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1.
J Virol ; 94(24)2020 11 23.
Article in English | MEDLINE | ID: mdl-32907977

ABSTRACT

Dengue virus (DENV) NS5 RNA-dependent RNA polymerase (RdRp), an important drug target, synthesizes viral RNA and is essential for viral replication. While a number of allosteric inhibitors have been reported for hepatitis C virus RdRp, few have been described for DENV RdRp. Following a diverse compound screening campaign and a rigorous hit-to-lead flowchart combining biochemical and biophysical approaches, two DENV RdRp nonnucleoside inhibitors were identified and characterized. These inhibitors show low- to high-micromolar inhibition in DENV RNA polymerization and cell-based assays. X-ray crystallography reveals that they bind in the enzyme RNA template tunnel. One compound (NITD-434) induced an allosteric pocket at the junction of the fingers and palm subdomains by displacing residue V603 in motif B. Binding of another compound (NITD-640) ordered the fingers loop preceding the F motif, close to the RNA template entrance. Most of the amino acid residues that interacted with these compounds are highly conserved in flaviviruses. Both sites are important for polymerase de novo initiation and elongation activities and essential for viral replication. This work provides evidence that the RNA tunnel in DENV RdRp offers interesting target sites for inhibition.IMPORTANCE Dengue virus (DENV), an important arthropod-transmitted human pathogen that causes a spectrum of diseases, has spread dramatically worldwide in recent years. Despite extensive efforts, the only commercial vaccine does not provide adequate protection to naive individuals. DENV NS5 polymerase is a promising drug target, as exemplified by the development of successful commercial drugs against hepatitis C virus (HCV) polymerase and HIV-1 reverse transcriptase. High-throughput screening of compound libraries against this enzyme enabled the discovery of inhibitors that induced binding sites in the RNA template channel. Characterizations by biochemical, biophysical, and reverse genetics approaches provide a better understanding of the biological relevance of these allosteric sites and the way forward to design more-potent inhibitors.


Subject(s)
Dengue Virus/genetics , Dengue Virus/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Allosteric Site , Antiviral Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Dengue/virology , HIV Reverse Transcriptase , High-Throughput Screening Assays , Humans , Models, Molecular , RNA-Dependent RNA Polymerase/drug effects , RNA-Dependent RNA Polymerase/genetics , Replicon , Sequence Alignment , Sequence Analysis, Protein , Viral Nonstructural Proteins/drug effects , Viral Nonstructural Proteins/genetics , Virus Replication/drug effects , Virus Replication/physiology
2.
Proc Natl Acad Sci U S A ; 117(30): 17992-18001, 2020 07 28.
Article in English | MEDLINE | ID: mdl-32669438

ABSTRACT

Dengue virus (DENV) was designated as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a "kissing" interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor's inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.


Subject(s)
Antiviral Agents/chemistry , Capsid Proteins/chemistry , Dengue Virus , Models, Molecular , Protein Conformation , Amino Acid Sequence , Antiviral Agents/pharmacology , Binding Sites , Capsid Proteins/genetics , Dengue Virus/drug effects , Mutation , Nucleocapsid/chemistry , Nucleocapsid/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Structure-Activity Relationship
3.
Adv Exp Med Biol ; 1062: 187-198, 2018.
Article in English | MEDLINE | ID: mdl-29845534

ABSTRACT

Flavivirus NS5 RNA-dependent RNA polymerase (RdRp) is an important drug target. Whilst a number of allosteric inhibitors have been described for Hepatitis C virus RdRp, few have been described for DENV RdRp. In addition, compound screening campaigns have not yielded suitable leads for this enzyme. Using fragment-based screening via X-ray crystallography, we identified a biphenyl acetic acid fragment that binds to a novel pocket of the dengue virus (DENV) RdRp, in the thumb/palm interface, close to its active site (termed "N pocket"). Structure-guided optimization yielded nanomolar inhibitors of the RdRp de novo initiation activity, with low micromolar EC50 in DENV cell-based assays. Compound-resistant DENV replicons exhibited amino acid mutations that mapped to the N pocket. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors and provides a significant opportunity for rational design of novel therapeutics against this proven antiviral target.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Dengue Virus/enzymology , Dengue/virology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Animals , Dengue/drug therapy , Dengue Virus/chemistry , Dengue Virus/drug effects , Dengue Virus/genetics , Drug Design , Humans , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Structure-Activity Relationship , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
4.
Nature ; 546(7658): 376-380, 2017 06 15.
Article in English | MEDLINE | ID: mdl-28562588

ABSTRACT

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.


Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Cryptosporidiosis/drug therapy , Cryptosporidiosis/parasitology , Cryptosporidium/drug effects , Cryptosporidium/enzymology , Pyrazoles/pharmacology , Pyridines/pharmacology , Animals , Animals, Newborn , Cattle , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Immunocompromised Host , Interferon-gamma/deficiency , Interferon-gamma/genetics , Male , Mice , Mice, Knockout , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyridines/chemistry , Pyridines/pharmacokinetics , Rats , Rats, Wistar
5.
J Med Chem ; 59(8): 3935-52, 2016 04 28.
Article in English | MEDLINE | ID: mdl-26984786

ABSTRACT

The discovery and optimization of non-nucleoside dengue viral RNA-dependent-RNA polymerase (RdRp) inhibitors are described. An X-ray-based fragment screen of Novartis' fragment collection resulted in the identification of a biphenyl acetic acid fragment 3, which bound in the palm subdomain of RdRp. Subsequent optimization of the fragment hit 3, relying on structure-based design, resulted in a >1000-fold improvement in potency in vitro and acquired antidengue activity against all four serotypes with low micromolar EC50 in cell-based assays. The lead candidate 27 interacts with a novel binding pocket in the palm subdomain of the RdRp and exerts a promising activity against all clinically relevant dengue serotypes.


Subject(s)
Antiviral Agents/pharmacology , Dengue Virus/enzymology , Enzyme Inhibitors/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Antiviral Agents/chemistry , Calorimetry , Cell Line , Drug Design , Enzyme Inhibitors/chemistry , Humans , Molecular Structure , Surface Plasmon Resonance
6.
J Biol Chem ; 291(16): 8541-8, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26872970

ABSTRACT

We performed a fragment screen on the dengue virus serotype 3 RNA-dependent RNA polymerase using x-ray crystallography. A screen of 1,400 fragments in pools of eight identified a single hit that bound in a novel pocket in the protein. This pocket is located in the polymerase palm subdomain and conserved across the four serotypes of dengue virus. The compound binds to the polymerase in solution as evidenced by surface plasmon resonance and isothermal titration calorimetry analyses. Related compounds where a phenyl is replaced by a thiophene show higher affinity binding, indicating the potential for rational design. Importantly, inhibition of enzyme activity correlated with the binding affinity, showing that the pocket is functionally important for polymerase activity. This fragment is an excellent starting point for optimization through rational structure-based design.


Subject(s)
DNA-Directed RNA Polymerases/chemistry , Dengue Virus/enzymology , Viral Proteins/chemistry , Catalytic Domain , Crystallography, X-Ray , Protein Structure, Tertiary
7.
Eur J Med Chem ; 106: 144-56, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26544629

ABSTRACT

Pyridone 1 was identified from a high-throughput cell-based phenotypic screen against Mycobacterium tuberculosis (Mtb) including multi-drug resistant tuberculosis (MDR-TB) as a novel anti-TB agent and subsequently optimized series using cell-based Mtb assay. Preliminary structure activity relationship on the isobutyl group with higher cycloalkyl groups at 6-position of pyridone ring has enabled us to significant improvement of potency against Mtb. The lead compound 30j, a dimethylcyclohexyl group on the 6-position of the pyridone, displayed desirable in vitro potency against both drug sensitive and multi-drug resistant TB clinical isolates. In addition, 30j displayed favorable oral pharmacokinetic properties and demonstrated in vivo efficacy in mouse model. These results emphasize the importance of 4-hydroxy-2-pyridones as a new chemotype and further optimization of properties to treat MDR-TB.


Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Pyridones/pharmacology , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiology , Animals , Antitubercular Agents/chemistry , Antitubercular Agents/metabolism , Biological Availability , Dose-Response Relationship, Drug , Drug Stability , Humans , Mice , Microbial Sensitivity Tests , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Pyridones/chemistry , Pyridones/metabolism , Rats , Structure-Activity Relationship
8.
Antiviral Res ; 119: 57-67, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25912817

ABSTRACT

The non-structural protein 5 (NS5) of flaviviruses is the most conserved amongst the viral proteins. It is about 900 kDa and bears enzymatic activities that play vital roles in virus replication. Its N-terminal domain encodes dual N7 and 2'-O methyltransferase activities (MTase), and possibly guanylyltransferase (GTase) involved in RNA cap formation. The C-terminal region comprises a RNA-dependent RNA polymerase (RdRp) required for viral RNA synthesis. Both MTase and RdRp activities of dengue virus NS5 are well characterized, structurally and functionally. Numerous crystal structures of the flavivirus MTase and RdRp domains have been solved. Inhibitors of both functions have been identified through screening activities using biochemical and cell-based assays, as well as via rational design approaches. This review summaries the current knowledge as well as prospective views on these aspects. This article forms part of a symposium on flavivirus drug discovery in Antiviral Research.


Subject(s)
Dengue Virus/chemistry , Drug Discovery , RNA-Dependent RNA Polymerase , Viral Nonstructural Proteins , Antiviral Agents/pharmacology , Dengue Virus/drug effects , Dengue Virus/genetics , Enzyme Inhibitors/pharmacology , Methyltransferases/antagonists & inhibitors , Methyltransferases/chemistry , Methyltransferases/metabolism , Prospective Studies , Protein Structure, Tertiary , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Severe Dengue/drug therapy , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication
9.
Sci Transl Med ; 7(269): 269ra3, 2015 Jan 07.
Article in English | MEDLINE | ID: mdl-25568071

ABSTRACT

New chemotherapeutic agents are urgently required to combat the global spread of multidrug-resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase InhA is one of the few clinically validated targets in tuberculosis drug discovery. We report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH (reduced form of nicotinamide adenine dinucleotide)-dependent manner and blocked the enoyl substrate-binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of Mycobacterium tuberculosis infection. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Oxidoreductases/antagonists & inhibitors , Animals , Antitubercular Agents/chemistry , Bacterial Proteins/metabolism , Biophysical Phenomena/drug effects , Crystallography, X-Ray , Disease Models, Animal , Drug Resistance, Multiple, Bacterial/drug effects , Enzyme Inhibitors/chemistry , Mice, Inbred BALB C , Models, Molecular , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Oxidoreductases/metabolism , Pyridines/chemistry , Pyridines/pharmacology , Reproducibility of Results , Sequence Analysis, DNA , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiology
10.
Antiviral Res ; 111: 78-81, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25241250

ABSTRACT

Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2'-O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket.


Subject(s)
Dengue Virus/enzymology , Methyltransferases/chemistry , Methyltransferases/metabolism , S-Adenosylmethionine/metabolism , Viral Proteins/chemistry , Binding Sites , Crystallography, X-Ray , Dengue Virus/chemistry , Dengue Virus/genetics , Methyltransferases/genetics , Models, Molecular , Protein Binding , Protein Footprinting , S-Adenosylmethionine/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
11.
Virology ; 450-451: 250-7, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24503088

ABSTRACT

Dengue virus (DENV) non-structural protein 4B (NS4B) has been demonstrated to be an attractive antiviral target. Due to its nature as an integral membrane protein, NS4B remains poorly characterized. In this study, we generated and characterized two monoclonal antibodies (mAb) that selectively bind to DENV NS4B protein. One mAb, 10-3-7, is specific for DENV-2 NS4B, and its epitope was mapped to residues 5-15 of NS4B. The other mAb, 44-4-7, cross-reacts with all the four serotypes of DENV NS4B, and its epitope was mapped to residues 141-147 of NS4B. Using the mAbs, we probed the intracellular orientation of the epitopes of NS4B by an epitope accessibility assay. The results showed that the N-terminus of NS4B is located in the ER lumen, whereas amino acids 130-148 of NS4B are located in the cytosol. The study demonstrates that the two anti-NS4B mAbs will be useful for future structural and functional analyses of DENV NS4B.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Dengue Virus/immunology , Dengue/immunology , Viral Nonstructural Proteins/immunology , Animals , Cross Reactions , Dengue/virology , Dengue Virus/chemistry , Dengue Virus/genetics , Epitope Mapping , Female , Humans , Mice , Mice, Inbred BALB C , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
12.
Antiviral Res ; 100(2): 500-19, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24076358

ABSTRACT

To combat neglected diseases, the Novartis Institute of Tropical Diseases (NITD) was founded in 2002 through private-public funding from Novartis and the Singapore Economic Development Board. One of NITD's missions is to develop antivirals for dengue virus (DENV), the most prevalent mosquito-borne viral pathogen. Neither vaccine nor antiviral is currently available for DENV. Here we review the progress in dengue drug discovery made at NITD as well as the major discoveries made by academia and other companies. Four strategies have been pursued to identify inhibitors of DENV through targeting both viral and host proteins: (i) HTS (high-throughput screening) using virus replication assays; (ii) HTS using viral enzyme assays; (iii) structure-based in silico docking and rational design; (iv) repurposing hepatitis C virus inhibitors for DENV. Along the developmental process from hit finding to clinical candidate, many inhibitors did not advance beyond the stage of hit-to-lead optimization, due to their poor selectivity, physiochemical or pharmacokinetic properties. Only a few compounds showed efficacy in the AG129 DENV mouse model. Two nucleoside analogs, NITD-008 and Balapiravir, entered preclinical animal safety study and clinic trial, but both were terminated due to toxicity and lack of potency, respectively. Celgosivir, a host alpha-glucosidase inhibitor, is currently under clinical trial; its clinical efficacy remains to be determined. The knowledge accumulated during the past decade has provided a better rationale for ongoing dengue drug discovery. Though challenging, we are optimistic that this continuous, concerted effort will lead to an effective dengue therapy.


Subject(s)
Antiviral Agents/isolation & purification , Dengue Virus/drug effects , Dengue/drug therapy , Dengue/virology , Drug Discovery/history , Drug Discovery/trends , History, 21st Century , Humans , Singapore
13.
J Biol Chem ; 288(43): 30883-91, 2013 Oct 25.
Article in English | MEDLINE | ID: mdl-24022489

ABSTRACT

Cyclomarin A (CymA) was identified as a mycobactericidal compound targeting ClpC1. However, the target was identified based on pulldown experiments and in vitro binding data, without direct functional evidence in mycobacteria. Here we show that CymA specifically binds to the N-terminal domain of ClpC1. In addition we have determined the co-crystal structure of CymA bound to the N-terminal domain of ClpC1 to high resolution. Based on the structure of the complex several mutations were engineered into ClpC1, which showed reduced CymA binding in vitro. The ClpC1 mutants were overexpressed in mycobacteria and two showed resistance to CymA, providing the first direct evidence that ClpC1 is the target of CymA. Phe(80) is important in vitro and in cells for the ClpC1-CymA interaction and this explains why other bacteria are resistant to CymA. A model for how CymA binding to the N-terminal domain of ClpC1 leads to uncontrolled proteolysis by the associated ClpP protease machinery is discussed.


Subject(s)
Bacterial Proteins/chemistry , Heat-Shock Proteins/chemistry , Models, Molecular , Mycobacterium tuberculosis/chemistry , Oligopeptides/chemistry , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Oligopeptides/metabolism , Protein Binding , Protein Structure, Tertiary
14.
J Biol Chem ; 288(18): 12891-900, 2013 May 03.
Article in English | MEDLINE | ID: mdl-23511634

ABSTRACT

The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a (1)H-(15)N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.


Subject(s)
Dengue Virus/enzymology , Multienzyme Complexes/chemistry , Viral Nonstructural Proteins/chemistry , Crystallography, X-Ray , Dengue Virus/genetics , Humans , Magnetic Resonance Spectroscopy , Multienzyme Complexes/genetics , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Quaternary , Protein Structure, Secondary , RNA Helicases/chemistry , RNA Helicases/genetics , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Viral Nonstructural Proteins/genetics
15.
J Virol ; 87(9): 5291-5, 2013 May.
Article in English | MEDLINE | ID: mdl-23408636

ABSTRACT

We report a highly reproducible method to crystallize the RNA-dependent RNA polymerase (RdRp) domain of dengue virus serotype 3 (DENV-3), allowing structure refinement to a 1.79-Å resolution and revealing amino acids not seen previously. We also present a DENV-3 polymerase/inhibitor cocrystal structure at a 2.1-Å resolution. The inhibitor binds to the RdRp as a dimer and causes conformational changes in the protein. The improved crystallization conditions and new structural information should accelerate structure-based drug discovery.


Subject(s)
Dengue Virus/enzymology , Enzyme Inhibitors/chemistry , RNA-Dependent RNA Polymerase/chemistry , Viral Proteins/chemistry , Crystallization , Dengue Virus/chemistry , Dengue Virus/genetics , Dimerization , Enzyme Inhibitors/metabolism , Models, Molecular , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/antagonists & inhibitors , Viral Proteins/genetics , Viral Proteins/metabolism
16.
Antiviral Res ; 96(2): 115-26, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22995600

ABSTRACT

Development of anti-dengue therapy represents an urgent un-met medical need. Towards antiviral therapy, recent advances in crystal structures of DENV enzymes have led to the possibility of structure-based rational design of inhibitors for anti-dengue therapy. These include (i) the structure of the 'active' form of the DENV protease in complex with a peptide substrate; (ii) the structure of DENV methyltransferase bound to an inhibitor that selectively suppresses viral methyltransferase, but not human methyltransferases; (iii) the structure of DENV RNA-dependent RNA polymerase in complex with a small-molecule compound. This review summarizes the structural biology of these three key enzymes (protease, methyltransferase, and polymerase) that are essential for DENV replication. The new structural information has provided new avenues for development of anti-dengue therapy.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Dengue Virus/enzymology , Drug Design , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry , Crystallography, X-Ray , Humans , Methyltransferases/antagonists & inhibitors , Methyltransferases/chemistry , Models, Molecular , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Protein Conformation , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/chemistry
17.
J Virol ; 86(1): 438-46, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22031935

ABSTRACT

Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic ß-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4' to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease.


Subject(s)
Dengue Virus/enzymology , Serine Endopeptidases/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallization , Dengue Virus/chemistry , Dengue Virus/genetics , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Peptide Hydrolases , Protein Binding , Protein Conformation , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
18.
Antiviral Res ; 85(3): 450-62, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20060421

ABSTRACT

Antiviral drug discovery is becoming increasingly important due to the global threat of viral disease pandemics. Many members of the genus Flavivirus are significant human pathogens, among which dengue virus (DENV) alone poses a public health threat to 2.5 billion worldwide, leading to 50-100 million human infections each year. Neither vaccine nor effective therapeutics is currently available for DENV. Development of a DENV vaccine has been challenging, because of the need to simultaneously immunize and induce a long-lasting protection against all four serotypes of DENV; an incompletely immunized individual may be sensitized to life-threatening dengue hemorrhagic fever or dengue shock syndrome. The challenges associated with vaccine development have underscored the importance of development of antiviral therapies for DENV and other flaviviruses. Here we review the strategies to identify inhibitors for DENV therapy. Both viral and host proteins essential for viral replication cycle are potential targets for antiviral development. Inhibitors could be identified by multiple approaches, including enzyme-based screening, viral replication-based screening, structure-based rational design, virtual screening, and fragment-based screening. The strategies discussed in this report should be applicable to antiviral development of other viruses.


Subject(s)
Antiviral Agents/pharmacology , Dengue Virus/drug effects , Drug Discovery/methods , Humans
19.
Biochemistry ; 48(19): 4040-9, 2009 May 19.
Article in English | MEDLINE | ID: mdl-19317456

ABSTRACT

Sterile alpha motif (SAM) domains are found in many different proteins and shown to play important roles in various biological processes. The N-terminal domain of deleted in liver cancer 1 (DLC1) protein is a SAM domain which exists in a monomeric form in aqueous solution and facilitates the distribution of EF1A1 to the membrane periphery and ruffles upon growth factor stimulation. Here, we report the structure of an N-terminal truncated DLC1 SAM domain (DLC1-SAM) and its urea-induced equilibrium unfolding investigated with various biophysical methods such as CD, fluorescence emission spectroscopy, and NMR. CD and tryptophan intrinsic fluorescence emission data imply that the unfolding of DLC1-SAM follows a simple two-state mechanism, yet the NMR data suggest the presence of at least one intermediate state. The intermediate cannot be detected by NMR, but it does not exist in large aggregates as shown by analytical ultracentrifugation experiments. Analysis of the free energy values for different residues shows that in the transition from the native state to non-native states the C-terminal helix is somewhat more stable than the other parts of the protein, whereas in the transition from the native and intermediate states to the denatured state, the stabilities of different residues are similar except for that of the region surrounding residues D37-F40 which has lower stability and is more readily denatured at high urea concentrations. Analysis of the midpoints of the transitions shows that the unfolding of the native state and formation of the denatured state are not cooperative and the unfolding of only a few residues seems to follow a two-state mechanism.


Subject(s)
Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Circular Dichroism , Dimerization , Dose-Response Relationship, Drug , Escherichia coli/genetics , GTPase-Activating Proteins , Histidine/chemistry , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Denaturation , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spectrometry, Fluorescence , Thermodynamics , Tumor Suppressor Proteins/genetics , Urea/pharmacology
20.
J Biol Chem ; 283(38): 26274-82, 2008 Sep 19.
Article in English | MEDLINE | ID: mdl-18641390

ABSTRACT

Beclin1 has a key regulatory role in the initiation of autophagy and is a tumor suppressor. We have examined the interplay between viral or human Bcl-2-like proteins and UVRAG and their opposite effects on Beclin1. We show that Beclin1 forms a dimer in solution via its coiled-coil domain both in vivo and in vitro. Viral Bcl-2 binds independently to two sites on the Beclin1 dimer, one with high affinity and one with lower affinity, whereas human Bcl-x(L) binds both sites equally with relatively low affinity. UVRAG disrupts the Beclin1-dimer interface, forming a heterodimer with Beclin1, suggesting that this is how UVRAG causes its effects on Beclin1 to activate autophagy. Both Bcl-2-like proteins reduce the affinity of UVRAG for Beclin1 approximately 4-fold, suggesting that they stabilize the Beclin1 dimer. Moreover, coimmunoprecipitation assays show that UVRAG substantially reduces Beclin1 dimerization in vivo. These data explain the concentration-dependent interplay between Bcl-2, UVRAG, and Beclin1, as both tumor suppressors, UVRAG and Beclin1, have single-copy mutations in human cancers. Furthermore, our data suggest that an alternative strategy for developing anti-cancer compounds would be to disrupt the Beclin1-dimer interface.


Subject(s)
Apoptosis Regulatory Proteins/chemistry , Membrane Proteins/chemistry , Tumor Suppressor Proteins/metabolism , bcl-X Protein/metabolism , Animals , Beclin-1 , Binding Sites , COS Cells , Calorimetry , Chlorocebus aethiops , Dimerization , Humans , Models, Biological , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Thermodynamics
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