KN-93: deciphering CaMKII inhibitor mechanism of action
Protein kinases are state-of-the art targets for developing therapeutic drugs, as they are involved in specific signaling pathways and regulate many cellular functions. Protein phosphorylation (and dephosphorylation) balance is the molecular switch for modulating these processes. The level and duration of phosphorylation/dephosphorylation states is a highly regulated process in normal cells and is often impaired in disease states.
Protein kinases transfer a phosphoryl group from ATP onto target proteins, offering easy definition and a quick view of enzymatic activity. This activity is the end point of a cascade of events involving other partners such as scaffolding and interacting proteins. A highly complex and synchronized sequence of protein-protein interactions fine-tunes the different signaling pathways, making it much more difficult to identify highly selective inhibitors.
Case studies are a convenient way to understand a kinase inhibitor’s mechanism of action. In this article, Marion H. Wong disclosed the strategies and techniques she used to characterize KN-93 a cell-permeable, reversible, and competitive inhibitor – calmodulin-dependent kinase type II (CaMKII).
She combined a variety of highly specific chemical techniques, including Surface Plasmon Resonance, NMR chemical shifts, and isothermal titration calorimetry. She revealed the specific binding sites of KN-93 on calcium-bound calmodulin (Ca2+/CaM), preventing its subsequent interaction with calmodulin kinase II (CaMKII) and impairing the onset of the phosphorylation process. Binding and kinetic properties of KN-93 (Kd, IC50 values), and other well-known CaMKII inhibitors, were studied in detail using a reliable in-vitro enzymatic assay.
See your kinase research from a different angle by exploring your inhibitors’ mechanism of action (MoA) in more detail, not only on your preferred kinase protein, but also on its protein partners. Open the door towards discovering even more potent and specific drugs!
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.
Journal of Molecular Biology. 2019 Mar 29;431(7):1440-1459.