Identification and Analysis of Flavivirus Protease and RNA Helicase Inhibitors

Summary

Principal Investigator: RADHAKRISHNAN K PADMANABHAN
Abstract: The mosquito-borne flaviviruses include the NIAID list of Class A, B, or C emerging human pathogens such as Yellow fever virus (YFV), West Nile virus (WNV) and Dengue (DEN1-4) viruses that cause serious illnesses with considerable morbidity and mortality. These viruses, in addition to flu-like symptoms, can cause hemorrhagic fevers (YFV and DEN) or encephalitis and/or meningo-encephalitis in susceptible humans. One of the long term goals of this laboratory has been to develop antiviral therapeutics through understanding of key pathways in the viral life cycle using in vitro enzymatic assays that mimic those utilized by these viruses in their natural hosts. Two of the nonstructural proteins, NS3 and NS5, have multiple enzyme activities that are required for the virus life cycle. NS3, in association with NS2B, is a serine protease that is required for polyprotein processing, a key early event in the virus life cycle. Moreover, NS3 exhibits an RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase (5'-RTPase) activities. The NTPase and 5'-RTPase activities are stimulated by interaction with NS5, the viral 5'-RNA methyltransferase and the RNA dependent RNA polymerase (RdRP). These enzyme activities are excellent targets for development of antiviral therapeutics. In Specific Aim 1, we propose test the hypothesis that small molecule compounds could potentially inhibit the interaction between the NS2B cofactor and the NS3 protease (NS3-pro) domain or by mimicking the substrate-protease interactions or both. To this hypothesis, we will screen approximately 175,000 compounds in the National Screening Laboratory using the in vitro protease assay. By using chemoinformatics and computer-assisted docking studies and the crystal structure of the DEN protease domain, we will select about 100 compounds with greater than or equal to 50% inhibition of (i) interaction between the NS2B cofactor-NS3-pro domain and (ii) interaction between the substrate-protease active site interaction by measuring the Kcat and Km values in the presence and absence of inhibitors. Further testing of selected compounds will be performed using cytotoxicity, viral infectivity and replicon-based assays. In Specific Aim 2, the hypothesis that small molecule compounds that could potentially inhibit NS3/NS5 interaction and block their individual functions will be tested. We will identify approximately 200 compounds using a similar strategy as in Aim 1 but by using the in vitro assay based on measurement of the Pi released from the hydrolysis of either NTP or the 5'-triphosphorylated RNA and the recently reported crystal structures of YFV and DEN2 RNA helicases and those of hepatitis C virus and bovine viral diarrhea virus (BVDV) RdRP for docking studies. Inhibitors of interaction between NS3-RNA, NS3-NTP, NS3-NS5 will be analyzed by immunoprecipitation, surface plasmon resonance and gel shift assays. Further testing of selected compounds will be performed using cytotoxicity, viral infectivity and replicon-based reporter assays. This proposed research is likely lead to identification of novel inhibitors that will be useful for co-crystallization and structure-function studies that could provide valuable insight into the nature of molecular interactions among flavivirus nonstructural proteins in the flavivirus life cycle.
Funding Period: 2009-09-01 - 2010-05-31
more information: NIH RePORT

Top Publications

  1. pmc Identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen
    Niklaus H Mueller
    Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, DC 20057, USA
    Antimicrob Agents Chemother 52:3385-93. 2008
  2. pmc Genome 3'-end repair in dengue virus type 2
    Tadahisa Teramoto
    Laboratory of Vector Borne Virus Diseases, Center for Biologics Evaluation and Review, Food and Drug Administration, Bethesda, Maryland 20892, USA
    RNA 14:2645-56. 2008
  3. ncbi Small molecule inhibitor discovery for dengue virus protease using high-throughput screening
    Mark Manzano
    Department of Microbiology Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
    Methods Mol Biol 1138:331-44. 2014
  4. ncbi Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination
    Mark Manzano
    Department of Microbiology Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
    Methods Mol Biol 1138:151-60. 2014
  5. ncbi Amodiaquine, an antimalarial drug, inhibits dengue virus type 2 replication and infectivity
    Siwaporn Boonyasuppayakorn
    Department of Microbiology and Immunology, Georgetown University, USA Electronic address
    Antiviral Res 106:125-34. 2014
  6. pmc Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome
    Joanna Sztuba-Solinska
    RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
    Nucleic Acids Res 41:5075-89. 2013
  7. pmc Characterization of 8-hydroxyquinoline derivatives containing aminobenzothiazole as inhibitors of dengue virus type 2 protease in vitro
    Huiguo Lai
    Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA
    Antiviral Res 97:74-80. 2013
  8. pmc Characterization of the 8-hydroxyquinoline scaffold for inhibitors of West Nile virus serine protease
    Manolya Ezgimen
    Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA
    Antiviral Res 94:18-24. 2012
  9. pmc Identification of cis-acting elements in the 3'-untranslated region of the dengue virus type 2 RNA that modulate translation and replication
    Mark Manzano
    Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, D C 20057, USA
    J Biol Chem 286:22521-34. 2011
  10. pmc Synthesis of a 6-methyl-7-deaza analogue of adenosine that potently inhibits replication of polio and dengue viruses
    Runzhi Wu
    Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66047, USA
    J Med Chem 53:7958-66. 2010

Scientific Experts

  • RADHAKRISHNAN K PADMANABHAN
  • Mark Manzano
  • Tadahisa Teramoto
  • Niklaus H Mueller
  • Huiguo Lai
  • Siwaporn Boonyasuppayakorn
  • Joanna Sztuba-Solinska
  • Erin D Reichert
  • Manolya Ezgimen
  • Bruce A Shapiro
  • Runzhi Wu
  • Sofia L Alcaraz-Estrada
  • Barry Falgout
  • Aruna Sampath
  • Manolya D Ezgimen
  • Janak Padia
  • Kuppuswamy Nagarajan
  • Stuart F J Le Grice
  • G Sridhar Prasad
  • Jason W Rausch
  • GREGORY CUNY
  • David A Ostrov
  • Kyungae Lee
  • Stephanie Polo
  • Wojciech Kasprzak
  • Craig E Cameron
  • Robin Levis
  • Hyung Suk Oh
  • Rosa M Del Angel
  • Mark Irvin M Manzano
  • Blake R Peterson
  • Eric D Smidansky
  • Ratree Takhampunya
  • Camilo Ansarah-Sobrinho
  • Lewis Markoff
  • Prasanth Viswanathan
  • Theodore C Pierson
  • Pravina Mattoo
  • Nagarajan Pattabiraman
  • Yukari Kohno

Detail Information

Publications13

  1. pmc Identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen
    Niklaus H Mueller
    Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, DC 20057, USA
    Antimicrob Agents Chemother 52:3385-93. 2008
    ..Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 +/- 0.4 microM; selectivity index, 100), presumably by inhibition of polyprotein processing...
  2. pmc Genome 3'-end repair in dengue virus type 2
    Tadahisa Teramoto
    Laboratory of Vector Borne Virus Diseases, Center for Biologics Evaluation and Review, Food and Drug Administration, Bethesda, Maryland 20892, USA
    RNA 14:2645-56. 2008
    ..Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3'-end sequences based on the best-fit RNA structure that can support viral replication...
  3. ncbi Small molecule inhibitor discovery for dengue virus protease using high-throughput screening
    Mark Manzano
    Department of Microbiology Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
    Methods Mol Biol 1138:331-44. 2014
    ..In this chapter, we describe an in vitro assay for the viral serine protease that has been successfully adapted to HTS format and has been used to screen several thousand compounds to identify inhibitors of the viral protease...
  4. ncbi Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination
    Mark Manzano
    Department of Microbiology Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
    Methods Mol Biol 1138:151-60. 2014
    ....
  5. ncbi Amodiaquine, an antimalarial drug, inhibits dengue virus type 2 replication and infectivity
    Siwaporn Boonyasuppayakorn
    Department of Microbiology and Immunology, Georgetown University, USA Electronic address
    Antiviral Res 106:125-34. 2014
    ..Both p-hydroxyanilino and diethylaminomethyl moieties are important for AQ to inhibit DENV2 replication and infectivity. Our results support AQ as a promising candidate for anti-flaviviral therapy. ..
  6. pmc Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome
    Joanna Sztuba-Solinska
    RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
    Nucleic Acids Res 41:5075-89. 2013
    ..In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region...
  7. pmc Characterization of 8-hydroxyquinoline derivatives containing aminobenzothiazole as inhibitors of dengue virus type 2 protease in vitro
    Huiguo Lai
    Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA
    Antiviral Res 97:74-80. 2013
    ..91±0.05μM was determined to be 2.36±0.13μM. This compound inhibits the DENV2 NS2B/NS3pro by a competitive mode of inhibition...
  8. pmc Characterization of the 8-hydroxyquinoline scaffold for inhibitors of West Nile virus serine protease
    Manolya Ezgimen
    Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA
    Antiviral Res 94:18-24. 2012
    ..01 ± 0.08 μM using the tetra-peptide substrate was determined to be 5.8 μM. This compound inhibits the WNV NS2B/NS3pro by a competitive mode of inhibition which is supported by molecular modeling...
  9. pmc Identification of cis-acting elements in the 3'-untranslated region of the dengue virus type 2 RNA that modulate translation and replication
    Mark Manzano
    Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, D C 20057, USA
    J Biol Chem 286:22521-34. 2011
    ..Taken together, our results indicate that the cis-acting RNA elements in the core region of DENV2 RNA that include two DB structures are required not only for RNA replication but also for optimal translation...
  10. pmc Synthesis of a 6-methyl-7-deaza analogue of adenosine that potently inhibits replication of polio and dengue viruses
    Runzhi Wu
    Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66047, USA
    J Med Chem 53:7958-66. 2010
    ....
  11. pmc Construction of a dengue virus type 4 reporter replicon and analysis of temperature-sensitive mutations in non-structural proteins 3 and 5
    Sofia L Alcaraz-Estrada
    Georgetown University Medical Center, Washington, DC, USA
    J Gen Virol 91:2713-8. 2010
    ..Furthermore, using in vitro methyltransferase assays, we identified that the N96I mutation in NS5 exhibited a ts phenotype for N7-methylation, but not for 2'-O-methylation...
  12. pmc Molecular targets for flavivirus drug discovery
    Aruna Sampath
    National Center for Natural Product Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA
    Antiviral Res 81:6-15. 2009
    ..The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research...
  13. pmc Effects of detergents on the West Nile virus protease activity
    Manolya D Ezgimen
    Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, DC 20057, United States
    Bioorg Med Chem 17:3278-82. 2009
    ..Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays...