Discovery and Optimization of ML417: A Potent and Highly Selective D3 Dopamine Receptor Agonist
Authors: Moritz Amy E. , Free R. Benjamin , Weiner Warren , Akano Emannuel , Bachani Muzna , Doyle Trevor , Southall Noel , Ferrer Marc , Javitch Jonathan A. , Steiner Joseph , Aubé Jeffrey , Frankowski Kevin , Sibley David R. 
Published: November 2023

Dopamine receptors (DARs) regulate diverse physiological functions including cognition, movement, and reward‐related behaviors, and are involved in the development and/or treatment of neuropsychiatric disorders including schizophrenia and Parkinson's disease (PD). They are composed of two classes based on structure and pharmacology: D1‐like (D1R and D5R) and D2‐like (D2R, D3R, and D4R). The D2R and D3R display high sequence homology within their orthosteric binding sites where the endogenous agonist dopamine binds, making discovery of truly subtype selective compounds difficult. In addition, known D2R‐like ligands can also exhibit high degrees of cross reactivity with closely related GPCRs, leading to the potential for side effects and uncertainty as to the roles each DAR subtype plays in normal and pathological processes. Thus, a highly selective D3R agonist that lacks efficacy at other DARs may have great utility for both therapeutic and basic science pursuits. As such, our lab sought to discover selective D3R agonists by employing high throughput screening paradigms using the NIH Molecular Libraries Program 400,000+ small molecule library and a D3R enzyme complementation‐based β‐arrestin recruitment assay. Hits were counter screened against the D2R to allow elucidation of compounds that activate the D3R without effects on the D2R. Orthogonal confirmation and counter‐screens were also performed to obtain an initial assessment of selectivity and mechanisms of action. We identified 62 potentially selective hit compounds. The most promising compound was chosen for a full optimization study and investigation of its structure‐activity relationships. 375 analogs were synthesized and screened in the β‐arrestin assay in an effort to increase both affinity and selectivity. The lead compound identified through this process, ML417, acts as a full agonist at the D3R with a potency of 36 nM, while having minimal effects on D2R‐mediated β‐arrestin translocation. ML417 also acts as a potent and selective D3R agonist in a separate BRET‐based assay of β‐arrestin recruitment. Importantly, the compound also exhibits potent and selective agonist activity in D3R‐G protein‐mediated signaling responses as demonstrated using Go‐BRET‐based assays and pERK assays. ML417 was further assessed for receptor cross‐reactivity using panels of other GPCRs, and was found to have limited liability for either functional interactions with, or displacement of a radioligand from numerous GPCRs. As D3R‐preferring orthosteric agonists show promise as neuroprotective and neurorestorative agents, we conducted preliminary studies using ML417 in a neuroprotective assay, and found that it displays neuroprotective properties. This highly selective and potent D3R agonist will prove useful as a research tool to identify biological processes governed by D3R activation, and may show utility as a therapeutic drug lead.

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