Discovery of antiviral candidates based on the yellow fever virus enzyme NS5 RNA-dependent RNA polymerase

Author(s)

Victor Gawriljuk Ferraro Oliveira

• Document type: Master's Dissertation
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2021-03-23

Examining board members

Glaucius Oliva; Carolina Borsoi Moraes; Maria Cristina Nonato

• Advisor: Glaucius Oliva; Andre Schützer de Godoy

Abstract

The yellow fever resurgence in non-endemic regions of Brazil and Africa between 2016 and 2018, put at risk urban regions with high population density and low vaccination coverage. The lack of a yellow fever treatment, as well as the need for drugs that can control emerging viruses, highlights the importance in the discovery of new antivirals. The goal of this work was the discovery of antiviral candidates with the yellow fever virus RNA dependent RNA polymerase as target. In order to achieve this goal, the production of the recombinant form of this enzyme as well as two RNA strand elongation assays were standardized and evaluated for inhibitor screening. The first used modified nucleotides coupled to an alkaline phosphatase reaction, while in the second, protein activity was measured with the intercalating dye SYBR Green I. The SGI assay was used for the screening and evaluation of inhibitors, since it had a higher fluorescence intensity and easier implementation in medium scale experiments. Afterwards, we screened the compound library Pandemic Response Box and identified 25 compounds as RdRp inhibitors. Several compounds had potency within the nano molar range, showing potential existence of false-positives in the screening. The molecules were validated with an SGI interference assay and 18 compounds significantly interfered with the dye. Among the inhibitors considered reliable, Clofazimine, TTP-8307 and MMV218827 had potency within 0.2 to 4 micromolar. Despite the high potency of MMV218827 (IC50 = 0.2 μM), the compound was disregarded as a promising candidate after destabilizing the RdRp in thermal shift assays and because it belongs to a class of compounds that shows nonspecific inhibition of proteins. Clofazimine (IC50 = 4 μM) and TTP-8307 (IC50 = 4 μM) increased the RdRp stability and have chemical scaffolds new to RdRp inhibitors, expanding the development of new inhibitors for this target. Crystallization assays were also performed; however, it was not possible to obtain suitable crystals. Thus, the results obtained herein show that the SGI elongation assay can be used to screen compounds for the yellow fever virus RdRp, and that Clofazimine as well as TTP-8307 are possible antiviral candidates for this virus. Future studies should evaluate the activity of those compounds in cell-based assays. (AU)

• FAPESP's process: 18/25773-6 - Antiviral candidates discovery based on the structure of the yellow fever NS5 RNA-dependent RNA polymerase enzyme
• Grantee: Victor Gawriljuk Ferraro Oliveira
• Support Opportunities: Scholarships in Brazil - Master