The CaMKIIα hub ligand Ph-HTBA promotes neuroprotection after focal ischemic stroke by a distinct molecular interaction.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a potential target for acute neuroprotection due to its key role in physiological and pathological glutamate signaling. The hub domain organizes the CaMKII holoenzyme into large oligomers, and additional functional effects on holoenzyme activation have lately emerged. We recently reported that compounds related to the proposed neuromodulator γ-hydroxybutyrate (GHB) selectively bind to the CaMKIIα hub domain and increase hub thermal stabilization, which is believed to have functional consequences and to mediate neuroprotection. However, the detailed molecular mechanism is unknown. In this study, we functionally characterize the novel and brain permeable GHB analog (E)-2-(5-hydroxy-2-phenyl-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (Ph-HTBA). Administration of a single dose of Ph-HTBA at a clinically relevant time point (3-6 h after photothrombotic stroke) promotes neuroprotection with a superior effect at low doses compared to the smaller GHB analog 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA). In contrast to HOCPCA, Ph-HTBA reduces Ca2+-stimulated CaMKIIα Thr286 autophosphorylation in primary cortical neurons and substrate phosphorylation of recombinant CaMKIIα, potentially contributing to its neuroprotective effect. Supported by previous in silico docking studies, we suggest that Ph-HTBA makes distinct molecular interactions with the hub cavity, which may contribute to its differential functional profile and superior neuroprotective effect compared to HOCPCA. Together, this highlights Ph-HTBA as a promising tool to study hub functionality, but also as a good candidate for clinical development.

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain.

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.