Boophilin D1, a Kunitz type protease inhibitor, as a source of inhibitors for the ZIKA virus NS2B-NS3 protease

Author(s)

Manzato, Veronica de Moraes ; Di Santo, Camila ; Torquato, Ricardo Jose Soares ; Coelho, Camila ; Gallo, Gloria ; Hardy, Leon ; Wurtele, Martin ; Tanaka, Aparecida Sadae

• Document type: Journal article
• Source: Biochimie; v. 214, p. 6-pg., 2023-07-03.

Abstract

Arboviruses are a global concern for a multitude of reasons, including their increased incidence and human mortality. Vectors associated with arboviruses include the mosquito Aedes sp., which is responsible for transmitting the Zika virus. Flaviviruses, like the Zika virus, present only one chymotrypsin-like serine protease (NS3) in their genome. Together with host enzymes, the NS2B co-factor NS3 protease complex are essential for the viral replication cycle by virus polyprotein process-ing. To search for Zika virus NS2B-NS3 protease (ZIKVPro) inhibitors, a phage display library was con-structed using the Boophilin domain 1 (BoophD1), a thrombin inhibitor from the Kunitz family. A BoophilinD1 library mutated at positions P1-P40 was constructed, presenting a titer of 2.9x106 (cfu), and screened utilizing purified ZIKVPro. The results demonstrated at the P1-P4' positions the occurrence of 47% RALHA sequence (mut 12) and 11.8% RASWA sequence (mut14), SMRPT, or KALIP (wt) sequence. BoophD1-wt and mutants 12 and 14 were expressed and purified. The purified BoophD1 wt, mut 12 and 14, presented Ki values for ZIKVPro of 0.103, 0.116, and 0.101 mM, respectively. The BoophD1 mutant in-hibitors inhibit the Dengue virus 2 protease (DENV2) with Ki values of 0.298, 0.271, and 0.379 mM, respectively. In conclusion, BoophD1 mut 12 and 14 selected for ZIKVPro demonstrated inhibitory activity like BoophD1-wt, suggesting that these are the strongest Zika inhibitors present in the BoophD1 mutated phage display library. Furthermore, BoophD1 mutants selected for ZIKVPro inhibit both Zika and Dengue 2 proteases making them potential pan-flavivirus inhibitors.& COPY; 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. (AU)

• FAPESP's process: 12/03657-8 - Inhibitor and proteases of ectoparasites: relationship of structure-function and identification of the role of these molecules in the interaction of diseases vector e their etiological agents
• Grantee: Aparecida Sadae Tanaka
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/03779-5 - Use of phage display as a tool in the diagnosis and control of diseases transmitted by hematophagous vectors
• Grantee: Aparecida Sadae Tanaka
• Support Opportunities: Research Projects - Thematic Grants