Structure-Based Discovery of a Novel Allosteric Inhibitor against Human Dopamine Transporter.

Human dopamine transporter (hDAT) regulates the reuptake of extracellular dopamine (DA) and is an essential therapeutic target for central nervous system (CNS) diseases. The allosteric modulation of hDAT has been identified for decades. However, the molecular mechanism underlying the transportation is still elusive, which hinders the rational design of allosteric modulators against hDAT. Here, a systematic structure-based method was performed to explore allosteric sites on hDAT in inward-open (IO) conformation and to screen compounds with allosteric affinity. First, the model of the hDAT structure was constructed based on the recently reported Cryo-EM structure of the human serotonin transporter (hSERT) and Gaussian-accelerated molecular dynamics (GaMD) simulation was further utilized for the identification of intermediate energetic stable states of the transporter. Then, with the potential druggable allosteric site on hDAT in IO conformation, virtual screening of seven enamine chemical libraries (∼440,000 compounds) was processed, resulting in 10 compounds being purchased for in vitro assay and with Z1078601926 discovered to allosterically inhibit hDAT (IC50 = 0.527 [0.284; 0.988] µM) when nomifensine was introduced as an orthosteric ligand. Finally, the synergistic effect underlying the allosteric inhibition of hDAT by Z1078601926 and nomifensine was explored using additional GaMD simulation and postbinding free energy analysis. The hit compound discovered in this work not only provides a good starting point for lead optimization but also demonstrates the usability of the method for the structure-based discovery of novel allosteric modulators of other therapeutic targets.

Molecular Mechanism for the Allosteric Inhibition of the Human Serotonin Transporter by Antidepressant Escitalopram.

Human serotine transporter (hSERT) is one of the most influential drug targets, and its allosteric modulators (e.g., escitalopram) have emerged to be the next-generation medication for psychiatric disorders. However, the molecular mechanism underlying the allosteric modulation of hSERT is still elusive. Here, the simulation strategies of conventional (cMD) and steered (SMD) molecular dynamics were applied to investigate this molecular mechanism from distinct perspectives. First, cMD simulations revealed that escitalopram's binding to hSERT's allosteric site simultaneously enhanced its binding to the orthosteric site. Then, SMD simulation identified that the occupation of hSERT's allosteric site by escitalopram could also block its dissociation from the orthosteric site. Finally, by comparing the simulated structures of two hSERT-escitalopram complexes with and without allosteric modulation, a new conformational coupling between an extracellular (Arg104-Glu494) and an intracellular (Lys490-Glu494) salt bridge was identified. In summary, this study explored the mechanism underlying the allosteric modulation of hSERT by collectively applying two MD simulation strategies, which could facilitate our understanding of the allosteric modulations of not only hSERT but also other clinically important therapeutic targets.