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

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 IC50 value of 6.2 +/- 1.0 mu M. 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. (AU)