An
amine-containing molecule called Compound A has been reported by a group from Bristol-Myers Squibb to act as a positive allosteric modulator (PAM) at the
dopamine D1 receptor. We synthesized the more active enantiomer of Compound A (BMS-A1) and compared it with the D1 PAMs DETQ and MLS6585, which are known to bind to intracellular loop 2 and the extracellular portion of transmembrane helix 7, respectively. Results from D1/D5 chimeras indicated that PAM activity of BMS-A1 tracked with the presence of D1 sequence in the N-terminal/extracellular region of the D1 receptor, a unique location compared with either of the other PAMs. In pairwise combinations, BMS-A1 potentiated the small allo-agonist activity of each of the other PAMs, while the triple PAM combination (in the absence of
dopamine) produced a cAMP response about 64% of the maximum produced by
dopamine. Each of the pairwise PAM combinations produced a much larger leftward shift of the
dopamine EC50 than either single PAM alone. All three PAMs in combination produced a 1000-fold leftward shift of the
dopamine curve. These results demonstrate the presence of three non-overlapping allosteric sites that cooperatively stabilize the same activated state of the human D1 receptor. SIGNIFICANCE STATEMENT: Deficiencies in
dopamine D1 receptor activation are seen in
Parkinson disease and other neuropsychiatric disorders. In this study, three positive allosteric modulators of the
dopamine D1 receptor were found to bind to distinct and separate sites, interacting synergistically with each other and
dopamine, with the triple combination causing a 1000-fold leftward shift of the response to
dopamine. These results showcase multiple opportunities to modulate D1 tone and highlight new pharmacological approaches for allosteric modulation of
G-protein-coupled receptors.