{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,19]],"date-time":"2025-11-19T17:13:29Z","timestamp":1763572409571,"version":"build-2065373602"},"reference-count":63,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2018,7,27]],"date-time":"2018-07-27T00:00:00Z","timestamp":1532649600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001807","name":"Funda\u00e7\u00e3o de Amparo \u00e0 Pesquisa do Estado de S\u00e3o Paulo","doi-asserted-by":"publisher","award":["CEPID Redoxoma (No. 2013\/07937-8)","2012\/06633-2, 2014\/07248-0, 2016\/12392-9, 2012\/50880-4"],"award-info":[{"award-number":["CEPID Redoxoma (No. 2013\/07937-8)","2012\/06633-2, 2014\/07248-0, 2016\/12392-9, 2012\/50880-4"]}],"id":[{"id":"10.13039\/501100001807","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003593","name":"Conselho Nacional de Desenvolvimento Cient\u00edfico e Tecnol\u00f3gico","doi-asserted-by":"publisher","award":["302352\/2014-7"],"award-info":[{"award-number":["302352\/2014-7"]}],"id":[{"id":"10.13039\/501100003593","id-type":"DOI","asserted-by":"publisher"}]},{"name":"NAP Rexoma (PRP-USP)","award":["."],"award-info":[{"award-number":["."]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>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\u2032-nucleotidase (ecto-5\u2032-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\u2032-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\u2032-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.<\/jats:p>","DOI":"10.3390\/molecules23081876","type":"journal-article","created":{"date-parts":[[2018,7,27]],"date-time":"2018-07-27T12:20:03Z","timestamp":1532694003000},"page":"1876","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Be Aware of Aggregators in the Search for Potential Human ecto-5\u2032-Nucleotidase Inhibitors"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4767-4825","authenticated-orcid":false,"given":"Lucas G.","family":"Viviani","sequence":"first","affiliation":[{"name":"Departamento de Qu\u00edmica Fundamental, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4094-3102","authenticated-orcid":false,"given":"Erika","family":"Piccirillo","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica Fundamental, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"},{"name":"Departamento de Bioqu\u00edmica, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arquimedes","family":"Cheffer","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leandro","family":"De Rezende","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica Fundamental, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"},{"name":"Departamento de Bioqu\u00edmica, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Henning","family":"Ulrich","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8500-2707","authenticated-orcid":false,"given":"Ana Maria","family":"Carmona-Ribeiro","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2937-5255","authenticated-orcid":false,"given":"Antonia T.-do","family":"Amaral","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica Fundamental, Instituto de Qu\u00edmica, Universidade de S\u00e3o Paulo, Av. Prof. Lineu Prestes, 748, S\u00e3o Paulo 05508-000, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2018,7,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1038\/nprot.2006.77","article-title":"A detergent-based assay for the detection of promiscuous inhibitors","volume":"1","author":"Feng","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4891","DOI":"10.1021\/jm100488w","article-title":"Complementarity between a docking and a high-throughput screen in discovering new cruzain inhibitors","volume":"53","author":"Ferreira","year":"2010","journal-title":"J. Med. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1021\/ci200528d","article-title":"Recognizing pitfalls in virtual screening: A critical review","volume":"52","author":"Scior","year":"2012","journal-title":"J. Chem. Inf. 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