title: A small molecule inhibitor of dengue virus type 2 protease inhibits the replication of all four dengue virus serotypes in cell culture.
abstract: BACKGROUND
  Dengue has emerged as the most significant of arboviral diseases in the 21st century. It is endemic to >100 tropical and sub-tropical countries around the world placing an estimated 3.6 billion people at risk. It is caused by four genetically similar but antigenically distinct, serotypes of dengue viruses. There is neither a vaccine to prevent nor a drug to treat dengue infections, at the present time. The major objective of this work was to explore the possibility of identifying a small molecule inhibitor of the dengue virus protease and assessing its ability to suppress viral replication in cultured cells.
  
  
  METHODS
  We cloned, expressed and purified recombinant dengue virus type 2 protease. Using an optimized and validated fluorogenic peptide substrate cleavage assay to monitor the activity of this cloned dengue protease we randomly screened ~1000 small molecules from an 'in-house' library to identify potential dengue protease inhibitors.
  
  
  RESULTS
  A benzimidazole derivative, named MB21, was found to be the most potent in inhibiting the cloned protease (IC₅₀ = 5.95 μM). In silico docking analysis indicated that MB21 binds to the protease in the vicinity of the active site. Analysis of kinetic parameters of the enzyme reaction suggested that MB21 presumably functions as a mixed type inhibitor. Significantly, this molecule identified as an inhibitor of dengue type 2 protease was also effective in inhibiting each one of the four serotypes of dengue viruses in infected cells in culture, based on analysis of viral antigen synthesis and infectious virus production. Interestingly, MB21 did not manifest any discernible cytotoxicity.
  
  
  CONCLUSIONS
  This work strengthens the notion that a single drug molecule can be effective against all four dengue virus serotypes. The molecule MB21 could be a potential candidate for 'hit-to-lead' optimization, and may pave the way towards developing a pan-dengue virus antiviral drug.
authors.affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India. rajeraut@gmail.com.|Department of Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi, 110062, India. rajeraut@gmail.com.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Raut
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science Pilani, Hyderabad Campus, Hyderabad, 500078, India. hbks.13@gmail.com.
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Beesetti
authors.affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India. poornima.icg@gmail.com.
authors.forename: Poornima
authors.initials: P
authors.lastname: Tyagi
authors.affiliation: Department of General Medicine, Lady Hardinge Medical College, Shaheed Bhagat Singh Marg, New Delhi, 110001, India. irakhanna07@gmail.com.
authors.forename: Ira
authors.initials: I
authors.lastname: Khanna
authors.affiliation: Department of Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi, 110062, India. skjain@jamiahamdard.ac.in.
authors.forename: Swatantra K
authors.initials: SK
authors.lastname: Jain
authors.affiliation: Department of Pharmacy, Birla Institute of Technology and Science Pilani, Hyderabad Campus, Hyderabad, 500078, India. jeanvariums@gmail.com.
authors.forename: Variam U
authors.initials: VU
authors.lastname: Jeankumar
authors.affiliation: Department of Pharmacy, Birla Institute of Technology and Science Pilani, Hyderabad Campus, Hyderabad, 500078, India. pyogee@hyderabad.bits-pilani.ac.in.|Centre for Infectious Disease Research, Birla Institute of Technology and Science Pilani, Hyderabad Campus, Hyderabad, 500078, India. pyogee@hyderabad.bits-pilani.ac.in.
authors.forename: Perumal
authors.initials: P
authors.lastname: Yogeeswari
authors.affiliation: Department of Pharmacy, Birla Institute of Technology and Science Pilani, Hyderabad Campus, Hyderabad, 500078, India. dsriram@hyderabad.bits-pilani.ac.in.|Centre for Infectious Disease Research, Birla Institute of Technology and Science Pilani, Hyderabad Campus, Hyderabad, 500078, India. dsriram@hyderabad.bits-pilani.ac.in.
authors.forename: Dharmarajan
authors.initials: D
authors.lastname: Sriram
authors.affiliation: Department of Biological Sciences, Birla Institute of Technology and Science Pilani, Hyderabad Campus, Hyderabad, 500078, India. swaminathan@hyderabad.bits-pilani.ac.in.|Centre for Infectious Disease Research, Birla Institute of Technology and Science Pilani, Hyderabad Campus, Hyderabad, 500078, India. swaminathan@hyderabad.bits-pilani.ac.in.
authors.forename: Sathyamangalam
authors.initials: S
authors.lastname: Swaminathan
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology* / Q000633:toxicity; D001562:Benzimidazoles / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D004353:Drug Evaluation, Preclinical; D007700:Kinetics; D062105:Molecular Docking Simulation; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology* / Q000633:toxicity; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000378:metabolism*; D065288:Serogroup; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects*; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D001562:Benzimidazoles; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; C031000:benzimidazole; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases