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Curcumin Allosterically Inhibits the Dengue NS2B-NS3 Protease by Disrupting Its Active Conformation.

ABSTRACT: Flaviviruses including dengue virus and Zika virus encode a unique two-component NS2B-NS3 protease essential for maturation/infectivity, thus representing a key target for designing antiflavivirus drugs. Here, for the first time, by NMR and molecular docking, we reveal that curcumin allosterically inhibits the dengue protease by binding to a cavity with no overlap with the active site. Further molecular dynamics simulations decode that the binding of curcumin leads to unfolding/displacing the characteristic ?-hairpin of the C-terminal NS2B and consequently disrupting the closed (active) conformation of the protease. Our study identified a cavity most likely conserved in all flaviviral NS2B-NS3 proteases, which could thus serve as a therapeutic target for the discovery/design of small-molecule allosteric inhibitors. Moreover, as curcumin has been used as a food additive for thousands of years in many counties, it can be directly utilized to fight the flaviviral infections and as a promising starting for further design of potent allosteric inhibitors.

SUBMITTER: Lim L

PROVIDER: S-EPMC7557217 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Curcumin Allosterically Inhibits the Dengue NS2B-NS3 Protease by Disrupting Its Active Conformation.

Lim Liangzhong L Dang Mei M Roy Amrita A Kang Jian J Song Jianxing J

ACS omega 20200929 40

Flaviviruses including dengue virus and Zika virus encode a unique two-component NS2B-NS3 protease essential for maturation/infectivity, thus representing a key target for designing antiflavivirus drugs. Here, for the first time, by NMR and molecular docking, we reveal that curcumin allosterically inhibits the dengue protease by binding to a cavity with no overlap with the active site. Further molecular dynamics simulations decode that the binding of curcumin leads to unfolding/displacing the ch ...[more]

PMID: 33073093

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Project description: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 Ki 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.

Project description:BACKGROUND: The dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is now intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme-substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of designing inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed. METHODS: Alanine substitutions were introduced by site-directed mutagenesis at residues L115, D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters Km, kcat and kcat/Km were determined. RESULTS: Kinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activity. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole. CONCLUSIONS: Kinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.

Dataset Information

Curcumin Allosterically Inhibits the Dengue NS2B-NS3 Protease by Disrupting Its Active Conformation.

Publications

Curcumin Allosterically Inhibits the Dengue NS2B-NS3 Protease by Disrupting Its Active Conformation.

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