Identification of influenza virus inhibitors targeting NS1A utilizing fluorescence polarization-based high-throughput assay.
This article describes the development of a simple and robust fluorescence polarization (FP)-based binding assay and adaptation to high-throughput identification of small molecules blocking dsRNA binding to NS1A protein (nonstructural protein 1 from type A influenza strains). This homogeneous assay employs fluorescein-labeled 16-mer dsRNA and full-length NS1A protein tagged with glutathione S-transferase to monitor the changes in FP and fluorescence intensity simultaneously. The assay was optimized for high-throughput screening in a 384-well format and achieved a z' score greater than 0.7. Its feasibility for high-throughput screening was demonstrated using the National Institutes of Health clinical collection. Six of 446 small molecules were identified as possible ligands in an initial screening. A series of validation tests confirmed epigallocatechine gallate (EGCG) to be active in the submicromolar range. A mechanism of EGCG inhibition involving interaction with the dsRNA-binding motif of NS1A, including Arg38, was proposed. This structural information is anticipated to provide a useful basis for the modeling of antiflu therapeutic reagents. Overall, the FP-based binding assay demonstrated its superior capability for simple, rapid, inexpensive, and robust identification of NS1A inhibitors and validation of their activity targeting NS1A.
Texas Institute for Drug and Diagnostic Development, University of Texas at Austin, Austin, TX 78712, USA. euncho@mail.utexas.edu
Eun Jeong
EJ
Cho
Shuangluo
S
Xia
Li-Chung
LC
Ma
Jon
J
Robertus
Robert M
RM
Krug
Eric V
EV
Anslyn
Gaetano T
GT
Montelione
Andrew D
AD
Ellington
D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005454:Fluorescence Polarization / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects; D015203:Reproducibility of Results; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000998:Antiviral Agents; C085820:INS1 protein, influenza virus; D017361:Viral Nonstructural Proteins