Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure−Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations

Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]​pyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptor–membrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies.