Be Aware of Aggregators in the Search for Potential Human ecto-5′-Nucleotidase Inhibitors

Abstract

Promiscuous inhibition due to aggregate formation has been recognized as a major concern in drug discovery campaigns. Here, we report some aggregators identified in a virtual screening (VS) protocol to search for inhibitors of human ecto-5′-nucleotidase (ecto-5′-NT/CD73), a promising target for several diseases and pathophysiological events, including cancer, inflammation and autoimmune diseases. Four compounds (A, B, C and D), selected from the ZINC-11 database, showed IC50 values in the micromolar range, being at the same time computationally predicted as potential aggregators. To confirm if they inhibit human ecto-5′-NT via promiscuous mechanism, forming aggregates, enzymatic assays were done in the presence of 0.01% (v/v) Triton X-100 and an increase in the enzyme concentration by 10-fold. Under both experimental conditions, these four compounds showed a significant decrease in their inhibitory activities. To corroborate these findings, turbidimetric assays were performed, confirming that they form aggregate species. Additionally, aggregation kinetic studies were done by dynamic light scattering (DLS) for compound C. None of the identified aggregators has been previously reported in the literature. For the first time, aggregation and promiscuous inhibition issues were systematically studied and evaluated for compounds selected by VS as potential inhibitors for human ecto-5′-NT. Together, our results reinforce the importance of accounting for potential false-positive hits acting by aggregation in drug discovery campaigns to avoid misleading assay results.

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

“… a All enzymatic assays were performed as previously described using the malachite green method to quantify phosphate release. All enzymatic assays were performed in the presence of a detergent (CHAPS 0.01%, w/v) to avoid aggregate formation. b Values represent the mean ± standard error of the mean (S.E.M.)…”

“…Human ecto-5′-NT inhibition assays were performed as previously described, , with minor modifications. In short, all assays were carried out in a reaction mixture containing the following: HEPES buffer (10 mM; pH 7.4), MgCl 2 (2 mM), CaCl 2 (1 mM), CHAPS (0.01%, w/v) as a detergent to avoid colloidal aggregation, human ecto-5′-NT (3.6 nM), AMP (500 μM), and each tested compound at various final concentrations depending on the compound solubility in the assay buffer.…”

NT5E is associated with unfavorable prognosis and regulates cell proliferation and motility in gastric cancer

“…Ecto-5′-nucleotidase is a glycosyl-phosphatidylinositol anchored membrane glycoprotein [13]. Originally, Ecto-5′-nucleotidase had been found to strengthen endothelial barrier function and depress leukokinesis, and be markedly affected by hypoxia and inflammatory mediators [14,15]. Subsequently, more and more human cancers have been suggested to display high Ecto-5′-nucleotidase expression, such as lung cancer [16,17], breast cancer [18], colorectal cancer [19,20], pancreatic cancer [21], gallbladder cancer [22], prostate cancer [23,24], thyroid cancer [25], and head and neck cancer [26].…”

Gallotannins are non‐specific inhibitors of α‐amylase: Aggregates are the active species taking part in inhibition

“…Based on the prediction of Aggregator Advisor software (Irwin et al, 2015), we presumed that Aleppo tannin was a non-specific aggregation-based inhibitor as it had a relatively high calculated logP value (2.8) as well as a similarity of 0.84 to an already reported aggregator (Ferreira et al, 2010). However, neither of these values exceeded the limits (logP > 3, similarity to a known aggregator >0.85) reported in the literature for aggregators (Viviani et al, 2018). In addition, the referenced article for the single hit in Aggregator Advisor did not contain any experimental data related to this compound.…”