Negative allosteric modulators of the D3 dopamine receptor as therapeutic leads for substance use disorders

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Title: Negative allosteric modulators of the D3 dopamine receptor as therapeutic leads for substance use disorders Summary: Dopamine receptors (DRs) play a critical role in cell signaling processes and modulation of information transfer within the nervous system. DRs comprise five distinct subtypes subdivided into two families, D1-like (D1R and D5R) and D2-like (D2R, D3R, and D4R), that produce profoundly diverse physiological effects. In particular, D3R antagonists have been investigated as a therapeutic approach to treating substance use disorders (SUDs), both in disrupting drug seeking motivation and preventing relapse. Drug overdose deaths have more than doubled in the past decade driven largely by rising opioid abuse, underscoring the urgent need to identify new SUD therapies that are effective for opioid SUD. Recent evidence suggests that D3R antagonism may be an especially effective treatment for opioid SUD. However, the high sequence homology shared by the D3R and other GPCRs within their orthosteric binding sites has made the discovery of highly selective compounds difficult, leading to the potential for off-target side effects due to simultaneous receptor modulation by such agents. It is now appreciated that, in addition to highly conserved orthosteric sites, many G protein-coupled receptors, including DRs, possess distinct and non-conserved allosteric sites. Thus, compounds that modulate receptors through the interaction with an allosteric site have the potential to be profoundly selective. Here we seek to develop structurally novel D3R negative allosteric modulators that possess exceptional D3R selectivity as new chemical probes and therapeutic leads, in an overarching goal to support development of a D3R antagonist SUD therapy. In an effort to discover highly selective allosteric antagonists for the D3R, we employed a high-throughput screen of the NIH small molecule library. The library was initially screened using a D3R-mediated β-arrestin recruitment assay. Antagonist hits were counter-screened for radioligand displacement using an orthosteric ligand, which led to the identification of three promising hit compounds with allosteric properties. In this study, we will develop flexible, robust synthetic routes to each scaffold to facilitate medicinal chemistry. Analogs of the three scaffolds will be evaluated for potency, D3R selectivity and preliminary in vitro pharmacokinetic properties to identify the most promising series to advance for iterative medicinal chemistry optimization. The most promising series will be further optimized with an emphasis on the requisite properties for an in vivo probe compound. The probe will be intensively characterized for D3R selectivity in several functional outputs including β-arrestin recruitment and G-protein activation. Schild-type functional assays will be used to confirm that this compound acts in a non- competitive manner at the D3R. Finally, in vivo pharmacokinetic experiments will provide a guide for compound dosing. The probe compound will be used to study the pharmacology of D3R allosteric sites, the therapeutic potential of D3R antagonists and provide new therapeutic leads for SUDs.