Molecular Mechanism of Chiral Recognition of R- and S-Methamphetamine by the Human Dopamine Transporter: Insights from Thermodynamic and Kinetic Analyses

Methamphetamine (METH) exerts its stimulant and addictive effects primarily through interactions with the human dopamine transporter (hDAT), yet the molecular origins of the pronounced stereoselectivity between its S- and R-enantiomers remain poorly understood. Here, we employed all-atom molecular dynamics simulations combined with free-energy calculations and kinetic analyses to elucidate the atomic-level mechanisms underlying the stereoselective recognition of METH by hDAT. Our results reveal that S-METH binds more strongly and forms more stable interaction networks with outward-facing and occluded conformations of hDAT than R-METH. Kinetic analyses further show that S-METH associates with the outward-facing transporter more rapidly than R-METH. Importantly, S-METH promotes a thermodynamically favorable and kinetically accelerated transition from the outward-facing to the occluded state while simultaneously increasing the free-energy barrier and slowing the transition toward the inward-facing state. This dual effect effectively traps hDAT in a long-lived occluded state. In contrast, R-METH exhibits weaker binding interactions and less pronounced modulation of transporter conformational dynamics. Together, these findings provide a comprehensive thermodynamic and kinetic framework for understanding the chiral selectivity of hDAT toward METH enantiomers and offer mechanistic insight into the markedly different pharmacological potencies of S- and R-METH. The mechanistic principles revealed here may inform the rational design of dopamine transporter modulators with optimized binding thermodynamics and conformational kinetic profiles.