Discovery of a new allosteric inhibitor for human dopamine transporter by physics-based modeling and experimental validation

The Dopamine Transporter (DAT) is a pivotal target in the management of central nervous system (CNS) disorders. Existing drugs that competitively bind to the orthosteric site on DAT are associated with several side effects. Although the concept of allostery in DAT has been acknowledged for years, the precise allosteric sites remain elusive, impeding the rational design of allosteric modulators for DAT. In this study, we utilized a conformation-guided strategy to discover new allosteric modulators for the human DAT (hDAT). Starting with the recently reported cryo-EM structure of hDAT in the occluded (OC) state, several potential allosteric sites were mapped on the transporter. Then, a druggable pocket located at the extracellular vestibule was selected to perform virtual screening against four Enamine libraries (106,083 compounds), which led to the selection of four compounds for fluorescence-based substrate uptake assays. Among these, Z236004662 (DAM-001) from Enamine CNS Library was found as a new allosteric inhibitor of hDAT with an IC₅₀ of 24.70 [6.472; 153.2] μM and 1.026 [0.616; 1.769] μM in the absence and presence of orthosteric inhibitor nomifensine. Further comprehensive molecular dynamics (MD) simulations and end-point binding free energy analysis were conducted to elucidate the synergistic effect of DAM-001 and nomifensine on hDAT allosteric inhibition. Additional supervised MD (SuMD) was used to reveal the specific recognition and spontaneously binding pathways of DAM-001 to the allosteric site on hDAT. This work demonstrates the potential of our method for identifying new allosteric modulators for related therapeutic targets.

Allosteric site; Dopamine transporter; Drug design; Molecular dynamics simulation; Virtual screening.