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Molecules 2018, 23(8), 1876; https://doi.org/10.3390/molecules23081876

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

1
Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo 05508-000, Brazil
2
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo 05508-000, Brazil
*
Author to whom correspondence should be addressed.
Academic Editor: Rebecca Wade
Received: 29 June 2018 / Revised: 22 July 2018 / Accepted: 26 July 2018 / Published: 27 July 2018
(This article belongs to the Special Issue Molecular Modeling in Drug Design)
Full-Text   |   PDF [5794 KB, uploaded 27 July 2018]   |  

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. View Full-Text
Keywords: aggregation; promiscuous mechanism; human ecto-5′-nucleotidase; virtual screening; enzymatic assays; turbidimetry; dynamic light scattering aggregation; promiscuous mechanism; human ecto-5′-nucleotidase; virtual screening; enzymatic assays; turbidimetry; dynamic light scattering
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Viviani, L.G.; Piccirillo, E.; Cheffer, A.; de Rezende, L.; Ulrich, H.; Carmona-Ribeiro, A.M.; Amaral, A. .-D. Be Aware of Aggregators in the Search for Potential Human ecto-5′-Nucleotidase Inhibitors. Molecules 2018, 23, 1876.

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