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.