Extrahelical binding site for a 1 H -imidazo[4,5-c]quinolin-4-amine A 3 adenosine receptor positive allosteric modulator on helix 8 and distal portions of transmembrane domains 1 and 7.

This study describes the localization and computational prediction of a putative binding site of an A 3 adenosine receptor (A 3 AR) positive allosteric modulator LUF6000 (2-cyclohexyl-1 H -imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1 H -imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by π-π stacking interactions with the side chains of Y284 7.55 in TMD 7 and Y293 8.54 in H8, and by π-NH bonding between Y284 7.55 and the exocyclic amine. The 2-cyclohexyl group is positioned 'upward' within a small hydrophobic sub-pocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N - 1 amine of the heterocycle and the carbonyl of G29 1.49 further stabilizes the interaction. MD simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y293 8.54 ; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1 H -imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A 3 AR PAMs. Significance Statement Orthosteric A 3 AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1 H -Imidazo[4,5-c]quinolin-4-amine class of A 3 AR positive allosteric modulators.