A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease.
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ABSTRACT:
SUBMITTER: Yang CC
PROVIDER: S-EPMC3910792 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dengue virus (DENV) causes disease globally, with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available, and treatment is supportive. In this study, we identified BP2109, a potent and selective small-molecule inhibitor of the DENV NS2B/NS3 protease, by a high-throughput screening assay using a recombinant protease complex consisting of the central hydrophilic portion of NS2B and the N terminus of the protease domain. BP2109 inhibited DENV (serotypes 1 to 4), but not Japanese encephalitis virus (JEV), replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with a 50% inhibitory concentration (IC(50)) of 15.43 ± 2.12 μM and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC(50)) of 0.17 ± 0.01 μM. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of the NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue virus NS2B/NS3 protease and a dengue virus replicon construct conferred 10.3- and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined to be the key mutation conferring dengue virus replicon resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect resistance to BP2109. Both the R55K and E80K mutations are located in the central hydrophilic portion of the NS2B cofactor, where extensive interactions with the NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.
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Project description:Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.
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Project description:Dengue virus (DENV) is a mosquito-borne flavivirus that has strained global healthcare systems throughout tropical and subtropical regions of the world. In addition to plaguing developing nations, it has re-emerged in several developed countries with recent outbreaks in the USA (CDC, 2010), Australia (Hanna et al., 2009), Taiwan (Kuan et al., 2010) and France (La Ruche et al., 2010). DENV infection can cause significant disease, including dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and death. There are no approved vaccines or antiviral therapies to prevent or treat dengue-related illnesses. However, the viral NS2B-NS3 protease complex provides a strategic target for antiviral drug development since NS3 protease activity is required for virus replication. Recently, we reported two compounds with inhibitory activity against the DENV protease in vitro and antiviral activity against dengue 2 (DEN2V) in cell culture (Tomlinson et al., 2009a). Analogs of one of the lead compounds were purchased, tested in protease inhibition assays, and the data evaluated with detailed kinetic analyses. A structure activity relationship (SAR) identified key atomic determinants (i.e. functional groups) important for inhibitory activity. Four "second series" analogs were selected and tested to validate our SAR and structural models. Here, we report improvements to inhibitory activity ranging between ∼2- and 60-fold, resulting in selective low micromolar dengue protease inhibitors.
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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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Project description:Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors.
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Project description:UNLABELLED:Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-viral activity of compounds present in the leaves of Carica papaya against dengue 2 virus (DENV-2). We analysed the anti-dengue activities of the extracts from Carica papaya by using bioinformatics tools. Interestingly, we find the flavonoid quercetin with highest binding energy against NS2B-NS3 protease which is evident by the formation of six hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results suggest that the flavonoids from Carica papaya have significant anti-dengue activities. ABBREVIATIONS:ADME - Absorption, distribution, metabolism and excretion, BBB - Blood brain barrier, CYP - Cytochrome P450, DENV - - Dengue virus, DHF - Dengue hemorrhagic fever, DSS - Dengue shock syndrome, GCMS - - Gas chromatography- Mass spectrometry, MOLCAD - Molecular Computer Aided Design, NS - Non structural, PDB - Protein data bank, PMF - Potential Mean Force.
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Project description:Zika virus is a mosquito-transmitted flavivirus that causes devastating fetal outcomes in the context of maternal infection during pregnancy. An important target for drugs combatting Zika virus pathogenicity is NS2B-NS3 protease, which plays an essential role in hydrolysis and maturation of the flavivirus polyprotein. We identify hydroxychloroquine, a drug that already has approved uses in pregnancy, as a possible inhibitor of NS2B-NS3 protease by using a Food and Drug Administration-approved drug library, molecular docking, and molecular dynamics simulations. Further, to gain insight into its inhibitory potential toward NS2B-NS3 protease, we performed enzyme kinetic studies, which revealed that hydroxychloroquine inhibits protease activity with an inhibition constant (K i) of 92.34 ± 11.91 μM. Additionally, hydroxychloroquine significantly decreases Zika virus infection in placental cells.
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Project description:Dengue virus (DENV) infection causes serious health problems in humans for which no drug is currently available. Recently, DENV NS2B-NS3 protease has been proposed as a primary target for anti-dengue drug discovery due to its important role in new virus particle formation by conducting DENV polyprotein cleavage. Triterpenoids from the medicinal fungus Ganoderma lucidum have been suggested as pharmacologically bioactive compounds and tested as anti-viral agents against various viral pathogens including human immunodeficiency virus. However, no reports are available concerning the anti-viral activity of triterpenoids from Ganoderma lucidum against DENV. Therefore, we employed a virtual screening approach to predict the functional triterpenoids from Ganoderma lucidum as potential inhibitors of DENV NS2B-NS3 protease, followed by an in vitro assay. From in silico analysis of twenty-two triterpenoids of Ganoderma lucidum, four triterpenoids, viz. Ganodermanontriol (-6.291 kcal/mol), Lucidumol A (-5.993 kcal/mol), Ganoderic acid C2 (-5.948 kcal/mol) and Ganosporeric acid A (-5.983 kcal/mol) were predicted to be viral protease inhibitors by comparison to reference inhibitor 1,8-Dihydroxy-4,5-dinitroanthraquinone (-5.377 kcal/mol). These results were further studied for binding affinity and stability using the molecular mechanics/generalized Born surface area method and Molecular Dynamics simulations, respectively. Also, in vitro viral infection inhibition suggested that Ganodermanontriol is a potent bioactive triterpenoid.
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Project description:Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 μM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.
| S-EPMC10723727 | biostudies-literature
Project description:West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH2 inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11 µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.
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