GHB analogs confer neuroprotection through specific interaction with the CaMKII alpha hub domain

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GHB analogs confer neuroprotection through specific interaction with the CaMKII alpha hub domain

Ca two plus calmodulin-dependent protein kinase II alpha subunit is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKII alpha 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 gamma-hydroxybutyrate bind selectively to CaMKII alpha. By means of a two point two-angstrom x-ray crystal structure of ligand-bound CaMKII alpha 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 CaMKII alpha 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. Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKII alpha 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.

The calcium calmodulin-dependent protein kinase II alpha subunit is a central mediator of synaptic plasticity and responds to minute fluctuations in calcium. The CaMKII alpha holoenzyme is a large protein assembly of twelve to fourteen subunits, each consisting of a kinase domain flexibly linked to the central hub domain. The hub domain is conserved through evolution. It organizes the holoenzyme into oligomeric structures, yet displays remarkable dynamics. This correlates well with an emerging functional importance in activation-triggered destabilization and release of vertical dimers that may enable spreading of activity. Furthermore, the hub domain has been reported to interact directly with the kinase domains to confer allosteric control of kinase activity. The importance of preserving hub integrity is further evident from a human patient with a mutation in the hub causing defective oligomerization and severe neurodevelopmental defects. Thus far, pharmacological modulation of the hub domain has never been demonstrated but would constitute an attractive approach to regulate overall kinase function in cases of CaMKII alpha aberrant activity.

Functionally, CaMKII alpha is activated in a highly cooperative manner, initiated by increases in intracellular calcium, calcium calmodulin binding, and autophosphorylation at residue Thr286 in the regulatory segment. This is then accompanied by translocation of CaMKII alpha to the postsynaptic density. In cases of excessive stimuli, such as ischemic brain injury or glutamate-mediated excitotoxicity, Thr286 autophosphorylation permits calcium calmodulin-independent

Significance

GHB is a natural brain metabolite of GABA, previously reported to be neuroprotective. However, the high-affinity binding site for GHB has remained elusive for almost forty years. We here unveil CaMKII alpha, a highly important neuronal kinase, as the long-sought-after GHB high-affinity target. Via a specific interaction within the central hub domain of CaMKII alpha, GHB analogs act to stabilize the hub oligomer complex. This interaction potentially explains pronounced neuroprotective effects of GHB analogs in cultured neurons exposed to a chemical insult and in mice exposed to ischemia. The postischemic treatment effects of GHB analogs underline these compounds as selective and high-affinity potential drug candidates and CaMKII alpha as a relevant pharmacological target for stroke therapy.

autonomous activity which can persist for hours and cause cell death.

The natural brain substance gamma-hydroxybutyrate is a metabolite of gamma-aminobutyric acid which has been reported to be neuroprotective in mammals. GHB binds with high affinity to an until-now unknown specific binding protein highly expressed in forebrain regions. This site is distinct from GABA B receptors also known to bind GHB, albeit with low affinity. We here reveal CaMKII alpha as the long-sought-after specific GHB high-affinity binding site. Moreover, we show that the highly selective and brain-penetrant GHB analog three-hydroxycyclopent-one-enecarboxylic acid confers significant neuroprotection in pathological states of CaMKII activation, such as after an ischemic injury. This is plausibly explained by the pronounced effect of GHB analogs on CaMKII alpha hub stabilization upon binding and consequently functional regulation of the holoenzyme, although a causal link remains to be fully proven.

Results

Discussion

Materials and Methods

Overview

The document details groundbreaking findings on the neuroprotective properties of GHB analogs through their specific interactions with the CaMKII alpha hub domain. These interactions promote enhanced stability and functionality of the holoenzyme, presenting GHB analogs as promising candidates for therapeutic applications in cerebral ischemia.

Key Points

1GHB analogs bind selectively to the CaMKII alpha hub domain
2This binding increases the stability of the holoenzyme complex
3The interaction provides significant neuroprotection in pathological states
4Detailed molecular insights were obtained through x-ray crystallography
5GHB analogs may serve as potential therapeutic agents for stroke therapy.

Details

Authors: Ulrike Leurs, Anders B. Klein, Ethan D. McSpadden, Nane Griem-Krey, Sara M. Ø. Solbak, Josh Houltone, Inge S. Villumsen, Stine B. Vogensena, Louise Hamborg, Stine J. Gauger, Line B. Palmela, Anne Sofie G. Larsen, Mohamed A. Shehata, Christian D. Kelstrup, Jesper V. Olsen, Anders Bacha, Robert O. Burnie, D. Steven Kerr, Emma K. Gowin, Selina M. W. Teurlings, Chris C. Chib, Christine L. Geeb, Bente Frølund, Birgitte R. Kornum, Geeske M. van Woerden, Rasmus P. Clausen, John Kuriyan, Andrew N. Clark, Petrine Wellendorph
Category: Biology and Natural Sciences

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