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Structure‐Activity Relationships of a Negative Allosteric Modulator of the D3 Dopamine Receptor and Investigation of its Binding Site

Dopamine receptors (DARs) are G protein‐coupled receptors (GPCRs) that regulate diverse physiological functions including movement, cognition, mood, and reward‐related behaviors, and are involved in the treatment or etiology of many neuropsychiatric disorders including schizophrenia, substance use disorder (SUD), and Parkinson’s disease. DARs are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. Antagonists of D2‐like DARs are currently used as drug therapies for many psychiatric disorders. While the D2R is highly expressed in the dorsal striatum, which regulates movement, the D3R has a more limited distribution to limbic regions associated with the control of mood and emotion. D3R‐selective antagonists may therefore be useful as schizophrenia or SUD therapeutics as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R‐preferring antagonists. Discovery of subtype selective compounds for the D3R and D2R has been challenging due to high sequence homology within their orthosteric binding sites. However, compounds that modulate receptor activity through interactions with a less conserved allosteric site of the receptor have the potential to be highly selective. To find highly selective allosteric antagonists of the D3R, our lab utilized a high‐throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library using a D3R‐mediated β‐arrestin recruitment assay. We found one compound, MLS6357, that was selective for the D3R versus the D2R in several functional outputs including β‐arrestin recruitment and G‐protein activation. Further, radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross‐reactivity with other GPCRs. Additionally, Schild‐type functional assays found that MLS6357 is a purely non‐competitive negative allosteric modulator of the D3R. We synthesized 46 analogs of MLS6357 using iterative medicinal chemistry and tested them for activity which revealed structure activity‐relationships and enabled further refinement of the scaffold. These efforts produced modulators that are 5‐ to 9‐fold more potent than the original hit compound. To identify the allosteric site on the D3R, we utilized D3R/D2R chimeras which revealed receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R‐related pathophysiology.