Machine Learning for Discovery of New ADORA Modulators

Adenosine (ADO) is an extracellular signaling molecule generated locally under conditions that produce ischemia, hypoxia, or inflammation. It is involved in modulating a range of physiological functions throughout the brain and periphery through the membrane-bound G protein-coupled receptors, called adenosine receptors (ARs) A₁AR, A_2AAR, A_2BAR, and A₃AR. These are therefore important targets for neurological, cardiovascular, inflammatory, and autoimmune diseases and are the subject of drug development directed toward the cyclic adenosine monophosphate and other signaling pathways. Initially using public data for A₁AR agonists we generated and validated a Bayesian machine learning model (Receiver Operator Characteristic of 0.87) that we used to identify molecules for testing. Three selected molecules, crisaborole, febuxostat and paroxetine, showed initial activity in vitro using the HEK293 A₁AR Nomad cell line. However, radioligand binding, β-arrestin assay and calcium influx assay did not confirm this A₁AR activity. Nevertheless, several other AR activities were identified. Febuxostat and paroxetine both inhibited orthosteric radioligand binding in the µM range for A_2AAR and A₃AR. In HEK293 cells expressing the human A_2AAR, stimulation of cAMP was observed for crisaborole (EC₅₀ 2.8 µM) and paroxetine (EC₅₀ 14 µM), but not for febuxostat. Crisaborole also increased cAMP accumulation in A_2BAR-expressing HEK293 cells, but it was weaker than at the A_2AAR. At the human A₃AR, paroxetine did not show any agonist activity at 100 µM, although it displayed binding with a K_i value of 14.5 µM, suggesting antagonist activity. We have now identified novel modulators of A_2AAR, A_2BAR and A₃AR subtypes that are clinically used for other therapeutic indications, and which are structurally distinct from previously reported tool compounds or drugs.