Virtual Screening Approach for the Identification of Hydroxamic Acids as Novel Human Ecto-5′-Nucleotidase Inhibitors

Abstract

Ecto-5′-nucleotidase (ecto-5′-NT, CD73) is a zinc-binding metallophosphatase that plays a key role in extracellular purinergic pathways, being implicated in several physiological and pathophysiological processes, such as immune homeostasis, inflammation, and tumor progression. As such, it has been recognized as a promising biological target for many diseases, including cancer, infections, and autoimmune diseases. Despite its importance, so far only a few inhibitors of this target enzyme are known, most of which are not suitable as drug candidates. Here, we aimed to search for hydroxamic acid-containing compounds as potential human ecto-5′-NT inhibitors, since this group is known to be a strong zinc chelator. To this end, we performed a hierarchical virtual screening (VS) search consisting of three consecutive steps (filtering for compounds bearing a hydroxamic acid group, shape-based matching, and docking followed by visual inspection), which were applied to screen the ZINC-14 database ("all purchasable subset"). Out of 25 compounds selected by this VS protocol, 12 were acquired and further submitted to enzymatic assays for VS experimental validation. Four of them (i.e., 33.3%) were found to inhibit human ecto-5′-NT in the low micromolar range. The most potent one showed an IC 50 value of 6.2 ± 1.0 μM. All identified inhibitors satisfy drug-like criteria and provide novel scaffolds to be explored in further hit-to-lead optimization steps. Furthermore, to the best of our knowledge, they are the first hydroxamic acid-containing inhibitors of human ecto-5′-NT described so far.

Decision Making in Structure-Based Drug Discovery: Visual Inspection of Docking Results

“…Thus, the location of hydrogen bonds is essential to achieve potent ligand binding and the effect of protein environment on this type of interaction should be carefully considered during visual analysis. In addition to formed hydrogen bonds, all unsatisfied hydrogen bond donor and acceptor groups without interacting counterparts, in particular those buried in the binding site, need to be evaluated, as they generally result in reduced binding free energies due to an uncompensated desolvation free energy penalty. , In metalloproteins, distinct electrostatic interactions with metal cofactors can be of major importance for ligand potency (Figure A). ,, Interestingly, none of the studies reviewed in this perspective reported the consideration of weak hydrogen bonds, halogen bonds, orthogonal dipole interactions, or amide−π interactions as a criterion during visual inspection (see also survey results for additional details).…”

“…While the displacement of water molecules is favorable due to an increase in entropy and sometimes enthalpy, some may form enthalpically strong interactions with the protein and bridge ligand–protein interactions favorably contributing to the binding free energy. Thus, solvation effects such as water-mediated interactions should be considered in visual inspection (Figure B,C). , However, the proper assessment of the multifaceted water behavior is by far not trivial even for extremely advanced experts. Moreover, the solvation scenario in the binding site in the apo state (no ligand present) is not routinely included in the visual comparison.…”

“…As we will describe below, visual inspection of docking results has been used in several successful discovery projects. ,,,− Additionally, visual inspection was also applied during the validation of docking protocols, in particular to evaluate pose prediction by redocking or cross-docking to supplement standard measures such as the root-mean-square deviation (RMSD) and native contacts. , Unfortunately, most of those studies did only vaguely specify applied criteria or did not mention them at all. ,,− , A recent review focused on decision making in medicinal chemistry in the evaluation of large data sets with respect to potency, selectivity, pharmacokinetics, structural biology, and computational results. With regard to compound prioritization based on such data sets, the authors reported a considerable disagreement among medicinal chemists, presumably arising from their different experience, background, and training. , This discrepancy among different researchers may also exist for visual evaluation of docking poses.…”

CD39 and CD73 as Promising Therapeutic Targets: What Could Be the Limitations?

“…For this reason, APCP became a prototype for drug development (Bhattarai et al, 2015;Corbelini et al, 2015;Bhattarai et al, 2020). In addition, the recent availability of the crystal structures of ecto-5′-nucleotidases has led to the development of numerous docking and virtual screening studies (Knapp et al, 2012;Bhattarai et al, 2019;Viviani et al, 2020).…”

“…This and another small molecule inhibitor (LY3475070) are currently being evaluated in phase one clinical trials. In addition to APCP based inhibitors of CD73, descriptions of numerous other inhibitors of the enzyme can be found in the literature (Figueiró et al, 2014;Baqi, 2015;Yang et al, 2017;Ghoteimi et al, 2019;Iqbal et al, 2020;Viviani et al, 2020). Finally, nucleoside analogues with two carboxylate groups and benzothiazine derivatives are CD73 inhibitors for treating cancer that have been patented (Gong et al, 2018;Ghoteimi et al, 2019).…”