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.

钙/钙调蛋白依赖性蛋白激酶II（CaMKII）是一种多功能的丝氨酸/苏氨酸蛋白激酶，可在各种细胞过程中传递钙信号。CaMKII被钙结合的钙调蛋白（Ca ²⁺ / CaM）通过涉及CaMKII中调节性C末端α-螺旋的直接结合机制激活。Ca ²⁺ / CaM结合触发接近CaM结合位点的关键苏氨酸残基的转磷酸化作用，从而导致CaMKII的自激活状态。CaMKII在病理生理过程中的关键作用已得到证明，已成为众多疾病管理中的关键目标。分子KN-93是研究细胞和体内最广泛使用的抑制剂CaMKII的功能。普遍认为，KN-93直接与CaMKII结合，从而通过与Ca ²⁺ / CaM竞争来阻止激酶活化。在这里，我们采用表面等离子体激元共振，NMR和等温滴定量热法来表征这种假定的相互作用。我们的结果表明，KN-93直接与Ca ²⁺ / CaM结合而不与CaMKII结合。这种结合会破坏Ca ²⁺ / CaM与CaMKII相互作用的能力，从而有效地抑制CaMKII的活化。我们的发现还表明，KN-93可以与CaMKIIδ衍生的肽竞争与Ca ²⁺ / CaM的结合。如表面等离振子共振和等温滴定热分析数据所示，显然至少有两个KN-93分子可以与Ca结合²⁺ / CaM。我们的发现为如何解释用KN-93获得的体外和体内数据提供了新的见解。他们进一步建议，在基于KN-93的作用机理研究和药物发现工作中，应考虑其他依赖Ca ²⁺ / CaM的非CaMKII活性。