Abstract
The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a β2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state β2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.
Highlights
Positive allosteric modulators (PAMs) are a promising approach for amplifying physiologic control circuits
We identified CID 2886111 from its close structural similarity to CID 2862078 [6-tert-butyl-2-(thiophene-2-carbonylamino)-4,5,6,7-tetrahydro1-benzothiophene-3-carboxamide], which was reported to be active in a D1 PAM assay by the Sibley group at the National Institutes of Health (Luderman et al, 2016)
Results with D1 receptor chimeras and mutants show that the D1 PAM DETQ occupies a cleft in intracellular loop 2 (IC2) as previously described for compound B (Lewis et al, 2015)
Summary
Positive allosteric modulators (PAMs) are a promising approach for amplifying physiologic control circuits. This study describes an intracellular binding site for DETQ [2-(2,6-dichlorophenyl)1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a PAM of the dopamine D1 receptor (Beadle et al, 2014; Svensson et al, 2017; Bruns et al, 2018). Binding of agonist to the activated state traps the receptor in this state, causing accumulation of activated receptors that mediate a downstream response. If a second allosteric site exists, ligands that bind there can act as positive or negative allosteric modulators (PAMs or NAMs). A PAM has higher affinity for the activated state than the inactive state and will synergize with an orthosteric agonist, increasing its affinity and/or efficacy. A NAM has higher affinity for the inactive state than the activated state and will decrease the affinity and/or efficacy of an orthosteric agonist. Evidence has accumulated that G protein–coupled receptors (GPCRs) can have different activated states that drive different signaling pathways
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