Lipid Trolling to Optimize A₃ Adenosine Receptor-Positive Allosteric Modulators (PAMs)

A₃ adenosine receptor (A₃AR) positive allosteric modulators (PAMs) (2,4-disubstituted-1H-imidazo[4,5-c]quinolin-4-amines) allosterically increase the E_max of A₃AR agonists, but not potency, due to concurrent orthosteric antagonism. Following mutagenesis/homology modeling of the proposed lipid-exposed allosteric binding site on the cytosolic side, we functionalized the scaffold, including heteroatom substitutions and exocyclic phenylamine extensions, to increase allosteric binding. Strategically appended linear alkyl-alkynyl chains with terminal amino/guanidino groups improved allosteric effects at both human and mouse A₃ARs. The chain length, functionality, and attachment position were varied to modulate A₃AR PAM activity. For example, 26 (MRS8247, p-alkyne-linked 8 methylenes) and homologues increased agonist Cl-IB-MECA's E_max and potency ([³⁵S]GTPγS binding). The putative mechanism involves a flexible, terminally cationic chain penetrating the lipid environment for stable electrostatic anchoring to cytosolic phospholipid head groups, suggesting "lipid trolling", supported by molecular dynamic simulation of the active-state model. Thus, we have improved A₃AR PAM activity through rational design based on an extrahelical, lipidic binding site.