Development of an anti-influenza drug screening assay targeting nucleoproteins with tryptophan fluorescence quenching.
Recent studies have shown that NP (nucleoprotein), which possesses multiple functions in the viral life cycle, is a new potential anti-influenza drug target. NP inhibitors reliably induce conformational changes in NPs, and these changes may confer inhibition of the influenza virus. The six conserved tryptophan residues in NP can be used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residues in the protein upon binding to an NP inhibitor. In the present study, we found that the fluorescence of recombinant NP proteins was quenched following the binding of available NP inhibitors (such as nucleozin) in a concentration- and time-dependent manner, which suggests that the inhibitor induced conformational changes in the NPs. The minimal fluorescence-quenching effect and weak binding constant of nucleozin to the swine-origin influenza virus H1N1pdm09 (SOIV) NP revealed that the SOIV is resistant to nucleozin. We have used the fluorescence-quenching property of tryptophans in NPs that were bound to ligands in a 96-well-plate-based drug screen to assess the ability of promising small molecules to interact with NPs and have identified one new anti-influenza drug, CSV0C001018, with a high SI value. This convenient method for drug screening may facilitate the development of antiviral drugs that target viruses other than the influenza virus, such as HIV and HBV.
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan.
Hui-Chen
HC
Hung
Chia-Lin
CL
Liu
John T-A
JT
Hsu
Jim-Tong
JT
Horng
Ming-Yu
MY
Fang
Su-Ying
SY
Wu
Shau-Hua
SH
Ueng
Min-Ying
MY
Wang
Cheng-Wen
CW
Yaw
Ming-Hon
MH
Hou
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D004285:Dogs; D004353:Drug Evaluation, Preclinical; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013552:Swine; D014364:Tryptophan / Q000737:chemistry*
D000998:Antiviral Agents; D009698:Nucleoproteins; D011994:Recombinant Proteins; D014364:Tryptophan