Structural basis for the functions of dopamine receptors, dopamine transporter, and sigma 1 receptor

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Dopamine Receptors Dopamine plays a major role in the regulation of cognitive, emotional and behavioral functions abnormalities in its regulation have been implicated in neuropsychiatric and substance use disorders. Dopamine receptors belong to G protein-coupled receptor (GPCR) family. The members of dopamine D2-like receptor subgroup (consisting of D2R, D3R, and D4R) are implicated in various vital physiological functions, including voluntary movement, reward, sleep, memory, learning, and pleasure. That D3R expression is elevated in response to drugs of abuse, has prompted efforts toward the development of D3R-selective agents for the treatment of drug addiction. Inhibition of D3R may be less prone to causing motor side effects that can result from D2R blockade. Currently we focus on investigating the intermediate conformational states between the crystallographically identified inactive and active states, and the changes in the interaction network that mediate the transitions among these states, in order to reveal the structural basis for the partial agonism of selected ligands, the allosteric property of a negative allosteric modulator (NAM) and the of Na+ sensitivity of selected antagonists and NAM. By using the experimental functional assays being adapted and developed in my group (see Method Development), we will achieve efficient synergy between the in vitro and in silico characterizations of i) ligand binding in D3R, D2R, and other dopamine receptors, and ii) the functional crosstalks between receptors and different signaling pathways, i.e., G-protein vs. -arrestin, and the selectivity among various types of G-proteins. Such efforts, in close collaboration with medicinal chemists, will lead towards achieving the goal of tailoring desired specificity and efficacy, not only for the receptor subtypes, but also for the targeted signaling pathways. Dopamine and Serotonin Transporters DAT and SERT belong to the Neurotransmitter:Sodium Symporter (NSS) family, and serve to terminate dopamine and serotonin neurotransmission respectively, by recycling released neurotransmitters back into the presynaptic neuron. DAT is the primary target for abused psychostimulants such as cocaine and methamphetamine, whereas 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) binds to SERT. An understanding of the full spectrum of functional states and their transitions in a transporter cycle is required to understand the functions of these proteins and the complexity of the ligand binding modes, in order to identify and eventually exploit the therapeutic opportunities in reducing the efficacy of the abused drugs. Thus, the varied inhibition mechanisms of inhibitors are of particular interest in developing targeted and effective therapeutic interventions for drug abuse and other psychiatric disorders. Currently we focus on investigating the structural basis of the atypical DAT inhibitors that may stabilize the transporter in inward-facing conformations, as well as the molecular mechanisms of the allosteric modulations of NSS by ligands targeting the EV, using MhsT and SERT as model systems. Similar to targeting the SBPs of D3R and D2R (see above), allosteric modulation achieved by targeting the S2 site has obvious advantages over competitive inhibitions of substrate binding in the canonical S1 site, in terms retaining the normal functions of DAT and SERT. The resulting molecular models stably bound with either S2:substrate or S2:inhibitor will serve to optimize the allosteric inhibitors. Indeed, in collaboration with medicinal chemists, we have begun to identify novel SERT S2-specific inhibitors with even higher affinities. Our mechanistic understanding of the allosteric communications between the S1 and S2 sites will guide to further refine the allosteric inhibition properties, e.g., either sensitive or insensitive to the ligand bound in the S1 site. Sigma 1 Receptor The sigma 1 receptor (1R) is a structurally unique transmembrane protein that functions as a molecular chaperone. It is located at the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM), and has been found to translocate to the plasma membrane and other parts of the cell, and modulates the functions of a number of ion channels, receptors, or kinases, including targets relevant to drug abuse such as dopamine receptors and DAT. Dysfunctions of such modulations are connected to many neurological disorders. In particular, 1R has been implicated in cocaine abuse. Cocaine shows biochemical affinity and pharmacological activity at both DAT and 1R. It has been demonstrated that compounds that can antagonize the action of cocaine at both sites may have therapeutic potential of cocaine abuse. The behavioral effects of the 1R drugs in the animal models of psychostimulant dependence are quite remarkable 1R agonists can substitute for cocaine in self-administration, and the antagonists can block the self-administration interestingly, a few DAT inhibitors have high affinities for both DAT and 1R (e.g., those described in and aforementioned JHW007). However, the 1R pharmacology and the synergy of the ligands on other targets, in particular DAT, are ill-defined at molecular and cellular levels. Currently we focus on using a combined experimental and computational approach to study the molecular interactions between 1R and its ligands based on the newly available crystal structure. We use molecular modeling and simulations and experimental molecular biophysics approaches to characterize ligand-induced conformational and oligomerizational changes of 1R, and to investigate its interactions with the client proteins that have high-resolution structural information, such as BiP and DAT. Method Development The research on drug abuse needs to accommodate new drugs and drug use trends, such as the designer drugs that contribute to the current opioid crisis. In order to develop therapeutic and/or overdose reversal procedures, efficient characterizing the efficacy of abused compounds requires adequate mechanistic understanding of the usual targets of the abused drugs, and the functional crosstalk among these targets from molecular and cellular levels. Thus, in addition to contributing to the corresponding scientific fields in general, the computational and experimental approaches being developed below will serve to streamline the characterization of the drug-protein interactions, to identify related mechanism features, and to eventually establish mechanistic interaction framework of the targets. MD simulations is the basic computational technique we are using to rigorously characterize ligand-protein interactions. While we have been using the MSM analysis to integrate the simulations data together, we have just started to guide MD simulations with MSM to efficiently sample the energy surfaces by avoiding well-sampled regions. Although the MSM-based adaptive sampling has been proposed, no infrastructure is publicly available, and such an infrastructure depends on the operating system setup and hardware composition. Thus, we are establishing an automated procedure to identify the under-sampled microstates for any given simulated condition from MSM analysis as the starting point for additional MD simulations. This is part of our efforts towards the long-term goal of high-throughput MD to efficiently study (ligand-induced) multiple conformational states of a protein target.