Molecular And Pharmacological Studies Of Dopamine Receptors

National Institute Of Neurological Disorders And Stroke

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Abstract

The D1 dopamine receptor (D1R) is a crucial regulator of dopaminergic signaling and is involved in neurological processes and diseases. It is an attractive target for treating neuropsychiatric disorders, however, the liabilities of orthosteric agonists have curtailed this treatment modality. Positive allosteric modulators (PAMs) of D1R are therefore an alternative drug development strategy. We discovered two structurally distinct D1R PAMs via a high-throughput screen: MLS1082 and MLS6585. Both PAMs potentiate agonist-stimulated G-protein and beta-arrestin-mediated signaling and increase dopamine affinity for the D1R, although with different maximum efficacy and estimated Kb values. Combination experiments and receptor mutagenesis studies indicated that MLS1082 acts via the previously described intracellular loop-2 (ICL2) allosteric site targeted by two known D1R PAMs, Compound B and DETQ. MLS6585, however, does not act via this ICL2 site. To identify the MLS6585 binding site, chimeras of the D1R and D2R were used. MLS6585 has no PAM activity at the D2R, so loss of potentiation from the introduction of D2R sequence was used to detect potential regions of interest. This chimeric approach identified transmembrane region 7 (TM7) of D1R as a potential site for mediating MLS6585 activity. Further, specific point mutations identified residues near the extracellular region of TM7 that are required for MLS6585 PAM activity. These mutations had no effect on the activities of other PAMs binding to the ICL2 site. We used analog sets of MLS6585 to begin to understand structure-activity relationships underlying D1R allosteric modulation. In addition to validating the MLS6585 scaffold as a D1R PAM, the analogs implicate structural moieties that are crucial for PAM activity and receptor selectivity. Together, these efforts increase our understanding of D1R allosteric modulation as a means for developing novel therapeutic interventions. Schizophrenia is a devastating illness characterized by both positive (hallucinations, delusions) and negative (flat affect, decreased motivation) symptoms coupled with cognitive impairment. Current antipsychotic medications are effective in treating the positive symptoms through antagonism of the D2 dopamine receptor (D2R). However, antipsychotic treatment is also hindered by side-effects due to off-target activities at other GPCRs and unfavorable binding kinetics at the D2R. We have identified and characterized a novel D2R antagonist with high selectivity against other GPCRs ML321. In functional profiling screens of up to 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Schild-type functional assays revealed that ML321 acts as a competitive antagonist of the D2R while kinetic studies showed that ML321 exhibits slow-on and fast-off receptor binding rates; properties that are believed to limit extrapyramidal side-effects that are commonly observed with antipsychotics. In fact, using doses that were maximally effective in antipsychotic-predictive behavioral assays in rodents, ML321 promoted little to no catalepsy, suggesting that ML321 may produce less extrapyramidal side-effects in patients. Importantly, no other D2R antagonist exhibits this pharmacological and behavioral profile supporting its development into an advanced drug lead. While a promising therapeutic, ML321 has a short metabolic half-life, impeding its clinical development. A metabolite study revealed that the primary site of metabolism involves oxidation of the alkyl-thiophene portion of ML321. To create more metabolically stable derivatives, we iteratively designed and synthesized over 100 analogs with modifications focused on the alkyl-thiophene moiety. These analogs were pharmacologically characterized for both D2R binding affinity and function, and were also tested for metabolic stability, permeability, and solubility. These efforts have led to the optimization of ML321 into a collection of lead candidates that show similar pharmacological characteristics of ML321, but with marked increases in metabolic stability and ADME properties. Molecular docking and mutagenesis studies have led to a better understanding of how ML321 binds to the D2R, which will further assist in analog design and development. Together, these findings have advanced our understanding of ML321 structure-activity relationships, particularly around the alkyl-thiophene moiety, and have identified lead candidates for in vivo pharmacokinetic studies, thus representing substantial progress in the development of a new antipsychotic treatment. Since its first use in treating Parkinsons disease (PD), L-DOPA has remained the gold standard of therapy for this disorder, defined by the progressive degeneration of dopaminergic neurons in the CNS leading to profound bradykinesia and tremor. The efficacy of L-DOPA wanes over time and is associated with increasing side effects, including motor fluctuations and dyskinesias. Several dopaminergic agonists have also been introduced to treat PD, including pramipexole and ropinirole, which exhibit fewer motor side effects but are associated with impulse control disorders such as excessive gambling and hypersexuality. Notably, the dopamine receptor subtype(s) mediating the therapeutic actions and/or side effects in PD therapy remain unknown. However, the preference of pramipexole and ropinirole for the D3 dopamine receptor (D3R) suggests the involvement of this subtype, although these drugs also activate the D2R at therapeutic doses. Importantly, no drug currently employed to treat PD alters the course of the disease and the discovery of neuroprotective agents remain an unmet need in PD therapeutics. Recently, we discovered a novel, potent and highly selective agonist for the D3R, ML417, that is brain penetrant and was found to protect against 6-OHDA-induced neurodegeneration of dopaminergic neurons (Moritz et al., J. Med. Chem. 63: 5526, 2020). In the current study, we used ML417 to probe the role of the D3R in a rat model of PD. We initially sought to investigate the role of the D3R in ameliorating bradykinesia in a hemi-parkinsonian rat model induced by 6-OHDA infusion into the medial forebrain bundle. Using a validated cylindrical treadmill test of locomotion, doses of ML417 up to 20 mg/kg had no effect on improving impairments in walking as assessed by step counts in the hemi-parkinsonian rats. In contrast, administration of L-DOPA (6 mg/kg) significantly improves locomotion in the same model. Further, pretreatment with a D3R-selective antagonist, SB277011A (30 mg/kg), did not attenuate the effects of L-DOPA in reducing bradykinesia. These results suggest that the D3R does not mediate the anti-bradykinetic effects of current PD therapeutics. However, we hypothesize that D3R stimulation may be beneficial for the treatment of L-DOPA-induced dyskinesias (LIDs) in PD. To test this, we used a chronic L-DOPA administration paradigm (12 mg/kg/day for 7 days) to induce dyskinesias in the hemi-parkinsonian rats. Subsequently, the effects of ML417 and SB277011A were assessed in these animals using an abnormal involuntary movement (AIMs) scoring method. Pretreatment with a single dose of ML417 (20 mg/kg) significantly reduced the intensity and duration of dyskinesias promoted with a single dose of L-DOPA (6 mg/kg). Further, co-administration of SB277011A (30 mg/kg) with ML417 attenuated the anti-dyskinetic effects of ML417, suggesting that the benefit is D3R-mediated. Overall, this study implies that D3R stimulation has no therapeutic effect on bradykinesia in PD, however, it may be beneficial in treating dyskinesias arising from L-DOPA therapy.