In Silico Evaluation of the Effects of Temperature on the Affinity of the SV2C Ligand UCB-1A to SV2 Paralogs

Abstract Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinson’s disease. In the attempt to develop a ligand selective for SV2C, and i in collaboration with UCB Biopharma, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from 4 to 37 °C. UCB-1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study, we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB-1A affinity for SV2A decreased significantly at 37 °C compared with 4 °C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 37 °C showed that UCB-1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 Å stability cutoff, whereas UCB-1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB-1A adopts a conserved binding pose across all paralogs stabilized by π–π stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB-1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.