Discovery, Characterization, and Optimization of a Novel Positive Allosteric Modulator-Antagonist of the D3 Dopamine Receptor

Abstract ID 92916 Poster Board 479 Dopamine receptors (DARs) are responsible for a plethora of physiologic functions, including cognition, mood, movement, and motivation. Not surprisingly, DARs play a role in the etiology and/or therapy of many neuropsychiatric disorders, including schizophrenia and substance use disorder (SUD). There are five DAR subtypes divided into two categories: D1-like (D1R and D5R) and D2-like (D2R, D3R, and D4R). Current antipsychotic medications are antagonists of the D2-like subfamily. However, the lack of selectivity among these D2R-preferring antagonists frequently produce motor-related extrapyramidal side effects, often leading to a lack of compliance among patients. Compounds that selectively antagonize the D3R may lead to fewer side effects while providing effective attenuation of drug craving and/or psychotic symptoms, making the discovery of novel D3R-selective antagonists a priority. A challenge to this goal is posed by the high sequence homology between the D2R and D3R within their orthosteric binding sites. Our lab aims to overcome these selectivity challenges by identifying D3R negative allosteric modulators (NAMs), compounds that inhibit the D3R via binding to an allosteric site. To this end, we screened the NIH Molecular Libraries Program 400,000+ small molecule library and identified MLS6357 as a promising hit compound that exhibited selectivity for the D3R over the D2R in multiple signaling outputs, while displaying an allosteric mechanism of action. Interestingly, radioligand binding assays demonstrated that MLS6357 increases D3R affinity for agonists, indicating that this scaffold is a D3R positive allosteric modulator-antagonist (PAM-antagonist), a special class of NAMs that potentiate agonist-receptor binding while concomitantly decreasing agonist-stimulated receptor signaling. Iterative medicinal chemistry and functional assays were used to synthesize and characterize >100 analogs of MLS6357, with several compounds displaying several-fold increases in potency as antagonists in both β-arrestin recruitment and G-protein activation assays, compared to the parent compound MLS6357. Among them, analogs UNC8747 and UNC6869 also maintained global D3R-selectivity. Additionally, both UNC8747 and UNC6869 appeared to recapitulate the activity of MLS6357 as PAM-antagonists and exhibited brain penetrance at sufficient concentrations to occupy the D3R in vivo, making them promising candidates for in vivo behavioral testing. In addition, several other analogs displayed functional selectivity for inhibiting G-protein activation versus β-arrestin recruitment and vice versa. Site-directed mutagenesis approaches are being utilized to determine the allosteric binding site for this PAM-antagonist scaffold at the D3R. The development of a novel, potent PAM-antagonist with high selectivity for the D3R may provide a therapeutic advance for many neuropsychiatric conditions, including SUD.