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

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 processing. To search for Zika virus NS2B-NS3 protease (ZIKV_Pro) inhibitors, a phage display library was constructed using the Boophilin domain 1 (BoophD1), a thrombin inhibitor from the Kunitz family. A BoophilinD1 library mutated at positions P1-P4' was constructed, presenting a titer of 2.9x10⁶ (cfu), and screened utilizing purified ZIKV_Pro. 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 ZIKV_Pro of 0.103, 0.116, and 0.101 μM, respectively. The BoophD1 mutant inhibitors inhibit the Dengue virus 2 protease (DENV2) with Ki values of 0.298, 0.271, and 0.379 μM, respectively. In conclusion, BoophD1 mut 12 and 14 selected for ZIKV_Pro 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 ZIKV_Pro inhibit both Zika and Dengue 2 proteases making them potential pan-flavivirus inhibitors.

Keywords: Kunitz domain; NS2B-NS3 proteases; Phage display; Zika virus.