Abstract by Stine Juul Gauger

The Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major mediator of synaptic plasticity underlying learning and memory. Despite its association with pathological conditions such as ischemic stroke and neurological disorders, CaMKIIα as a potential drug target has been largely under-explored. Recently, it was discovered that the neuromodulator γ-hydroxybutyrate (GHB) and GHB analogues selectively target the CaMKIIα hub domain, responsible for holoenzyme organization. These small molecules are first in class to show isozyme selectivity and serve as a promising strategy to modulate CaMKIIα functionality. By interaction in the bottom of the hub cavity, GHB analogues mediate stabilization of recombinant CaMKIIα hub, postulated to affect CaMKIIα functionality and contribute to the neuroprotective properties shown in vivo.
This PhD project aimed to investigate molecular interactions in the hub cavity and the consequence of GHB analogue binding under native conditions. First, development of a new tritium labelled tool ([3H]O-5-HDC) and site-directed mutagenesis, pinpointed residues essential for selective CaMKIIα binding of GHB analogues. These were localized in two separate regions of the CaMKIIα hub cavity, directly or indirectly affecting ligand interaction. The hub cavity space and regional interplay was further explored by chemical extension of GHB analogues to accommodate additional binding interactions in the upper part of the hub cavity. Two compounds showed a noticeable binding mode with molecular interactions that yielded low nanomolar binding affinity and induced a unique hub conformation yet to be further explored. Based on these insights, development of conformationally selective GHB analogues could potentially be relevant in conditions of different CaMKIIα dysregulation to induce distinct modulatory actions. Although the GHB analogues interact via the same CaMKIIα binding pocket, a conformational shift in the hub domain structure has only been reported for the larger sized GHB analogues, highlighting a potentially different functional consequence dependent on the ligand type. Yet, the smaller ligand HOCPCA and the bicyclic analogue Ph-HTBA were both unable to improve behavioural phenotypes in a mouse model of the neurodevelopmental disorder Angelman Syndrome, where CaMKIIα dysregulation has been reported. To elucidate the functional relevance and unveil the therapeutical potential of the various types of GHB analogues, still further studies on both the molecular and cellular level are needed.