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| Author(s): |
Erika Piccirillo
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2017-09-06 |
| Examining board members: |
Antonia Tavares do Amaral;
Josef Wilhelm Baader;
Chuck Shaker Farah;
Elizabeth Igne Ferreira;
Rafael Victório Carvalho Guido
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| Advisor: | Antonia Tavares do Amaral |
| Abstract | |
Dengue and Food-and-mouth disease are viral infections that occur in Brazil and in the world, causing a huge socioeconomic impact. Viral proteases are recognized as targets for antiviral design, because they are crucial for the life cycle of many viruses, such as flavivirus (Dengue virus DENV) and picornavirus (Food-and-mouth disease virus FMDV). In order to discovery novel inhibitors of viral proteases (of NS2B/NS3pro of DENV or of Lbpro of FMDV) virtual screening models were proposed comprising a sequence of different filters (e.g. pharmacophore, drug-like, similarity and docking) applied to databases of commercial compounds (280x103 to 23x106 compounds). In all models, the final selection of compounds was always done by visual inspection. For DENV NS2B/NS3pro, four virtual screening models were proposed (Model I-DENV to Model IV-DENV). Model I-DENV was built, based on the crystal structure bound to a peptidemimetic inhibitor, and applied to ZINC database. Finally, ten compounds were purchased and submitted to enzymatic assays against this protease to the experimental validation of this model. Two compounds showed some inhibitory activity (IC50 150 - 300 µM). In order to improve these results, NS2B/NS3pro flexibility was included, using molecular dynamics (MD) simulations, and a novel database was built (ZINC-Curated). Throughout an exhaustively analysis of ZINC-Curated, using statistical/chemometrics tools, we confirmed that this new database was enriched with drug like compounds. Other three virtual screening models were built including different information from MD simulations. Model II-DENV was built using docking and applied to the ZINC-Curated database, selecting sixteen compounds. None of them showed a significant inhibitory activity against DENV NS2B/NS3pro. Models III-DENV and IV-DENV were built using pharmacophore models, which have their performance previously evaluated using literature data, and applied to NCI and ZINC-Curated databases, respectively. Model III-DENV selected fifteen compounds, showing four of them inhibitory activity (IC50 30 - 100 µM). Model IV-DENV selected eighteen compounds. Four of them were active against this protease (IC50 4 - 90 µM), representing a hit rate of ~22 %. Moreover, a set of thirteen structural analogues of the most active compound were built, being three of them also active. Thus, the modifications done in the virtual screening procedure really improved our results. For FMDV Lbpro, two virtual screening models were built (Models I-FMDV and II-FMDV). The first one was based on the crystal structure, without ligands, and used a set of in house potential covalent inhibitors. Six of the in house compounds were selected and tested against this protease. Two of them showed a weak inhibitory activity (IC50 300 - 600 µM). Later on, the Lbpro bound with ligands was available being therefore used to build another model. Model II FMDV was applied to the ZINC-Curated database, selecting fifteen compounds that were purchased and also tested against the target protease. But none of them showed a significant inhibition. Thus, the incorporated changes were not enough to retrieve active compounds. However, these models/results contributed to better understand Lbpro binding site interactions (AU) | |
| 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) |