Nardilysin (NRDC) is a zinc metallopeptidase of the M16 family that participates in diverse biological processes, including ectodomain shedding and antigen processing. NRDC also functions as a transcriptional coregulator, and this coregulatory activity depends on its metallopeptidase catalytic function. However, small-molecule inhibitors targeting NRDC have not been well characterized, limiting pharmacological investigation of its enzymatic function. Here we established a fluorescence-quenched peptide substrate assay suitable for high-throughput screening of NRDC protease activity. Using this assay, we screened 33,305 compounds and identified 140 candidate NRDC inhibitors. Through secondary screening, orthogonal validation and structure-activity relationship analysis, three inhibitor scaffolds were identified, from which compound R5-1 was selected for further characterization. Enzyme kinetic analysis demonstrated that R5-1 inhibits NRDC activity in a non-competitive manner, indicating reduced catalytic turnover rather than directly competing with substrate binding. Structural modeling and docking analysis further suggested that R5-1 binds to a distal allosteric site in NRDC. Pharmacological inhibition of NRDC with R5-1 attenuated NRDC-dependent transcriptional repression of PGC-1α, phenocopying the effect of catalytic inactivation. Furthermore, administration of R5-1 significantly ameliorated arthritis severity in a mouse model of autoimmune arthritis. Together, these findings identify R5-1 as a small-molecule inhibitor of NRDC and provide mechanistic insights linking NRDC protease activity to transcriptional regulation and inflammatory disease.
Although affinity prediction of protein–ligand binding remains an important challenge, cofolding models are expected to make virtual screening more effective for drug discovery and development. To verify the effectiveness of selecting a tractable number of candidates from a compound library using cofolding models, we strove to identify a novel inhibitory active compound using Boltz-2, a representative cofolding model, for aldo-keto reductase 1B10 (AKR1B10), which is highly expressed in various cancers. Consequently, of the 40 candidate compounds obtained after narrowing-down 867 candidates from a chemically diverse library based on prediction results by Boltz-2 and candidate selection with sufficient diversity, 70% (28 of 40 tested) compounds at IC50 < 10 μM were found to have inhibitory activity and to provide identification of multiple submicromolar inhibitors exhibiting novel scaffolds. Our results demonstrate that our sparse selection approach using Boltz-2 is helpful for enhancing AI-driven drug discovery. Moreover, the findings highlight its potential applicability for translating AI-generated predictions into experimentally actionable hits.
Shinya Kawano, Akira Ikari, Satoshi Endo et al.· Journal of Chemical Informat...· 0 citations
Claudin-14 (CLDN14) is a tight junction protein that contributes to the development of chemoresistant phenotypes in human colorectal cancer (CRC) cells. CLDN14 may represent a potential therapeutic target for CRC. In this study, we identified velpatasvir (VEL), a clinically approved hepatitis C virus nonstructural protein 5A (NS5A) inhibitor, as a potent suppressor of CLDN14 protein expression using in silico screening and Western blot analysis. CLDN14 expression was not decreased by other NS5A inhibitors, including ledipasvir, pibrentasvir, and daclatasvir. Quartz crystal microbalance analysis revealed that VEL directly binds to recombinant CLDN14 protein with a dissociation constant of 1.8 ± 0.2 μM. VEL treatment did not affect CLDN14 mRNA levels, suggesting that CLDN14 expression is regulated at a post-transcriptional level. Pharmacological inhibition of clathrin-mediated endocytosis and lysosomal degradation significantly reversed the VEL-induced reduction in CLDN14 protein levels, suggesting the involvement of endocytosis-lysosomal degradation pathway. Functionally, VEL increased paracellular permeability to mineral ions and enhanced the transepithelial flux of aqueous fluorescent tracers. In DLD-1 spheroids, VEL attenuated intracellular oxidative stress and reduced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), a central regulator of redox homeostasis. Consequently, VEL promoted the intracellular accumulation of doxorubicin and significantly potentiated its cytotoxic effects. Moreover, VEL enhanced the antitumor efficacy of other chemotherapeutic agents, including oxaliplatin and SN-38, the active metabolite of irinotecan. These findings suggest that VEL enhances anticancer drug sensitivity in CRC cells through lysosome-dependent downregulation of CLDN14 protein and suppression of Nrf2-dependent oxidative stress responses.
Tomoka Ando, Yuko Mizukami, Shiemi Tosaki et al.· European Journal of Pharmaco...· 0 citations
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