Full text
| |
| Author(s): |
Gabriela Dias Noske
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) |
| Defense date: | 2023-10-24 |
| Examining board members: |
Glaucius Oliva;
Richard Charles Garratt;
Beatriz Gomes Guimarães;
Germán Gustavo Sgro;
Daniela Barretto Barbosa Trivella
|
| Advisor: | Glaucius Oliva; Andre Schützer de Godoy |
| Abstract | |
The recent advances in research have enabled a greater understanding of the mechanisms of several viral diseases that supported the development of vaccines and antiviral drugs. However, viral diseases still represent one of the major public health and economic concerns worldwide, highlighting the constant need for the development of new antivirals. Among the viruses that still lack specific drugs for their treatment are those belonging to the flavivirus genus, which includes the viruses that cause dengue, zika and yellow fever. The yellow fever virus (YFV) has a genome composed of a single RNA strand that encodes a polyprotein containing three structural and seven non-structural proteins. NS3 has two domains: a protease domain and a helicase/NTPase domain. The NS3 protease acts together with another non-structural protein as a cofactor, NS2B, which helps the correct folding of NS3pro and allows it to assume its active form. The NS2B-NS3pro complex assists in the cleavage of the immature polyprotein, releasing the individual proteins that form the viral replication complex. Considering the importance of this complex in the viral replication, it is clear that it represents an important target in the design of antiviral candidates. Therefore, the objective of this project was to determine the crystallographic structure of the NS2B-NS3pro enzyme and use it in the search for inhibitors. The coding sequence was cloned, expressed and the protein purified. It was also possible to obtain crystals of the enzyme, used to solve the 3D structure, resulting in a final model at 2.9 Å resolution. Furthermore, we evaluated the activity of the protein using a fluorescence-based assay. It was possible to determine its kinetic constants, in addition to the screening for inhibitors. Among approximately 1000 compounds screened, 18 hits were found, 6 of them with IC50 values below 1.0 μM. The most promising ligands had antiviral activity tested in cells containing the YFV replicon, in which 3 compounds presented EC50 at low micromolar. In conclusion, we were able to move forward with the cloning, expression and purification of the YFV NS2B-NS3 protease complex. Furthermore, we obtained crystals of the protein, which made it possible to resolve its crystallographic structure. The enzyme demonstrated high proteolytic activity against the synthetic fluorescent peptide, which was of paramount importance for screening inhibitors. Additionally, due to the pandemic caused by the new coronavirus, SARS-CoV-2, and as part of the collaborative project led by CIBFar/CEPID as an emergency effort to develop antivirals against SARSCoV- 2, studies were also developed with the coronaviral proteases. One of the virus proteases, Mpro, is a dimeric protein widely studied and explored for the development of antivirals. However, details about its self-maturation process remained unknown. In this work, Mpro was expressed and purified in three different constructs (IMT-Mpro, Mpro and C145S Mpro). Both constructs were crystallized and had their crystallographic structures solved, as well as characterized in solution. Furthermore, the structure of the C145S Mpro enzyme was determined at 3.5Å, in complex with the endogenous N-terminal peptide, using cryo-electron microscopy. We also analyzed the influence of different inhibitors on the enzyme maturation process. Finally, we were able to infer that the N-terminal cleavage is not critical for the dimerization of the enzyme, but rather, the conformational changes are caused by the adjustment induced after covalent binding of the substrate. The elucidation of the functional and structural changes that occur during the maturation process obtained in this work provides important information for understanding this mechanism and for proposing specific inhibitors, which target intermediate stages of the enzyme maturation process. Furthermore, this work helps us gain a greater understanding of the proteases of yellow fever and SARS-CoV-2 viruses and supports the development of new antivirals. (AU) | |
| FAPESP's process: | 18/25600-4 - Discovery and development of antiviral candidates against Yellow Fever virus based on the structure of the NS2B-NS3 protease complex |
| Grantee: | Gabriela Dias Noske |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |