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

Human dopamine transporter (hDAT) regulates the reuptakeof extracellulardopamine (DA) and is an essential therapeutic target for central nervoussystem (CNS) diseases. The allosteric modulation of hDAT has beenidentified for decades. However, the molecular mechanism underlyingthe transportation is still elusive, which hinders the rational designof allosteric modulators against hDAT. Here, a systematic structure-basedmethod 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 therecently reported Cryo-EM structure of the human serotonin transporter(hSERT) and Gaussian-accelerated molecular dynamics (GaMD) simulationwas further utilized for the identification of intermediate energeticstable states of the transporter. Then, with the potential druggableallosteric site on hDAT in IO conformation, virtual screening of sevenenamine chemical libraries (& SIM;440,000 compounds) was processed,resulting in 10 compounds being purchased for in vitro assay and with Z1078601926 discovered to allosterically inhibithDAT (IC50 = 0.527 [0.284; 0.988] & mu;M) when nomifensinewas introduced as an orthosteric ligand. Finally, the synergisticeffect underlying the allosteric inhibition of hDAT by Z1078601926and nomifensine was explored using additional GaMD simulation andpostbinding free energy analysis. The hit compound discovered in thiswork not only provides a good starting point for lead optimizationbut also demonstrates the usability of the method for the structure-baseddiscovery of novel allosteric modulators of other therapeutic targets.

Automated summary

The study investigates the molecular mechanism underlying the transportation of human dopamine transporter (hDAT), which regulates the reuptake of extracellular dopamine (DA) and is an essential target for CNS diseases. Using a structure-based method, allosteric sites on hDAT in inward-open conformation were explored and compounds with allosteric affinity were screened. The discovery of a new allosteric inhibitor for hDAT provides a starting point for lead optimization and demonstrates the usability of the method for discovering novel allosteric modulators of other therapeutic targets.