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Abstract
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 various 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, guided by in silico approaches, we synthesized 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.
Supplementary materials
Title
Exploring the NCS-382 Scaffold for CaMKIIα Modulation: Synthesis, Pharmacology, and Biophysical Characterization of Ph-HTBA as a Novel High-Affinity Brain-Penetrant Stabilizer of the CaMKIIα Hub Domain
Description
Concentration-response curves of [3H]NCS-382 displacement (Figure S1), surface plasmon resonance binding of NCS-382, 1b and Ph-HTBA (1i) (Figure S2), thermal shift assessment of NCS-382 and 1b (Figure S3), intrinsic tryptophan fluorescence measurement of 1k (Figure S4), in vivo metabolic evaluation of Ph-HTBA after intravenous administration to mice (Figure S5), analytical HPLC traces of 1b, 1k, Ph-HTBA and the sodium salt of Ph-HTBA (Figure S6), and summarized SPR kinetic parameters for NCS-382, 1b, and Ph-HTBA (Table S1).
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