TY - JOUR
T1 - Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609
AU - Ohtsuka, Satomi
AU - Ozeki, Yui
AU - Fujiwara, Moeno
AU - Miyagawa, Tomoyuki
AU - Kanayama, Naoki
AU - Magari, Masaki
AU - Hatano, Naoya
AU - Suizu, Futoshi
AU - Ishikawa, Teruhiko
AU - Tokumitsu, Hiroshi
N1 - Funding Information:
This work was supported by Grants-in-aid for Scientific Research (JP18K06113 and JP19H03447 to H.T.) from the Japan Society for the Promotion of Science.
Publisher Copyright:
Copyright © 2020 American Chemical Society.
PY - 2020/5/5
Y1 - 2020/5/5
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.biochem.0c00149
DO - 10.1021/acs.biochem.0c00149
M3 - Article
C2 - 32298102
AN - SCOPUS:85084271892
VL - 59
SP - 1701
EP - 1710
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 17
ER -