Characterization of the Binding Pocket for a Novel Positive Allosteric Modulator of the D1 Dopamine Receptor

Abstract ID 90950 Poster Board 344 The D1 dopamine receptor (D1R) is a G protein-coupled receptor essential for motivation, movement, and reward, among other functions. Deficits in dopamine (DA) signaling at the D1R are linked to several neurological disorders, such as Parkinson's disease and ADHD. Additionally, drugs that enhance DA signaling at the D1R may be beneficial for the treatment of cognitive impairment. Unfortunately, orthosteric D1R agonists have largely failed in the clinic due to poor oral bioavailability, rapid metabolism, and tolerance. However, positive allosteric modulators (PAMs) may provide an alternative and more effective treatment approach. PAMs may exhibit higher receptor selectivity by binding to less conserved regions of the D1R and maintain the tone of endogenous DA signaling. Previously, our lab conducted a high throughput screen of 380,000 small molecules to identify potential PAMs of the D1R. Following hit validation and optimization, we identified MLS6585 as the lead compound. MLS6585 potentiates DA-mediated β-arrestin signaling at the D1R by 5-fold and increases DA affinity to a similar extent in radioligand binding assays. This PAM was also found to bind to a unique, but unidentified allosteric site in the D1R. Chemical optimization of MLS6585 was performed to increase its efficacy for potentiating DA affinity and signaling at the D1R. Over 110 analogs of MLS6585 were evaluated, and UNC9815 was identified as producing greater PAM efficacy than MLS6585. In functional assays, UNC9815 increases DA potency (EC50) by 11-fold and by 6-fold in radioligand binding assays. We used UNC9815 to identify its allosteric binding site in the D1R through mutagenesis approaches. Preliminary studies suggested that the binding site for this PAM scaffold likely involves the transmembrane 7 (TM7) region of the D1R. Furthermore, Phe 319 (F319) in TM7 was identified as an important residue for PAM activity. Mutation of this residue showed a functional loss of PAM activity by UNC9815. However, radioligand binding assays suggested that UNC9815 was still binding to the mutated D1R. Together, these results suggest that F319 may be required for propagation of PAM signaling, but not for binding to the D1R. We then used molecular docking and dynamics simulations to predict and model other potential binding sites. Our results predicted a binding pocket comprised of residues from TM1, TM7 and Helix 8 (H8) of the D1R. We created three cluster mutants by mutating the predicted UNC9815 contacting residues from either TM1, TM7, or H8 together. The TM1 cluster mutant exhibited a loss of UNC9815-mediated PAM activity suggesting that these residues may be important for PAM binding. Single point mutations are currently underway to further analyze the binding pocket model involving TM1, TM7, and H8. Delineation of this site may allow for rational structure-guided analog design to aid in the discovery of therapeutically viable compounds.