System-level mapping of kinase inhibitors’ polypharmacology can uncover opportunities for drug repurposing and identify starting points for medicinal chemistry campaigns aimed at novel targets. Here, we describe a large-scale profiling of 257 kinase inhibitors that revealed domains of promiscuity, a broad region of uninhibited targets and discrete pockets of selectivity. Focusing on a subcluster enriched for AGC group kinases and using the AKT inhibitor AT-7867 (an antiproliferative agent) as a starting point, we employed scaffold hopping and property-based optimization to discover NCGC-6782, a LATS1/2 inhibitor with robust proproliferative activity. In biochemical and cell-based assays, NCGC-6782 inhibited LATS1/2 activity, reduced YAP phosphorylation, and promoted rapid YAP nuclear translocation and transactivation of YAP-target genes. Kinome-wide profilings demonstrated good in vitro selectivity and exquisite chemoproteomic and kinetic selectivity. In vivo, NCGC-6782 exhibited favorable pharmacokinetic (PK) profiles following intraperitoneal (IP), intravenous (IV), or oral (PO) dosing, leading to rapid inhibition of YAP-phosphorylation in murine liver samples. Transcriptomics and proteomics following NCGC-6782 revealed upregulation of YAP-dependent genes (4 h) followed by activation of cell cycle programs (24 h). Consistently, NCGC-6782 accelerated proliferative regeneration in both cell-based and in vivo wound healing models of full-thickness excisional skin injury, significantly shortening wound-closure time without evidence of systemic toxicity. Altogether, we describe an optimized, potent, and selective LATS1/2 kinase inhibitor with desirable drug-like properties and broad potential for regenerative medicine applications following acute injury of nonproliferative, postmitotic tissues.

Endoplasmic reticulum-associated degradation (ERAD) must dynamically adapt to cellular protein-folding demands, yet mechanisms regulating ERAD-associated adaptors remain incompletely defined. Here, we identify the E3 ubiquitin ligase RNF5 as a regulator of the ERAD adaptors HERPUD1 (HERP1) and OS9. RNF5 depletion increased HERP1 and OS9 protein abundance, while semiquantitative endpoint RT-PCR did not reveal obvious changes in their transcript signals. Available cycloheximide-chase experiments were consistent with increased adaptor persistence and more rapid loss of the ERAD-L substrate null Hong Kong α1-antitrypsin (NHK) after RNF5 depletion. In contrast, expression of the catalytically inactive RNF5 C42S mutant was associated with slower NHK loss, indicating that RNF5 depletion and catalytic inactivation produce distinct functional outcomes. Proteomic screening and co-immunoprecipitation supported association of RNF5 with HERP1- and OS9-containing complexes. RNF5 depletion reduced compound 225-induced HERP1 ubiquitination and adaptor loss, while wild-type RNF5 produced stronger HERP1 ubiquitination than C42S, supporting a contribution of RNF5 catalytic activity. RNF5 depletion also attenuated selected tunicamycin-induced UPR markers and 225-associated cytotoxic/apoptosis-related readouts while increasing SA-β-galactosidase- and γH2AX-associated phenotypes. Together, these findings identify RNF5 as a regulator of ERAD-adaptor abundance and NHK processing, while leaving the causal relationship between adaptor regulation and the RNF5-depletion phenotype to be resolved by formal rescue and epistasis studies.