Positive Allosteric Modulators of the D 1 Dopamine Receptor Act at Diverse Binding Sites

The D 1 dopamine receptor (D1R) and its signaling is associated with several important neurological processes and neuropsychiatric disorders. Enhancing D1R signaling in the prefrontal cortex is associated with increased cognition and therefore is an appealing treatment strategy for the cognitive decline observed in schizophrenia, Alzheimer's disease, and other disorders. Because of the clinical liability inherent with D1R agonists, positive allosteric modulators of the D1R have been proposed as an alternative, due to their potential for high selectivity and larger therapeutic windows. Currently, the location and characteristics of potential binding site(s) for allosteric modulators on the D1R is unknown. We have identified two structurally diverse D1R positive allosteric modulators, MLS1082 and MLS6585, via a high throughput screen of the NIH Molecular Libraries Program. The compounds potentiate dopamine‐stimulated G‐protein‐ (cAMP stimulation, 3–5 fold) and β‐arrestin‐mediated (6–8 fold) signaling pathways and increase the binding affinity of dopamine for the D1R (3–6 fold). Neither compound displayed agonist activity in the absence of dopamine. Taking advantage of our structurally distinct potentiators, G‐protein signaling and β‐arrestin recruitment experiments using maximally effective concentrations of MLS6585 and MLS1082 in combination were used to determine if the compounds act at similar or separate sites. In combination, MLS1082 + MLS6585 caused an additive potentiation of the potency of dopamine beyond that caused by either compound alone for both β‐arrestin recruitment (11–20 fold) and cAMP accumulation (4–6 fold). This suggests that the two compounds are acting at separate sites on the receptor. We also observed similar results using analogs of the two compounds, with analogs of MLS6585 having additive activity with MLS1082 and vice‐versa. However, analogs are not additive with their parent compound, meaning the parent compounds and their analogs act at similar sites. The combination experiments were repeated with Compound B, a known D1R positive allosteric modulator. Compound B was additive with MLS6585 but not MLS1082, further suggesting that the D1R has two separate positive allosteric modulator binding sites. Point mutation studies are currently underway to identify the location of these putative sites as well as whether these sites may contribute separately to potentiation of signaling efficacy versus dopamine binding affinity. Support or Funding Information NINDS IRP This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .