Molecular And Pharmacological Studies Of Dopamine Receptors

National Institute Of Neurological Disorders And Stroke

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Abstract

Schizophrenia is a devastating neuropsychiatric illness impacting approximately 1% of the global population and is the 15th leading cause of disability worldwide. Current therapies treat mostly the positive symptoms and are associated with a plethora of off-target side effects such as sedation, weight gain, and diabetes, among others. Almost all FDA-approved antipsychotic medications target the D2 dopamine receptor (D2R) but also exhibit polypharmacology with other receptors. Further, there are few D2R antagonists that can selectively inhibit the D2R without also antagonizing the D3 and D4 dopamine receptors (D3R, D4R, respectively). Our lab conducted a high-throughput screen to identify D2R-selective antagonists with reduced activity at other GPCRs. This screen identified a promising hit compound that was chemically optimized into the lead drug candidates, NCGC1360 and NCGC1366. These drug leads have relatively high binding affinities at the D2R (~80 and ~50 nM, respectively), while displaying >100-fold selectivity over the D3R and >24-fold selectivity over the D4R. A β-arrestin recruitment assay revealed even higher D2R selectivity (>100-fold) of these compounds over the D3R and D4R. In a screen against an array of 46 GPCRs, channels, and transporters using radioligand binding assays, NCGC1360 and NCGC1366 only inhibited radioligand binding to the D2R. A functional screen of 168 GPCRs further determined that NCGC1360 and NCGC1366 were exceptionally selective with each compound completely antagonizing β-arrestin recruitment to the D2R with only minor effects at a few other receptors. Schild-type analyses using dopamine-stimulated β-arrestin recruitment revealed that both compounds are competitive antagonists. Pharmacokinetic studies of NCGC1360 in mice revealed a half-life of 1.8 hr in plasma and 1 hr in brain with excellent brain penetration. Oral administration to rats demonstrated excellent bioavailability and CNS penetrance with a half-life of 5.6 hr. NCGC1360 was also characterized in animal models that are predicative of antipsychotic efficacy and on-target side effects. NCGC1360 dose-dependently decreased amphetamine-induced hyperlocomotion in mice and also rescued deficits in pre-pulse inhibition induced by amphetamine. Importantly, NCGC1360 did not induce catalepsy up to the highest dose tested (10 mg/kg), suggesting that it would not induce extrapyramidal side effects in patients. Taken together, these studies provide support for continuing the development of this scaffold with preclinical and IND-enabling studies. While antagonists of D2Rs are currently used as antipsychotics, D3R-selective antagonists might serve as therapeutics for substance use disorder (SUD) as they could attenuate drug craving symptoms without the motor side effects produced by D2R antagonists, making the discovery of novel D3R-selective antagonists a priority. A challenge to this goal is posed by the high amino acid sequence homology between the D2R and D3R within their orthosteric binding sites. Our lab has 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. Cryo-EM approaches are currently being utilized to identify 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. The D1 dopamine receptor (D1R) is a G protein-coupled receptor involved in dopamine (DA) signaling and is crucial for cognition, reward, and movement. Thus, it represents a drug target for treating a plethora of neuropsychiatric disorders including Parkinson’s Disease and schizophrenia. Current therapeutic approaches mostly target the orthosteric site of the D1R to improve DA signaling, leading to the reduction of motor symptoms and/or enhanced cognition. However, orthosteric agonists of the D1R exhibit cardiovascular side effects and they can develop tolerance in patients. Due to these liabilities, we have explored the use of positive allosteric modulators (PAMs) to selectively target the D1R and enhance endogenous signaling in order to overcome the pitfalls of current D1R agonists. Previously, our lab conducted a high throughput screen of 400,000+ compounds and identified compound MLS6585 as a novel D1R PAM. Using maximally effective concentrations, MLS6585 increased the potency (EC50) of DA for the D1R by ~5-fold but had little to no effect on DA efficacy (Emax) as observed using functional assays measuring beta-arrestin recruitment to the receptor. To improve and optimize MLS6585 potency and efficacy, we synthesized and tested over 100 analogs and identified two compounds with improved PAM activity, UNC9815 and UNC10062. In beta-arrestin recruitment assays, UNC9815 increased DA potency ~10-fold but exhibited minimal efficacy enhancement. In contrast, UNC10062 increased DA potency by ~4-fold but increased the maximum efficacy (Emax) of DA by up to 200%. We utilized both UNC9815 and UNC10062 in attempts to identify the D1R binding site of this scaffold using molecular modeling, receptor chimeras, and site-directed mutagenesis experiments. Taken together, our modeling and mutagenesis data suggest that UNC9815 and UNC10062 bind to an allosteric pocket formed by residues derived from TM1and TM7 of the D1R. We have recently obtained cryo-EM structures of the D1R in complex with our D1 PAM scaffolds to delineate and validate this proposed allosteric binding site. These cryo-EM data indicated that our PAMs indeed associate with a binding pocket formed by TM1 and TM7 of the D1R. Ultimately, this knowledge will aid in the design of more potent and efficacious D1R PAMs using structure-guided analog design.