Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity.

γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub-maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high-resolution X-ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild-type hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.

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.

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.