From Empirical Screening to Lipid Trolling: the Evolution of Extrahelical Allosteric Modulators of A1 and A3 Adenosine Receptors

An early GPCR positive allosteric modulator (PAM) was the 2-amino-3-benzoylthiophene derivative PD81,723, which enhances Gi-coupled A1 adenosine receptor (AR) agonist effects. Cryo-EM studies located its extrahelical binding site on TMs 5 and 6, and extensive empirical SAR is consistent with the experimental A1AR structure. At the anti-inflammatory A3AR, a series of 1H-imidazo[4,5-c]quinolin-4-amine PAMs, originally derived from an A1AR antagonist scaffold, was recently found, using mutagenesis and molecular modeling, to bind at an extrahelical, lipid-exposed site on the receptor. The N4 and C2 positions on the scaffold were modified with substituted phenyl and cycloalkyl/branched alkyl groups, respectively, as in the prototypical PAM LUF6000. Before the structural binding hypothesis, extensive synthetic optimization failed to introduce hydrophilic groups needed for aqueous compatibility. Eventually, secondary anchoring of these A3AR PAMs within the phospholipid bilayer (lipid trolling) succeeded in improving the pharmacological profile, based on the attraction of appended terminal cationic chains on the PAM with anionic phospholipid head groups. Molecular dynamics (MD) simulation has predicted a rearrangement of anionic lipids around H8 when long-amino chain PAMs bind, and an upward movement of H8 that is reported to be associated with Class A GPCR activation. N1 alkylation with benzyl groups removed residual A3AR antagonism and produced substantial enhancement of agonist potency as well as Emax. Both A1AR and A3AR PAMs can activate partially in the absence of orthosteric agonist. Thus, at both receptors, potent PAMs have been discovered and optimized for selective enhancement of endogenous adenosine and potentially for treatment of conditions such as chronic pain. Collectively, extrahelical, lipid-exposed pockets are druggable allosteric sites in Class A GPCRs and amenable to deliberate engagement of the membrane environment to optimize receptor modulation. The evolution from empirical screening to structure-guided and membrane-aware design provides a generalizable framework for targeting signaling across the GPCR superfamily beyond ARs.