GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation[1][1],[2][2]. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers[3][3]-[6][4]. Recent findings have suggested that the hub is also an allosteric determinant of kinase activity[7][5], and is thus an emerging target for therapies to correct CaMKIIα dysregulation[8][6],[9][7]. However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKIIα hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite γ-hydroxybutyrate (GHB)[10][8] we reveal that CaMKIIα is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 Å crystal structure of CaMKIIα with bound ligand. Using differential scanning fluorimetry, we show that binding of ligands to the hub interior increases the thermal stability of hub oligomers in a concentration-dependent manner. Moreover, we demonstrate the functional significance of this hub stabilization by showing substantial neuroprotective effects in cellular excitotoxicity assays and in a mouse model of cerebral ischemia. Together, our results reveal that CaMKIIα hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKIIα-selective ligands have therapeutic potential. ### Competing Interest Statement The University of Copenhagen and Otago Innovation Ltd. have licensed the patent rights for GHB derivatives and their uses (WO/2019/149329) to Ceremedy Ltd. of which B.F., B.R.K. and P.W. are co-founders. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-6 [5]: #ref-7 [6]: #ref-8 [7]: #ref-9 [8]: #ref-10