Structure–Activity Relationships of a Brain-Penetrant D3 Dopamine Receptor Negative Allosteric Modulator and Investigation of its Binding Site

Abstract

Poster Board 281Dopamine receptors (DARs) are G-protein coupled receptors (GPCRs) that regulate diverse physiological functions including cognition, mood, movement, and reward-related behaviors, and are involved in the treatment or etiology of many neuropsychiatric disorders including schizophrenia and substance use disorder (SUD). DARs are classified as either D1-like (D1R and D5R) or D2like (D2R, D3R, and D4R) based on structural homology and pharmacological profiles. Antagonists of D2-like DARs are currently used in the therapies for many neuropsychiatric disorders, but D3R-selective antagonists may be better therapeutics for schizophrenia or SUD as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R-preferring antagonists due to limited distribution of the D3R in the brain. However, discovery of D3R-selective compounds is challenging due to high sequence homology of the D2R and D3R within their orthosteric binding sites, leading to the potential for off-target side effects produced by currently available compounds due to simultaneous antagonism of both subtypes or other closely related receptors. Our lab has endeavored to overcome the selectivity challenges posed by orthosteric antagonists by utilizing a D3R-mediated b-arrestin recruitment assay to screen the NIH Molecular Libraries Program 400,000+ small molecule library for compounds that inhibit the D3R via binding to less conserved allosteric sites. The most potent hit compound, MLS6357, was selective for the D3R versus the D2R and D4R in several functional outputs including b-arrestin recruitment and G-protein activation. 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 showed that MLS6357 acts as a purely non-competitive negative allosteric modulator (NAM) of the D3R. We synthesized and characterized > 70 analogs of MLS6357 using iterative medicinal chemistry approaches which produced analogs that are 100-fold and 60-fold more potent than the parent compound in D3R-mediated b-arrestin recruitment and G-protein activation assays, respectively, and revealed structure-activity relationships for further optimization of the scaffold. Moreover, some analogs appear to display functional selectivity for inhibition of G-protein activation versus inhibition of b-arrestin recruitment, and vice versa, and some also display inverse agonist activity in G-protein signaling assays. Using in vivo pharmacokinetic experiments in mice via i.p. administration, one of the lead analogs was found to be brain penetrant and achieved sufficient concentrations to occupy the D3R in vivo. To identify the allosteric binding site for the MLS6357 scaffold on the D3R, we utilized various D3R/D2R chimeras, receptor mutants, and molecular modeling techniques to reveal and characterize receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will ...