Exploring the NCS-382 Scaffold for CaMKIIα Modulation: Synthesis, Biochemical Pharmacology, and Biophysical Characterization of Ph-HTBA as a Novel High-Affinity Brain-Penetrant Stabilizer of the CaMKIIα Hub Domain.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a brain-relevant kinase and an emerging drug target for ischemic stroke and neurodegenerative disorders. Despite reported CaMKIIα inhibitors, their usefulness is limited by low subtype selectivity and brain permeability. (E)-2-(5-Hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (NCS-382) is structurally related to the proposed neuromodulator, γ-hydroxybutyric acid, and is a brain-penetrating high nanomolar-affinity ligand selective for the CaMKIIα hub domain. Herein, we report the first series of NCS-382 analogs displaying improved affinity and preserved brain permeability. Specifically, we present Ph-HTBA (1i) with enhanced mid-nanomolar affinity for the CaMKIIα binding site and a marked hub thermal stabilization effect along with a distinct CaMKIIα Trp403 flip upon binding. Moreover, Ph-HTBA has good cellular permeability and low microsomal clearance and shows brain permeability after systemic administration to mice, signified by a high Kp, uu value (0.85). Altogether, our study highlights Ph-HTBA as a promising candidate for CaMKIIα-associated pharmacological interventions and future clinical development.

Discovery and Optimization of 5-Hydroxy-Diclofenac toward a New Class of Ligands with Nanomolar Affinity for the CaMKIIα Hub Domain.

The Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) is a brain-relevant kinase involved in long-term potentiation and synaptic plasticity. We have recently pinpointed the CaMKIIα hub domain as the long-sought-after high-affinity target of γ-hydroxybutyrate ligands substantiated with a high-resolution cocrystal of 5-hydroxydiclofenac (3). Herein, we employed in silico approaches to rationalize and guide the synthesis and pharmacological characterization of a new series of analogues circumventing chemical stability problems associated with 3. The oxygen-bridged analogue 4d showed mid-nanomolar affinity and notable ligand-induced stabilization effects toward the CaMKIIα hub oligomer. Importantly, 4d displayed superior chemical and metabolic stability over 3 by showing excellent chemical stability in phosphate-buffered saline and high resistance to form reactive intermediates and subsequent sulfur conjugates. Altogether, our study highlights 4d as a new CaMKIIα hub high-affinity ligand with enhanced pharmacokinetic properties, representing a powerful tool compound for allosteric regulation of kinase activity with subtype specificity.