In silico reconstruction of human dopamine transporter and design of novel neuroprotective drugs for Parkinson’s disease

Abstract

Abnormally folded alpha-synuclein protein, dysfunctional mitochondria, increased oxidative stress and reduced dopamine neurotransmitter synthesis are a ll extremely well characterized phenomena in Parkinson’s disease (PD) and are thought to be interconnected. While direct targeting of these areas has demonstrated neuroprotection in vitro and in vivo, there has been a major lack of success in clinical trials. A critical component in the failure of these clinical trials is the inability to specifically target drugs to dopamine producing neurons in the brain. New drugs targeting the dopaminergic neurons by specific uptake through the human dopamine transporter (hDAT) could represent a viable strategy for establishing selective neuroprotection. Molecules able to increase the bioactive amount of extracellular dopamine, thereby enhancing and compensating a loss of dopaminergic neurotransmission, and to exert neuroprotective response because of their accumulation in the cytoplasm, are required. By means of homology modeling, molecular docking and molecular dynamics simulations, we have generated 3D structure models of hDAT in complex with substrate and inhibitors. Our results clearly reveal differences in binding kinetics of these compounds to the hDAT in the open and closed conformations, critical for future drug design. The established in silico approach allowed the identification of three promising substrate compounds that were subsequently analyzed for their efficiency in inhibiting hDAT-dependent fluorescent substrate uptake, through in vitro live cell imaging experiments. Taken together, our work presents the first implementation of a combined in silico/in vitro-approach enabling the selection of promising dopaminergic neuron specific substrates.