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Myricetin Allosterically Inhibits the Dengue NS2B-NS3 Protease by Disrupting the Active and Locking the Inactive Conformations.

ABSTRACT: The dengue NS2B-NS3 protease existing in equilibrium between the active and inactive forms is essential for virus replication, thus representing a key drug target. Here, myricetin, a plant flavonoid, was characterized to noncompetitively inhibit the dengue protease. Further NMR study identified the protease residues perturbed by binding to myricetin, which were utilized to construct the myricetin-protease complexes. Strikingly, in the active form, myricetin binds to a new allosteric site (AS2) far away from the active site pocket and the allosteric site (AS1) for binding curcumin, while in the inactive form, it binds to both AS1 and AS2. To decipher the mechanism for the allosteric inhibition by myricetin, we conducted molecular dynamics simulations on different forms of dengue NS2B-NS3 proteases. Unexpectedly, the binding of myricetin to AS2 is sufficient to disrupt the active conformation by displacing the characteristic NS2B C-terminal β-hairpin from the active site pocket. By contrast, the binding of myricetin to AS1 and AS2 results in locking the inactive conformation. Therefore, myricetin represents the first small molecule, which allosterically inhibits the dengue protease by both disrupting the active conformation and locking the inactive conformation. The results enforce the notion that a global allosteric network exists in the dengue NS2B-NS3 protease, which is susceptible to allosteric inhibition by small molecules such as myricetin and curcumin. As myricetin has been extensively used as a food additive, it might be directly utilized to fight the dengue infections and as a promising starting material for further design of potent allosteric inhibitors.

SUBMITTER: Dang M

PROVIDER: S-EPMC8793048 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Myricetin Allosterically Inhibits the Dengue NS2B-NS3 Protease by Disrupting the Active and Locking the Inactive Conformations.

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