Search for drug candidates against epidemic and pandemic viruses, targeting the RNA-dependent RNA polymerase (nsP4) of the Chikungunya virus and the papain-like protease (PL-Pro) of the SARS-CoV-2 virus

Author(s)

M. C. L. C. FREIRE

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2022-06-21

Examining board members

Glaucius Oliva; Antônio José da Costa Filho; Richard Charles Garratt; Roberto Dias Lins Neto; Flávio Henrique da Silva

• Advisor: Glaucius Oliva

Abstract

The development of control strategies for viral diseases has shown notable growth in recent decades. However, the development of specific treatments remains challenging and necessary for many infections. In this context, the detailed knowledge of the viral replication steps and the components involved in this process contributes significantly to the development of new antivirals. Among the viral diseases that remain without specific treatments, Chikungunya Fever is caused by the Chikungunya virus (CHIKV) and transmitted by the bite of female mosquitoes of the Aedes genus. CHIKV is a positive-sense single-stranded RNA virus belonging to the Alphavirus genus. Its genome encodes two polyproteins, which, after cleaved, give rise to five structural and four non-structural proteins. Among the non-structural proteins, nsP4 (nsP4-CHIKV) is the RNA-dependent RNA polymerase (RdRp). It has a crucial role in the viral RNA replication process, thus being considered a promising target for developing new antiviral drugs. In this project, nsP4-CHIKV was expressed, purified and characterized using biophysical methods, generating structural and dynamics information. A series of compounds were evaluated in the search for new molecules with antiviral potential for this target. A compound named LabMol-309 was identified, and biophysical and computational methods evaluated its interaction with the protein. The inhibitory activity of the compound LabMol-309 was confirmed by cellular assays using a replicon system and reporter virus. In addition, given the pandemic caused by the new coronavirus SARS-CoV-2, and within the emergency research effort conducted by CIBFar/CEPID, studies were also carried out together with collaborating groups that came together to seek solutions to this serious global problem of public health. Among SARS-CoV-2 proteins, the protease PLpro (papain-like protease) is responsible for processing the viral polyprotein, acting on replication and is considered a key target for searching for drugs. In this project, PLpro was expressed, purified, and enzymatic activity assays were performed to screen a series of compounds and peptides. Among the evaluated series, the peptides derived from the C-terminal region of Bothropstoxin-I were promising and were assessed in cell assays with the virus, confirming its inhibitory potential. This project is part of the CIBFar/CEPID/FAPESP and aims to establish a base in the group, which already has experience with other arboviruses, for CHIKV and SARS-CoV-2 studies. (AU)

• FAPESP's process: 18/17095-8 - Structural characterization of the Chikungunya Virus NSP4 RNA-dependent RNA polymerase and search for antiviral agents
• Grantee: Marjorie Caroline Liberato Cavalcanti Freire
• Support Opportunities: Scholarships in Brazil - Doctorate