BPS2026 – Discovery of novel VGLUT inhibitors through structure-based virtual screening, molecular dynamics simulations, and experimental validation

The vesicular glutamate transporters (VGLUTs) load glutamate, the brain’s principal excitatory neurotransmitter, from the cytosol into the synaptic vesicles. Despite their central role in glutamatergic neurotransmission, the mechanism and regulation of VGLUTs are incompletely understood, and no robust pharmacological tools are available for targeting them. Several compounds have been reported as VGLUT inhibitors, but none have been conclusively demonstrated to bind selectively to VGLUTs. The discovery of novel, potent, and selective VGLUT inhibitors will help elucidate their mechanism and might open therapeutic opportunities for neurologic disorders associated with excessive glutamate release. We used the published structure of rat VGLUT2 in the open lumen-facing (OLF) state and AlphaFold-predicted open cytosol-facing (OCF) state to launch the structure-based drug design. We developed a virtual screening pipeline by integrating molecular docking, machine-learning tools, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann surface area calculations. For experimental validation, we developed a VGLUT construct with robust heterologous expression. We used the stable purified protein in thermal-shift and surface plasmon resonance binding assays, confirming several hits with micromolar affinities (3-10 uM). These compounds represent promising leads for advancing the discovery and development of potent VGLUT inhibitors.