Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side

Abstract

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 M against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV.IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti, continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors.KEYWORDS dengue fever, Zika, antimicrobial peptides, antiviral agents, drug design, protease inhibitors, receptor-ligand interaction, structural biology, substrate D engue virus (DENV), the causative agent of the disease dengue fever, is endemic in more than 110 countries, with approximately 390 million people infected yearly, leading to about 20,000 deaths (1-3). Currently, no direct-acting antiviral drugs are available either in clinics or in development to combat dengue virus infections. Thus, a better understanding of the causative virus and potential viral drug targets is needed to develop effective therapies.DENV is a member of the family Flaviviridae and is an enveloped virus with a positive single-stranded RNA genome. There are four different serotypes (DENV1 to DENV4), and each serotype shares 65 to 70% seque...

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