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Selection of inhibitors for the NS2/NS3 proteases from Dengue and Zika viruses by cellular high-throughput assays coupled with cyclic peptide libraries

Grant number: 20/09678-3
Support type:Regular Research Grants
Duration: December 01, 2020 - November 30, 2022
Field of knowledge:Biological Sciences - Biochemistry - Enzymology
Principal Investigator:Vitor Marcelo Silveira Bueno Brandão de Oliveira
Grantee:Vitor Marcelo Silveira Bueno Brandão de Oliveira
Home Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
Assoc. researchers:Débora Noma Okamoto ; Marcelo Ferreira Marcondes Machado


For both Zika and Dengue viruses the NS2/NS3 protease, which is formed during replication process into the host cell, is essential for the processing of the viral polyprotein. The inhibition of this enzyme blocks the replication of the virus in the host cell. Therefore, inhibitors for these proteases are potential antiviral drugs.The main objective of this project is the development of inhibitors for the NS2B/NS3 protease of the Dengue virus as well as the Zika virus. Our proposal is to use cellular assays to select potential inhibitors of these enzymes generated in a library of cyclic peptides. The rigidity of the cyclic chain confers greater resistance to cleavage by proteases when compared to the same linear peptide sequence, thus, cyclic peptides are being pointed out as prototype inhibitors or even as potential protease inhibitors.We intend to use a technique based on the properties of divided inteins, abbreviated by SICLOPPS (Split Intein-mediated Circular Ligation Of Peptides and ProteinS), associated with codon randomization. This technique allows the generation of cell libraries of thousands of cyclic peptides. Ex: a library with 6 random codons can generate up to 64,000,000 different cyclic peptides. To select a possible inhibitor, the library must be coupled with a cell assay for the selection of positive clones (a cell with the NS2B/NS3 inhibited). For this, in addition to the cyclic peptide, inside the host cell it is necessary to express also: the NS2B/NS3 protease and a substrate for this enzyme, whose product is toxic to the cell. When the cyclic peptide (in one of the library clones) inhibits the protease by preventing the formation of the toxic product, the cell survives (positive clones). However, if the cyclic peptide does not inhibit the protease, the cell will not survive (negative clones). After the selection of positive clones, the corresponding cyclic peptides, identified by DNA sequencing, will be produced for in vitro kinetic tests, to confirm the screening. (AU)