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 dopamines affinity for the D1R, though 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. A small number of seemingly inactive analogs appear to act as silent allosteric modulators (SAMs) or neutral allosteric ligands (NALs), in that they blocked the activity of the parent PAM compound, presumably by competing for the same binding site. Together, these efforts increase our understanding of D1R allosteric modulation as a means for developing novel therapeutic interventions. The D2 dopamine receptor (D2R) is one of the most validated drug targets in psychiatry and there is a strong correlation between the clinical doses of antipsychotics and their potencies for blocking D2Rs. However, current FDA-approved antipsychotic drugs typically cross-react with other GPCRs, leading to many deleterious side-effects. We have now identified and characterized a lead D2R antagonist with high GPCR selectivity, ML321. ML321 shows little GPCR cross-reactivity beyond inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Behavioral assays in rodents demonstrate ML321's efficacy in models that are predictive of antipsychotic effects in humans (e.g., attenuation of both amphetamine and PCP-induced locomotor activity and pre-pulse inhibition). Importantly, using doses that are maximally effective in antipsychotic-predictive assays, ML321 promotes little to no catalepsy, suggesting that ML321 may produce fewer extrapyramidal side effects in patients (an on-target side effect). This property may be due to unique binding kinetics of ML321 at the D2R. Importantly, no other D2R antagonist exhibits this pharmacological and behavioral profile, which supports ML321's promise as a superior therapeutic. No concerns were noted during safety/toxicity studies including hERG channel activation, cytotoxicity, an AMES test, and a CYP inhibition study. ML321 demonstrates good brain penetrability (20% vs. plasma), but also exhibits a relatively short half-life in vivo of about 2 hr. The latter property may represent an impediment to the advancement of this antipsychotic candidate into clinical studies. Approximately 75 ML321 analogs have been synthesized and are being characterized pharmacologically using D2R radioligand binding and functional assays. In parallel, these structural analogs are being assessed in vitro for ADME in order to determine their metabolic stability, solubility and predicted CNS penetrability. Overall, our expectation is that the ML321 scaffold can be optimized into an advanced drug candidate with the potential for targeting schizophrenia and other psychotic syndromes involving the D2R. Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons resulting in bradykinesia, tremor, gait abnormalities, and numerous non-motor complications. Knowledge of the molecular mechanisms underlying dopaminergic neuron death remains limited, partially due to the lack of accurate disease models. Here, we report the development of a high-throughput assay for monitoring dopaminergic neurodegeneration in Caenorhabditis elegans (C. elegans). Two strains of C. elegans containing human PD-linked genes were used, one expressing mutant (A53T) alpha-synuclein, and the other expressing mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2). Both strains express GFP in their dopaminergic neurons and were crossed into a neuronal-RNAi sensitive background expressing mCherry under a pharyngeal promoter. This allows for the accurate measurement of neuronal viability (via GFP) and a normalization and sorting control of the total number of worms plated (via mCherry). Daily laser cytometry readings of GFP/mCherry fluorescence intensity revealed that robust temporal degeneration occurs within the first eight days of adulthood in both PD models, but not in a wild-type control strain. We determined a signal (cell loss) to baseline ratio of approximately 4-fold, sufficient for screening applications. Particularly in the case of the mutant LRRK2 model, neurodegeneration is severe: between day 3 and day 8 of adulthood, total GFP fluorescence intensity drops by 75-85%. In the LRRK2 mutants, administration of selective LRRK2 inhibitors (LRRK2-IN-1 and CZC 25146) confers neuroprotection in a dose-dependent manner. Similarly, using high content imaging, we found that the LRRK2 mutant-expressing worms displayed susceptibility to RNA interference. Administration of RNAi targeting LRRK2 slowed the course of the neurodegeneration in these worms. Similarly, administration of RNAi targeting GFP resulted in about 50% reduction of the mean GFP intensity at day 5. Our results indicate that this assay provides a reproducible, high-throughput measurement of dopaminergic neurodegeneration using an in vivo model. Future studies may exploit this model to conduct quantitative high-throughput screens to identify small molecules that inhibit neurodegeneration or use RNAi libraries to identify genes mediating the neurodegenerative response, and hence new drug targets for PD treatment.