PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons

Related Articles
Design and Evolution: Molecular Sleuthing Reveals Drug Selectivity
June 2015
Families in Gene Neighborhoods
June 2015
Ryanodine Receptor
April 2015
CCR5 and HIV Infection
January 2015
Drug Targets: Bile Acids in Motion
September 2014
Drug Targets: S1R's Ligands and Partners
September 2014
P2Y Receptors and Blood Clotting
September 2014
Bacterial CDI Toxins
June 2014
Glucagon Receptor
April 2014
March 2014
Microbial Pathogenesis: Targeting Drug Resistance in Mycobacterium tuberculosis
February 2014
Design and Discovery: Virtual Drug Screening
January 2014
Cancer Networks: IFI16-mediated p53 Activation
November 2013
G Proteins and Cancer
November 2013
Drug Discovery: Antidepressant Potential of 6-NQ SERT Inhibitors
October 2013
Drug Discovery: Finding Druggable Targets
October 2013
Drug Discovery: Identifying Dynamic Networks by CONTACT
October 2013
Drug Discovery: Modeling NET Interactions
October 2013
Membrane Proteome: GPCR Substrate Recognition and Functional Selectivity
August 2013
Infectious Diseases: Determining the Essential Structome
May 2013
NDM-1 and Antibiotics
May 2013
Microbial Pathogenesis: Computational Epitope Prediction
January 2013
Microbial Pathogenesis: Influenza Inhibitor Screen
January 2013
Microbial Pathogenesis: Measles Virus Attachment
January 2013
Cytochrome Oxidase
November 2012
Membrane Proteome: The ABCs of Transport
November 2012
Bacterial Phosphotransferase System
October 2012
Regulatory insights
September 2012
Solute Channels
September 2012
Pocket changes
July 2012
Receptor bias
July 2012
Anthrax Stealth Siderophores
June 2012
G Protein-Coupled Receptors
May 2012
Substrate specificity sleuths
April 2012
Reading out regioselectivity
December 2011
Superbugs and Antibiotic Resistance
December 2011
Terminal activation
December 2011
A change to resistance
November 2011
Docking and rolling
October 2011
Breaking down the defenses
September 2011
A2A Adenosine Receptor
May 2011
Cell wall recycler
May 2011
Subtly different
March 2011
January 2011
Subtle shifts
January 2011
ABA receptor diversity
November 2010
COX inhibition: Naproxen by proxy
November 2010
Zinc Transporter ZntB
July 2010
Peptidoglycan binding: Calcium-free killing
June 2010
Treating sleeping sickness
May 2010
Bacterial spore kinase
April 2010
Antibiotics and Ribosome Function
March 2010
Safer Alzheimer's drugs?
March 2010
Anthrax evasion tactics
September 2009
GPCR subunits: Separate but not equal
September 2009
Antibiotic target
August 2009
Salicylic Acid Binding Protein 2
August 2009
July 2009
Tackling influenza
June 2009
Bacterial Leucine Transporter, LeuT
May 2009
Anthrax stealth molecule
March 2009
Drug targets to aim for
February 2009
High-energy storage system
February 2009
Transporter mechanism in sight
February 2009
Scavenger Decapping Enzyme DcpS
November 2008
Blocking AmtB
September 2008

Research Themes Drug discovery

Microbial Pathogenesis: Influenza Inhibitor Screen

SBKB [doi:10.1038/sbkb.2012.119]
Technical Highlight - January 2013
Short description: A cell-free binding assay is optimized step by step and applied in high throughput to screen for small molecule inhibitors of an influenza protein.

Schematic representation of the FP-based assay to monitor biological interaction. Two ssRNA molecules anneal to form dsRNA and, upon binding of NS1A protein (blue), the FP value increases. If a chemical compound (purple) disrupts this interaction, the FP value decreases. Figure courtesy of Eun Jeong Cho. View full sized image.

Fluorescence polarization (FP) is a robust approach for probing molecular interactions. It is essentially a measure of the tumbling rate of a fluorescently labeled molecule, which increases when the labeled probe binds a large ligand. The measurements are less variable than those based on fluorescence intensity, and the assay is cell-free, rapid and nonradioactive. As such, it has been employed to identify leads in inhibitor screens. The introduction of plate readers with the capacity for high-throughput FP analyses has enabled effective application of this technique in drug discovery.

Ellington, Cho and colleagues, in collaboration with Guy Montelione (PSI NESG), have now developed a FP-based screen to identify inhibitors of nonstructural protein 1 from influenza A virus (NS1A), a key player in viral infection. The N-terminal domain of NS1A nonspecifically binds double-stranded RNA (dsRNA), including viral dsRNAs, preventing their degradation by the human immune system. First, with a fluorescein-labeled dsRNA probe, the authors titrated in a number of NS1A constructs and control proteins, settling on glutathione S-transferase-tagged NS1A for the assay, which exhibited a dose-dependent increase in FP signal. tRNA was included to reduce background, reaction temperature and incubation time were optimized, and the effect of detergent was considered.

Following assay validation, 446 small molecules from the National Institutes of Health Clinical Collection were screened in two 384-well plates, in duplicate. The data were reproducible, with large signal-to-noise ratios. Subsequent stringent hit validation left just one compound, epigallocatechine gallate. This compound, which is found in green tea extract, has been shown to have anti-flu activity in several experiments, corroborating its identification in this screen. Since a structure of the dsRNA-biding site of NS1A is available, the authors suggest structure-based design to optimize the identified inhibitor molecule. As this FP-based assay was robust and reproducible, it should prove effective in the identification of other NS1A inhibitors.

Irene Kaganman


  1. E.J. Cho et al. Identification of influenza virus inhibitors targeting NS1A utilizing fluorescence polarization-based high-throughput assay.
    J. Biomol. Screen. 17, 448-459 (2012). doi:10.1177/1087057111431488

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health