In silico evaluation of the effects of temperature on the affinity of the SV2C ligand UCB-1A to SV2 isoforms
Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, 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 degrees. UCB1A 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 UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} 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 UCB1A 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.