Novel Pyrazolo[3,4-c]pyridine Antagonists with Nanomolar affinity for A1 / A3 Adenosine Receptors: Binding Kinetics and Exploration of their Binding Profile Using Mutagenesis Experiments, MD simulations and TI/MD calculations

Abstract

Drugs targeting the four adenosine receptor (AR) subtypes can provide “soft" treatment of various significant diseases. Even for the two experimentally resolved AR subtypes the description of the orthosteric binding area and structure-activity relationships of ligands remains a demanding task due to the high similar amino acids sequence but also the broadness and flexibility of the ARs binding area. The identification of new pharmacophoric moieties and nanomolar leads and the exploration of their binding area with mutagenesis and state-of-the-art computational methods useful also for drug design purposes remains a challenging aim for all ARs. Here, we identified several low nanomolar ligands and potent competitive antagonists against A1R / A3R, containing the novel pyrazolo[3,4-c]pyridine pharmacophore for ARs, from a screen of an in-house library of only 52 compounds, originally designed for anti-proliferative activity. We identified L2-L10, A15, A17 with 3-aryl, 7-anilino and a electronegative group at 5-position as low micromolar to low nanomolar A1R / A3R antagonists. A17 has for A1R Kd = 5.62 nM and a residence time (RT) 41.33 min and for A3R Kd = 13.5 nM, RT = 47.23 min. The kinetic data showed that compared to the not potent or mediocre congeners the active compounds have similar association, for example at A1R Kon = 13.97 x106 M-1 (A17) vs Kon = 3.36 x106 M-1 (A26) but much lower dissociation rate Koff = 0.024 min-1 (A17) vs 0.134 min-1 (A26). Using molecular dynamics (MD) simulations and mutagenesis experiments we investigated the binding site of A17 showing that it can interact with an array of residues in transmembrane helix 5 (TM5), TM6, TM7 of A1R or A3R including residues E5.30, E5.28, T7.35 in A1R instead of Q5.28, V5.30 , L7.35 in A3R. A striking observation for drug design purposes is that for L2506.51A the binding affinity of A17 significantly increased at A1R. A17 provides a lead representative of a promising series and by means of the Thermodynamics Integration coupled with MD simulations (TI/MD) method, first applied here on whole GPCR- membrane system and showing a very good agreement between calculated and experimental relative binding free energies for A1R and A3R (spearman rank correlation p = 0.82 and 0.84, respectively), and kinetic experiments can lead to ligands with improved profile against ARs.

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

“…The biggest effect in this study was observed for the Y271 7.46 A A 1 R mutation, which caused a ∼10-fold reduction in the binding affinity of 10b ( Table 2 ). This effect is in contrast to that observed previously for pyrazolo[3,4- c ]pyridines 14 for which we showed that the Y271 7.46 A mutation caused a slight increase in binding affinity. Since the MD simulations showed contacts with H278 7.43 and not Y271 7.46 , the Y271 7.46 A mutation in A 1 R might affect the binding of 10b through contact with H278 7.43 .…”

“…This contrasts with our studies using 3-phenyl-7-anilinopyrazolo- [3,4-c]pyridines which showed a 1.5-fold decrease in affinity due to the E172 5.30 A mutation. 14 In addition, mutation of H251 6.2 A has been reported to reduce antagonist affinity against A 3 R 20,21 although here it did not have any effect on 10b affinity at A 1 R. Other residues of interest to mutate were T91 3.36 A and S267 7.42 A, which are deep in the orthosteric pocket. Interestingly, we found that mutation to alanine of these residues also did not have a significant effect on the binding affinity of 10b (Table 2).…”

“…This contrasts with our studies using 3-phenyl-7-anilinopyrazolo[3,4- c ]pyridines which showed a 1.5-fold decrease in affinity due to the E172 5.30 A mutation. 14 …”

“…By the repurposing of antiproliferative aromatic condensed nitrogen heterocycles, we previously identified nanomolar affinity pyrazolo­[3,4- c ]­pyridine A 1 R/A 3 R antagonists . It has been reported that non-xanthine pyrazolo derivatives that potently bind ARs are pyrazolo­[4,3- d ]­pyrimidines, pyrazolo­[1,5- c ]­quinazolines, pyrazolo­[3,4- b ]­pyridines, , pyrazolo­[3,4- b ]­pyridines, pyrazolo­[4,3- e ]-1,2,4-triazolo­[1,5- c ]­pyrimidines, pyrazolo­[3,4- c ]- or -[4,3- c ]­quinolines, pyrazolo­[4,3- d ]­pyrimidinones, pyrazolo­[3,4- d ]­pyrimidines, and pyrazolo­[1,5- a ]­pyridines .…”

“…By the repurposing of antiproliferative aromatic condensed nitrogen heterocycles, we previously identified nanomolar affinity pyrazolo[3,4- c ]pyridine A 1 R/A 3 R antagonists. 14 It has been reported that non-xanthine pyrazolo derivatives that potently bind ARs are pyrazolo[4,3- d ]pyrimidines, 3 pyrazolo[1,5- c ]quinazolines, 15 pyrazolo[3,4- b ]pyridines, 16 , 17 pyrazolo[3,4- b ]pyridines, pyrazolo[4,3- e ]-1,2,4-triazolo[1,5- c ]pyrimidines, pyrazolo[3,4- c ]- or -[4,3- c ]quinolines, pyrazolo[4,3- d ]pyrimidinones, pyrazolo[3,4- d ]pyrimidines, and pyrazolo[1,5- a ]pyridines. 18 After we previously identified the potent pyrazolo[3,4- c ]pyridine A 1 R/A 3 R antagonists 14 and observed that certain substituted pyrazolo[3,4- b ]pyridines had antagonistic potency against A 3 R or A 1 R, 16 , 17 we quantified the novel pyrazolo[3,4- d ]pyridazine scaffold for activity at ARs.…”