A new mechanism of allostery in a G protein–coupled receptor dimer

Abstract

SB269652 (1) is the first drug-like allosteric modulator of the dopamine D2 receptor (D2R), but contains structural features associated with orthosteric D2R antagonists. Using a functional complementation system to control the identity of individual protomers within a dimeric D2R complex, we converted the pharmacology of the interaction between SB269652 and dopamine from allosteric to competitive by impairing ligand binding to one of the protomers, indicating that the allostery requires D2R dimers. Additional experiments identified a “bitopic” pose for SB269652 extending from the orthosteric site into a secondary pocket at the extracellular end of the transmembrane (TM) domain, involving TM2 and TM7. Engagement of this secondary pocket was a requirement for the allosteric pharmacology of SB269652. This suggests a novel mechanism whereby a bitopic ligand binds in an extended pose on one G protein-coupled receptor protomer to allosterically modulate the binding of a ligand to the orthosteric site of a second protomer.

Discovery of a Novel Class of Negative Allosteric Modulator of the Dopamine D2 Receptor Through Fragmentation of a Bitopic Ligand

“…Until recently, examples of small molecule allosteric modulators were lacking for the D 2 R. We previously demonstrated that 1 bound the D 2 R in a bitopic mode and acted as a negative allosteric modulator of dopamine binding and function across a D 2 R dimer. 13 Studies at the mAChRs have used ligand fragmentation approaches to isolate orthosteric and allosteric pharmacophores from a bitopic ligand. 10,11 In this study we were able to isolate 3 an allosteric fragment of 1, derived from the indole-2-carboxamide moiety of the parent compound.…”

“…Our previous study identified two residues at the top of TM2 (Val91 2.61 and Glu95 2.65 ) that interact with the indole-2-carboxamide moiety of 1 and mutation of these residues to alanine reduced the affinity and/or negative cooperativity of 1 (Figure 5a, Table 2). 13 We hypothesised that if 3 occupies the same binding pocket as the 1H-indole-2-carboxamide moiety of 1, then one would expect a similar sensitivity to mutation of these residues. Mutation of Val91 2.61 Ala removed any effect of 3 up to a concentration of 300 µM upon a dopamine in a pERK1/2 assay, indicating this residue is key for 3 binding or function (Figure 5b, Table 2).…”

“…In previous work we demonstrated that 1 binds as a bitopic ligand to one protomer of a homodimer of the D 2 R to modulate the binding of a ligand to the orthosteric site of a second protomer. 13 We previously identified purely orthosteric fragments of 1 containing the 7-CTHIQ moiety that displayed competitive pharmacology with dopamine. This core group comprises the key elements expected to interact with the orthosteric binding site of aminergic receptors (i.e.…”

“…In addition we wanted to examine whether the approach of dividing bitopic ligands into discrete fragments can unmask novel purely allosteric scaffolds. Our previous study demonstrated that the indole-2-carboxamide moiety of 1 extended into a secondary 'allosteric' pocket within the D 2 R. 13 Accordingly, we first synthesised the fragment 3, efficacy and values less than 1 indicate negative cooperativity, Table 1). This pattern was distinct from that observed in an assay measuring ERK1/2 phosphorylation, in which 3 caused both a reduction in dopamine potency and a significant decrease in maximal response (Figure 2c).…”

“…13 We generated fragments of 1 that included the 7-cyano-1,2,3,4-tetrahydroisoquinoline (7-CTHIQ) head-…”