaces, remodeling of bone and other joint tissues, low-grade inflammation, and pain. It is a leading cause of disability in the aging population. We do not have effective disease-modifying drugs because we lack a comprehensive understanding of the genetic pathways underlying OA development. Our recent work demonstrated that dominant hypermorphic mutations in the osmotic stress sensing kinase, WNK2, are associated with OA susceptibility. To explore WNK2 inhibition as a potential disease-modifying osteoarthritis drug, we identified a WNK2 inhibitor by using a combination of computational and experimental approaches. We used AlphaFold2 to model the WNK2 kinase domain with high confidence (pLDDT score >90), enabling virtual screening of 4.8 million compounds in the ZINC database. Structure-based filtering prioritized 53 candidates interacting with WNK2-specific residues Glu245 and Glu249, with binding energies from -4 to -8 kcal/mol. M04, (1-(6-((3,5-bis-(trifluoromethyl)-benzyl)-(methyl)-amino)-pyrimidin-4-yl)-pyrrolidin-2-yl)-methanol, emerged as a partially selective WNK2 inhibitor with minimal cytotoxicity in human T/C-28a2 chondrocyte cells (cell viability IC₅₀ = 416 μM). M04 inhibited IL1β-mediated proinflammatory gene expression in primary human chondrocytes. RNaseq analysis comparing M04 vs a pan-WNK inhibitor in primary human chondrocytes indicated that M04 was a more effective at reducing expression of IL1β-induced OA-associated pathways. Further studies indicated the R-enantiomer is the active form of the molecule. These findings position M04 as a promising lead for WNK2-targeted OA therapy, supporting further in vivo studies for optimization and validation.