American Chemical Society Acta Medica Okayama 00062960 59 17 2020 Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609 1701 1710 EN Satomi Ohtsuka Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Yui Ozeki Graduate School of Education, Okayama University Moeno Fujiwara Faculty of Education, Okayama Universit Tomoyuki Miyagawa Graduate School of Education, Okayama University KanayamaNaoki Kanayama Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Masaki Magari Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama Universityayama University Naoya Hatano Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Futoshi Suizu Institute for Genetic Medicine, Hokkaido University Teruhiko Ishikawa Graduate School of Education, Okayama University Hiroshi Tokumitsu Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5′AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKβ) with a similar potency (Ki = 0.35 μM for CaMKKα, and Ki = 0.2 μM for CaMKKβ) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50 of ∼0.3 μM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases. No potential conflict of interest relevant to this article was reported.