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

Author(s)

Viviani, Lucas G. ; Piccirillo, Erika ; Cheffer, Arquimedes ; de Rezende, Leandro ; Ulrich, Henning ; Carmona-Ribeiro, Ana Maria ; T-do Amaral, Antonia

• Document type: Journal article
• Source: Molecules; v. 23, n. 8, p. 15-pg., 2018-08-01.

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) under bar, (B) under bar, (C) under bar and (D) under bar), 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) under bar. 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. (AU)

• FAPESP's process: 16/12392-9 - 21st European Symposium on Quantitative Structure-Activity Relationship Where Molecular Simulations Meet Drug Discovery
• Grantee: Antonia Tavares Do Amaral
• Support Opportunities: Research Grants - Meeting - Abroad
• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 12/50880-4 - Stem cells: from basic studies of kinin and purinergic receptor roles towards therapeutical applications
• Grantee: Alexander Henning Ulrich
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/14871-7 - 22nd European Symposium on Quantitative Structure-Activity Relationships
• Grantee: Antonia Tavares Do Amaral
• Support Opportunities: Research Grants - Meeting - Abroad
• FAPESP's process: 12/06633-2 - Rational search for inhibitors of Dengue and Foot-and-Mouth Disease proteases
• Grantee: Erika Piccirillo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 14/07248-0 - Virtual screening search for inhibitors of human ecto-5'-nucleotidase and of Mycobacterium tuberculosis thiorredoxin reductase: models generation and experimental validation
• Grantee: Lucas Gasparello Viviani
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)