Ensemble Docking Interrogates Structural Determinants of Ligand-Protein Interactions
Final Report Abstract
The aim of the project was to investigate the interaction of small molecule allosteric modulators of the human Y4 receptor subtype. We aimed to identify the binding site of several small molecules by receptor mutagenesis and varying the chemical structure of the ligands. These small molecules have been identified in previous studies by high throughput screening approaches in collaboration with the lab of Prof. C. D. Weaver (Vanderbilt University, Nashville, USA). In collaboration with Prof. J. Meiler (Vanderbilt University, Nashville, USA), we aimed to further specify the binding site of the small molecule ligands at the receptor by computation modeling of the receptor and ligand docking. We used a number of niclosamide derivatives to elucidate the chemical properties needed for a potent modulation of the Y4 receptor signal and for receptor subtype selectivity. We identified modifications at the nitroaniline ring of niclosamide that directs the selectivity towards the Y1 and Y5 receptor, whereas modification on the benzoyl ring alter the potency of the compound at the Y4 receptor and modulate the potency of the compound at the Y1 receptor subtype. The hydroxyl group may play a role in further studies as a potential modification site, as even larger modifications did not affect the potency at the Y 4 receptor. Additionally, we could identify tertbutyl-phenoxy-cyclohexanol (tBPC) as a novel small molecule positive allosteric modulator of the human Y4 receptor. In vitro studies showed a high selectivity of this compound towards the Y 4 receptor subtype. On the NPY receptor subtypes Y1, Y2 or Y5 no detectable effect of tBPC could be observed. tBPC was shown to improve the G-protein mediated signaling and the β-arresting signaling without changing the affinity of the receptor towards the native ligand PP (pancreatic polypeptide). The positive modulatory effect could also be observed for the less potent ligands NPY (neuropeptide Y) and PYY (peptide YY). To elucidate whether tBPC is also active in a more native environment, mouse colon mucosa tissue preparations were used to examine the effect of tBPC. Mouse colon mucosa endogenously express Y4 and the receptor response was monitored by using an electrophysiological approach. The compound could potentiate the effect of PP and was inactive in combination with PYY or VIP, underlining its selectivity towards the Y4 receptor and the native ligand PP.
