title: A screening assay for neuraminidase inhibitors using neuraminidases N1 and N3 from a baculovirus expression system.
abstract: BACKGROUND
  Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors.
  
  
  OBJECTIVE
  Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay.
  
  
  METHODS
  Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor.
  
  
  RESULTS
  The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments.
  
  
  CONCLUSIONS
  The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.
authors: Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
authors: Jarinrat
authors: J
authors: Kongkamnerd
authors: Adelaide
authors: A
authors: Milani
authors: Giovanni
authors: G
authors: Cattoli
authors: Calogero
authors: C
authors: Terregino
authors: Ilaria
authors: I
authors: Capua
authors: Luca
authors: L
authors: Beneduce
authors: Andrea
authors: A
authors: Gallotta
authors: Paolo
authors: P
authors: Pengo
authors: Giorgio
authors: G
authors: Fassina
authors: Stanislav
authors: S
authors: Miertus
authors: Wanchai
authors: W
authors: De-Eknamkul
mesh_terms: D016367:Baculoviridae / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D057916:Influenza A Virus, H7N1 Subtype / Q000201:enzymology* / Q000235:genetics; D057920:Influenza A Virus, H7N3 Subtype / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D053139:Oseltamivir / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D053139:Oseltamivir; D009439:Neuraminidase; C535162:oseltamivir carboxylate