Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode

Abstract

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A and AARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A/A, ARs, or AAR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A/A, A/A, A/A/A, and A). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A receptor.

Pharmacological Characterisation of Novel Adenosine Receptor A3R Antagonists

“…A number of compounds previously documented (K5, K9, K21, K22 and K24; Lagarias et al, 2018) or determined in this study (K26, K27 and K34) to have micromolar binding affinity for A3R showed no activity in our functional screen (Table 1, Supplementary Table 1). These compounds, with a Ki in the low micromolar range, were further tested to ensure our functional screen was robust.…”

“…2 and Supplementary Table 2). K8, despite showing no binding at any AR subtype (Lagarias et al, 2018), showed a reduced cAMP accumulation. We tested K8 for agonist activity at the A3R but was found to be no different to DMSO ( Supplementary Fig.…”

“…We set out to conduct a functional screen of 39 compounds for the identification of A3R antagonists, some of which have previously been identified to bind one of the three AR subtypes; A1R, A3R or A2AR (Lagarias et al, 2018). Our screen was conducted using A3R expressing Flp-In™-Chinese hamster ovary (CHO) cells where cAMP accumulation was detected following a combined stimulation of 1 µM forskolin (to allow A3R mediated Gi/o response to be observed), 1 µM tested compound and the predetermined IC80 concentration of NECA (3.16 nM).…”

“…Although the remaining AR subtypes have proven more difficult to crystallise with the A3R structure yet to be resolved, there are a number of A1R structures published including the adenosine-bound A1R-Gi complex (Draper-Joyce et al, 2018) and antagonist bound structures; DU172 (Glukhova et al, 2017) and PSB36 (Cheng et al, 2017). Thus, structuralbased drug design offers huge potential in the development of highly selective ligands (Carlsson et al, 2010, Katritch et al, 2010, Lenselink et al, 2016, Lagarias et al, 2018. The limited availability of diverse high-resolution structures of the remaining AR subtypes bound to pharmacologically distinct ligands has meant there is a discrepancy between the capability to predict compound binding versus pharmacological behaviour; partial agonism, inverse agonism, biased agonist, antagonism, allosteric modulation etc (Sexton and Christopoulos, 2018).…”

“…The limited availability of diverse high-resolution structures of the remaining AR subtypes bound to pharmacologically distinct ligands has meant there is a discrepancy between the capability to predict compound binding versus pharmacological behaviour; partial agonism, inverse agonism, biased agonist, antagonism, allosteric modulation etc (Sexton and Christopoulos, 2018). With this in mind, the potential antagonists (K1-K25, K28 and K35) identified in our previously published virtual screening investigation and binding experiments (Lagarias et al, 2018) and some newly identified potential antagonists (K26, K27, K29-K34 and K36-K39) were pharmacologically characterised using cAMP accumulation and ERK1/2 phorphorylation assays. We were able to identify a potent and selective A3R antagonist, K18 (O4-{[3-(2,6dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl}-2-methyl-1,3-thiazole-4carbohydroximamide) and, using molecular dynamic (MD) simulations combined with sitedirected mutagenesis, elude to the potential binding site.…”