Discovery of a High Affinity Adenosine A₁/A₃ Receptor Antagonist with a Novel 7-Amino-pyrazolo[3,4-d]pyridazine Scaffold

Abstract

Here we describe the design and synthesis of pyrazolo[3,4- d ]pyridazines as adenosine receptor (AR) ligands. We demonstrate that the introduction of a 3-phenyl group, together with a 7-benzylamino and 1-methyl group at the pyrazolopyridazine scaffold, generated the antagonist compound 10b , which displayed 21 nM affinity and a residence time of ∼60 min, for the human A 1 R, 55 nM affinity and a residence time of ∼73 min, for the human A 3 R and 1.7 μΜ affinity for the human A 2B R while not being toxic. Strikingly, the 2-methyl analog of 10b , 15b , had no significant affinity. Docking calculations and molecular dynamics simulations of the ligands inside the orthosteric binding area suggested that the 2-methyl group in 15b hinders the formation of hydrogen bonding interactions with N 6.55 which are considered critical for the stabilization inside the orthosteric binding cavity. We, therefore, demonstrate that 10a is a novel scaffold for the development of high affinity AR ligands. From the mutagenesis experiments the biggest effect was observed for the Y271 7.46 A mutation which caused an ∼10-fold reduction in the binding affinity of 10b .

Discovery and Structure–Activity Relationship Studies of Novel Adenosine A1 Receptor-Selective Agonists

“…We also included the mutant T257 6.58 A since we have previously shown that this residue is a good discriminator between different A 1 R agonists . We did not consider mutations of N254 6.55 or F171 ELC2 since these are known to prevent ligand binding to the A 1 R (including CA200645) and therefore cannot be studied. , Furthermore, we did not consider mutating T270 7.35 since, when we compared the sequences of the hA 1 R and the rA 1 R, we observed that, in the rA 1 R, the equivalent residue at position T270 7.35 is an Ile. Comparison of the binding affinities (p K i ) for 20 and 27 between the hA 1 R and the rA 1 R shows that 20 is equipotent between the two species while 27 has reduced affinity at the rA 1 R (p K i (hA 1 R) = 7.55 ± 0.11; p K i (rA 1 R) = 6.94 ± 0.08).…”

Computational Workflow for Refining AlphaFold Models in Drug Design Using Kinetic and Thermodynamic Binding Calculations: A Case Study for the Unresolved Inactive Human Adenosine A3 Receptor

“…Antagonism of hA 3 R could have important clinical implications as observed by clinical trials for the treatment of glaucoma, ulcerative colitis, psoriasis, and nonalcoholic steatohepatitis (NASH) 9 . As such, we have sought a specific workflow that enables further optimization of hA 3 R antagonists, , e.g., A17 and other leads that we have previously described …”

“…Regarding the binding interactions of ligands and the orthosteric binding area exploration, in previous works, we used homology models of the intermediate-active or -inactive hA 3 R generated from the experimental structure of the inactive hA 2A R in complex with either an agonist or antagonist . We were able to investigate the binding profiles of high efficacy agonists and subtype-selective antagonists. ,,, Furthermore, through the combination of MD simulations and mutagenesis experiments, we were able to explore the interactions between these ligands and both “wild-type” (WT) or mutated hA 3 Rs …”

A Computational Workflow for Refining AF2 Models in Drug Design Using Kinetic and Thermodynamic Binding Calculations: A Case Study for the Unresolved Inactive human Adenosine A3 Receptor

“…As such, we have sought a specific workflow that enables further optimization of hA3R antagonists, 86,102 e.g., A17 45 and other leads that we have previously described. 85 We have retrospectively applied a computational drug binding analysis for the 3,5-disubstituted-7-(phenylamino)-pyrazolo[3,4-c]pyridine antagonists L3-L6, L9, Α17 to describe their kinetic and thermodynamic binding profile and to select an appropriate homology model. We explored the thermodynamic and kinetic binding SARs of these antagonists against inactive hA3R using TI/MD calculations of relative binding free energies and τRAMD calculations of relative RTs in comparison with RTexps and by testing available homology models of inactive hA3R.…”

“…42 We were able to investigate the binding profiles of high efficacy agonists 47 and subtype selective antagonists. 34,48,49,85 . Furthermore through combination of MD simulations and mutagenesis experiments we were able to explore the interactions between these ligands and both 'wild-type' (WT) or mutated hA3Rs.…”