Structuring, organization and development of a search platform for antiviral agents against the arboviruses that cause Zika fever, Chikungunya fever and Yellow fever using the enzymatic domains of the non-structural proteins

• Grant number: 23/09637-3
• Support Opportunities: Scholarships in Brazil - Support Program for Fixating Young Doctors
• Start date: August 01, 2023
• End date: June 30, 2025
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Agreement: CNPq
• Principal Investigator: Glaucius Oliva
• Grantee: Nathalya Cristina de Moraes Roso Mesquita
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 23/01046-6 - Structuring, organization and development of a search platform for antiviral agents against the arboviruses that cause Zika fever, Chikungunya fever and Yellow fever using the enzymatic domains of the non-structural proteins, AP.R

Abstract

The arboviruses that cause Zika, Chikungunya and Yellow fever have been a major public health challenge in Brazil due to climatic conditions conducive to the dissemination of their vectors, the mosquitoes of the Aedes and Haemagogus gender. Despite all the efforts of the academic community, there are still no vaccines or therapeutic agents available for ZIKV and CHIKV, in addition to the existence of recent vaccine leaks against YFV, thus motivating the search for possible drug candidates for these viruses. In this context, detailed knowledge of viral replication steps and the components involved in this process significantly contribute to the development of new antivirals. While the genome of ZIKV and YFV is made up of a positive strand of RNA that encodes a single polyprotein, which after being processed, originates 10 proteins, 3 of which are structural (capsid, envelope and membrane protein) and 7 are non-structural (NSs - NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5); the CHIKV genome, also made up of a single positive strand of RNA, encodes two polyproteins that, after being cleaved, give rise to five structural proteins (envelope 1, envelope 2, envelope 3, capsid and 6k) and four non-structural proteins (nsP1, nsP2 , nsP3 and nsP4). The replication process essentially depends on the non-structural proteins encoded by the viral genomes, among the non-structural proteins, only NS3 and NS5 of ZIKV and YFV and nsP1, nsP2, and nsP4 of CHIKV have enzymatic functions, awakening great potential as a pharmacological target. In ZIKV and YFV, NS3 has two protein domains with enzymatic functions: an N-terminal serine protease (NS3Pro) and a C-terminal NTP-dependent RNA-helicase (NS3Hel), responsible, respectively, for the cleavage of polyproteins after translation (together with the C-terminal portion of NS2B) and by unwinding an RNA double strand prior to RNA polymerization and capping. In these same viruses, NS5 also has two enzymatic protein domains: an N-terminal RNA-dependent RNA polymerase (RdRp) that polymerizes RNA strands to construct new viral particles, and a C-terminal Methyltransferase (Mtase) that is involved in methylation. from the 5' end of the RNA genome. In CHIKV, nsP1 has two domains with enzymatic functions: Methyltransferase and Guanylyltransferase, carrying out the capping of the viral RNA, carrying out, and also carrying out, the anchoring of the replication complex in the cell membrane; nsP2 also has two domains with enzymatic functions: in its N-terminal region it has NTPase and helicase functions and in the C-terminal it has cysteine-protease activity, responsible for the processing of non-structural polyprotein and nsP4 has a single RNA-dependent RNA polymerase protein domain acting in the synthesis and elongation of viral RNA. Specific blocking of any of these functions is lethal to the replication process of these three viruses. Recently, some protocols for expression, purification and functional and structural characterization assays of the Protease (NS2B-NS3Pro), NS3 Helicase (NS3Hel), Polymerase (NS5Pol) domains of ZIKV; Protease (NS2B-NS3Pro) and Polymerase (NS5Pol) from YFV and Polymerase (NSP4Pol) from CHIKV, by several graduate students who were part of our laboratory. These protocols were useful for identifying some molecular hits that are in the development phase. Thus, the main objective of this project is to structure, organize and review the protocols already existing in the laboratory, to clone and develop the protocols that have not yet been established for the Methyltransferase protein domains of the three viruses, Yellow Fever and Chikungunya Helicase and Chikungunya Protease with the aim of establishing a complete search and development platform for antiviral agents against Zika fever, Chikungunya fever and Yellow fever using the enzymatic domains of their non-structural proteins.