Identification and Characterization of ‘Biased’ A3 Adenosine Receptor Allosteric Modulators
Authors: Fisher Courtney L. , Bitant Amelia A. , Rothwell Amy C. , Wan Tina C. , Fallot Lucas B. , Suresh R. Rama , Guo-Gao Zan , Jacobson Kenneth A. , Auchampach John A. 
Published: April 2020

IntroductionA3 adenosine receptor agonists are currently being evaluated in clinical trials for treatment of inflammation, cancer, and neuropathic pain. To circumvent complications associated with the use of direct agonists of GPCRs, we have pursued development of A3 adenosine receptor positive allosteric modulators (PAMs).MethodsIn prior studies, we synthesized a series of 1H‐imidazo[4,5]quinoline‐4‐amines that display allosteric modulatory activity versus the human A3 receptor. In prior screens, members of this class of modulators were found to increase the maximal efficacy of orthosteric agonists ~2–3‐fold in G protein‐dependent signaling assays, although they had the tendency to reduce agonist potency. In addition, lead compounds (LUF6000) were discovered to be substantially less potent at rodent A3ARs. The purpose of the present investigation was to re‐evaluate our expanded compound series based on LUF6000 in greater detail, with emphasis on examining effects on orthosteric ligand potency, G protein‐dependent versus G protein‐independent signaling, and efficacy versus mouse A3 adenosine receptors. Based on known species differences, human/mouse chimeric receptors were prepared to explore the possibility extracellular loops 1 & 2 that form an extracellular vestibule comprise the allosteric ligand binding site.ResultsOur evaluation centered on a series of 4‐(3,4‐dichlorophenylamino) compounds with 2‐substitutions consisting of 3–8 cycloalkyl rings. This was based on our observation during an initial screen that the 2‐cycloheptyl and 2‐cyclooctyl analogues increased affinity of the orthosteric radioligand ([125I]I‐AB‐MECA) in equilibrium binding assays in a manner that did not slow its (apparent) association binding rate. Among this series, compounds with larger 2‐substituents (c4–8) were found to increase the maximal efficacy of the orthosteric agonist Cl‐IB‐MECA in [35S]GTPgS binding assays ~2‐fold. Most prominently, the cyclooctyl derivative increased the efficacy of Cl‐IB‐MECA (~1.5‐fold) while also increasing its potency. A similar pattern was evident in cAMP assays, which also detected allo‐agonist activity. In contrast to these findings, analogues within the series with bulkier 2‐substituents substantially reduced (up to 95%), rather than enhanced, the efficacy of Cl‐IB‐MECA in arrestin2 recruitment assays (TANGO assay). This occurred at potencies that mirrored those observed in assays of G protein‐dependent signaling. Unfortunately, none of the derivatives investigated displayed improved activity versus mouse A3 adenosine receptors. Studies with human/mouse chimeric receptors indicated that modulators based on the imidazoquinolinamine template do not function as ‘vestibule’ modulators.ConclusionsWe have identified improved PAMs for the human A3 receptor that: 1) not only increase agonist efficacy, but also increase agonist potency (and binding affinity), 2) display allo‐agonist activity, and 3) may bias towards G protein‐dependent signaling.Support or Funding InformationNIH R01HL133589 and Therapeutic Accelerator Program