Conformation-Dependent Reversible Interaction of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

Abstract

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), a Ca2+/CaM-dependent enzyme that phosphorylates and activates multifunctional kinases, including CaMKI, CaMKIV, protein kinase B/Akt, and 5′AMP-activated protein kinase, is involved in various Ca2+-signaling pathways in cells. Recently, we developed an ATP-competitive CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo­[de]­benzo­[4,5]­imidazo­[2,1-a]­isoquinolin-7-one, Ohtsuka et al. Biochemistry 2020, 59, 1701–1710). To gain mechanistic insights into the interaction of CaMKK with TIM-063, we prepared TIM-063-coupled sepharose (TIM-127-sepharose) for association/dissociation analysis of the enzyme/inhibitor complex. CaMKKα/β in transfected COS-7 cells and in mouse brain extracts specifically bound to TIM-127-sepharose and dissociated following the addition of TIM-063 in a manner similar to that of recombinant GST–CaMKKα/β, which could bind to TIM-127-sepharose in a Ca2+/CaM-dependent fashion and dissociate from the sepharose following the addition of TIM-063 in a dose-dependent manner. In contrast to GST–CaMKKα, GST–CaMKKβ was able to weakly bind to TIM-127-sepharose in the presence of EGTA, probably due to the partially active conformation of recombinant GST–CaMKKβ without Ca2+/CaM-binding. These results suggested that the regulatory domain of CaMKKα prevented the inhibitor from interacting with the catalytic domain as the GST–CaMKKα mutant (residues 126–434) lacking the regulatory domain (residues 438–463) interacted with TIM-127-sepharose regardless of the presence or absence of Ca2+/CaM. Furthermore, CaMKKα bound to TIM-127-sepharose in the presence of Ca2+/CaM completely dissociated from TIM-127-sepharose following the addition of excess EGTA. These results indicated that TIM-063 interacted with and inhibited CaMKK in its active state but not in its autoinhibited state and that this interaction is likely reversible, depending on the concentration of intracellular Ca2+.

Development of a novel AAK1 inhibitor via Kinobeads-based screening

“…1 a). Consistent with results of a previous report 12 , CaMKKα/1 and β/2 were readily identified as the main targets of TIM-063 and two potential off-target protein kinases, AAK1 and ERK2, were detected by the proteomic approach (Table 1 and Supplemental Information S1 ). This was confirmed by immunoblot analyses with the corresponding antibodies, indicating that the protein kinases interacted with the TIM-063 motif on the inhibitor-immobilized sepharose but not with the control sepharose (Fig.…”

“…TIM-063 (2-hydroxy-3-nitro-7 H -benzo[de]benzo[4, 5]-imidazo[2,1- a ]isoquinolin-7-one, Fig. 1 a) was originally developed as a CaMKK inhibitor via screening of an STO-609–derived chemical library 11 and TIM-063–immobilized sepharose (TIM-127-sepharose) was used to characterize the Ca 2+ /CaM-dependent interaction of CaMKK and the inhibitor 12 . Because TIM-063 is an ATP-competitive inhibitor similar to STO-609 31 , the inhibitor may interact and inhibit off-target kinases.…”

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Figure 1 Identification and characterization of AAK1 as a TIM-063–interactant. ( a ) Protocol for identification of TIM-063–interactants using TIM-063–immobilized sepharose (TIM-127-sepharose 12 ) combined with mass spectrometry analysis. Mouse cerebrum extracts were applied onto TIM-127-sepharose, and after extensive washing of resin, TIM-063–interactants were eluted by the addition of 100 µM TIM-063 containing buffer, followed by identification via mass spectrometry analysis as described in the “ Methods ”.

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“…However, the fact that a single protein kinase inhibitor suppresses the activities of multiple off-target protein kinases may allow the development of off-target kinase inhibitors by modifying the inhibitor as a lead compound. We recently prepared Kinobeads 9 , 10 in which a Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) inhibitor (TIM-063, 2-hydroxy-3-nitro-7 H -benzo[de]benzo[4, 5] -imidazo[2,1- a ]isoquinolin-7-one) was immobilized on sepharose beads to characterize the interaction between TIM-063 and CaMKK 11 , 12 . In this study, we searched for TIM-063—interacting protein kinases using the Kinobeads technology 9 , 10 and successfully identified candidates of multiple off-target protein kinases for TIM-063, including AP2-associated protein kinase 1 (AAK1), as well as the main targets, CaMKK isoforms.…”

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction

“…The inhibitory properties of TIM-063 are similar to STO-609, except that TIM-063 can similarly inhibit both CaMKKα/1 (0.63 µM) and β/2 (0.96 µM). Moreover, TIM-063 has been shown to interact with and inhibit CaMKK in its active state (Ca 2+ /CaM-bound form) but not in its autoinhibited state (Ca 2+ /CaM-unbound form); this interaction is likely reversible, depending on the intracellular Ca 2+ concentration [ 130 ]. TIM-063, but not TIM-062, suppresses the Ca 2+ -induced phosphorylation of AMPK, CaMKI, and CaMKIV in cultured cells.…”