Abstract A088: Revealing intrinsic ATPase activity in kinases: Implications for drug discovery using ADP-Glo assay

Abstract Kinases are pivotal regulators of cellular signaling and are among the most targeted enzymes in drug discovery. Their downstream phosphorylation of substrates is a key step in many disease-associated signaling cascades. A kinase’s biochemical activity, and the effect of its cognate inhibitors, is traditionally assessed through measurement of the phosphorylation of a relevant substrate. A kinase’s intrinsic ATPase activity—ATP hydrolysis independent of substrate—remains an underexplored biochemical aspect of kinases which may require closer attention in assay development, to ensure that inhibitor activity and profiling are properly assessed. In this study, we employed the ADP-Glo™ assay to systematically evaluate ATPase activity across approximately 300 of human recombinant kinases produced in house. In this system, about 10% of kinases exhibited substantial intrinsic ATPase activity in the absence of substrate, suggesting that under these conditions and for a number of kinases, significant ATP turnover may occur independently of canonical phosphorylation events. Non-specific inhibitors (e.g., Staurosporine and K252a) and specific kinase inhibitors were used to examine and confirm the origin of the substrate-phosphorylation and ATPase activity. Select kinase mutants and their mutant-specific inhibitors were used to further examine the catalytic and ATPase activities. Our observations provide a deeper insight into kinase biochemistry and underscore the importance of understanding kinase-substrate catalytic activity from background ATP hydrolysis, particularly in high-throughput screening and lead optimization, so as not to mischaracterize inhibitor potency. Citation Format: Yusuke Kawase, Yugo Narumi, Etsuko Ishibushi, Tomoe Bunno, Hiromi Akimichi, Adam Shutes. Revealing intrinsic ATPase activity in kinases: Implications for drug discovery using ADP-Glo assay [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A088.