start-ver=1.4
cd-journal=joma
no-vol=59
cd-vols=
no-issue=17
article-no=
start-page=1701
end-page=1710
dt-received=
dt-revised=
dt-accepted=
dt-pub-year=2020
dt-pub=20200416
dt-online=
en-article=
kn-article=
en-subject=
kn-subject=
en-title=
kn-title=Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609
en-subtitle=
kn-subtitle=
en-abstract=
kn-abstract=Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5ŒAMP-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 (CaMKKƒ¿ and CaMKKƒÀ) with a similar potency (Ki = 0.35 ƒÊM for CaMKKƒ¿, and Ki = 0.2 ƒÊM for CaMKKƒÀ) 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 AMPKƒ¿ in HeLa cells with an IC50 of ?0.3 ƒÊM, 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.
en-copyright=
kn-copyright=

en-aut-name=OhtsukaSatomi
en-aut-sei=Ohtsuka
en-aut-mei=Satomi
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=1
ORCID=

en-aut-name=OzekiYui
en-aut-sei=Ozeki
en-aut-mei=Yui
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=2
ORCID=

en-aut-name=FujiwaraMoeno
en-aut-sei=Fujiwara
en-aut-mei=Moeno
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=3
ORCID=

en-aut-name=MiyagawaTomoyuki
en-aut-sei=Miyagawa
en-aut-mei=Tomoyuki
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=4
ORCID=

en-aut-name=KanayamaKanayamaNaoki
en-aut-sei=Kanayama
en-aut-mei=KanayamaNaoki
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=5
ORCID=

en-aut-name=MagariMasaki
en-aut-sei=Magari
en-aut-mei=Masaki
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=6
ORCID=

en-aut-name=HatanoNaoya
en-aut-sei=Hatano
en-aut-mei=Naoya
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=7
ORCID=

en-aut-name=SuizuFutoshi
en-aut-sei=Suizu
en-aut-mei=Futoshi
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=8
ORCID=

en-aut-name=IshikawaTeruhiko
en-aut-sei=Ishikawa
en-aut-mei=Teruhiko
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=9
ORCID=

en-aut-name=TokumitsuHiroshi
en-aut-sei=Tokumitsu
en-aut-mei=Hiroshi
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=10
ORCID=

affil-num=1
en-affil=Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
kn-affil=

affil-num=2
en-affil=Graduate School of Education, Okayama University
kn-affil=

affil-num=3
en-affil=Faculty of Education, Okayama Universit
kn-affil=

affil-num=4
en-affil= Graduate School of Education, Okayama University
kn-affil=

affil-num=5
en-affil=Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
kn-affil=

affil-num=6
en-affil=Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama Universityayama University
kn-affil=

affil-num=7
en-affil=Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
kn-affil=

affil-num=8
en-affil= Institute for Genetic Medicine, Hokkaido University
kn-affil=

affil-num=9
en-affil=Graduate School of Education, Okayama University
kn-affil=

affil-num=10
en-affil=Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
kn-affil=
END