Discovery of Small Molecule Probes for H1N1 Influenza NS1A
Influenza viruses are negative-sense, single-stranded RNA viruses that infect the upper, and occasionally the lower, respiratory tracts In the US. Illness from infections by influenza virus has caused an average of approximately 36,000 deaths from 1990-1999 and 226,000 hospitalizations during 1979-2001 (Thompson, W. W., et al. PMID: 15367555), (Thompson, W. W., et al. PMID:12517228). Influenza A more ..
BioActive Compounds: 1010
Influenza viruses are negative-sense, single-stranded RNA viruses that infect the upper, and occasionally the lower, respiratory tracts In the US. Illness from infections by influenza virus has caused an average of approximately 36,000 deaths from 1990-1999 and 226,000 hospitalizations during 1979-2001 (Thompson, W. W., et al. PMID: 15367555), (Thompson, W. W., et al. PMID:12517228). Influenza A viruses (IAV), which also infect a wide number of avian and mammalian species, pose a considerable public health burden with epidemic and pandemic potential. This is particularly evident with the new strain of "swine flu virus" which emerged recently in Mexico and spread to different parts of the North America (MMWR Morb Mortal Wkly Rep PMID:19390508). The most devastating IAV pandemic occurred in 1918 which resulted in approximately 30 million deaths worldwide (Reid, A. H., et al. PMID: 11226857).
Antivirals are widely used in treatment of epidemic and pandemic IAV infections. Certainly, the lack of availability of vaccines early in IAV pandemics has driven the search for effective antivirals. One potential and largely unexplored IAV target is to disarm viral proteins that modulate the host's antiviral response. NS1A protein is a critical viral protein in this regard. During viral infection, infected cells of the host mount a potent and diverse antiviral defense response to prevent virus proliferation (Randall, R. E., et al. PMID: 18089727). To perpetuate, IAV have evolved multiple mechanisms to circumvent these host defense mechanisms. Strategies include those which are strain-specific such as increased replication speed (Grimm, D., et al. PMID: 17426143), (Kurokawa, M., et al. PMID: 10202186) or those that reduce sensitivity to host-cell antiviral defense mechanisms (Dittmann, J., et al. PMID: 17970694). The NS1A protein inhibits the interferon-alpha-beta-induced 2'-5'-oligo synthetase. Drugs that target the NS1A double-stranded (ds) RNA binding domain would inhibit IAV replication.
The N terminus of the NS1A protein (NS1A) of IAV is a highly conserved, multifunctional viral protein which interacts with host dsRNA (Cheng, A., et al. PMID: 18813227). We have developed an AlphaScreen (Perkin Elmer)-based 1536-well high throughput screen (HTS) for NS1A to identify small molecule inhibitors of the dsRNA binding activity. Identification of probes for this activity would provide valuable tools for probing its dsRNA binding function within the cell. Further, this domain could also serve as a target for antiviral drug discovery. To further validate the assay we developed for NS1A dsRNA binding activity, we screened a small library (Prestwick) of 1120 compounds using HTS and identified several compounds that were reported to have nucleic acid binding properties.
Primary Assay Protocol: In 1536-well white non-binding plates (Corning), 1 ul of dsRNA diluted in 1X Assay Buffer (25 mM HEPES pH 7.4, 100 mM NaCl; 0.1% BSA) was added to each test well (125 nM final concentration). Three (3) ul of NS1A diluted in 1X assay Buffer (H1N1) (7.2 ng/ul) was added to each well (21.6 ng/well). The final concentration of DMSO was 2% in a total volume of 4ul. The plates were covered with adhesive film and incubated at 37C. After 15 minutes, plates were cooled to RT and two (2) ul Acceptor beads (Perkin Elmer, 20 ug/ml final) in 1X Assay Buffer added per well. The plates were centrifuged at 300xg for 30 seconds and incubated at RT for 30 minutes. Two (2) ul of Donor beads (Perkin Elmer, 20 ug/ml final) in 1X Assay Buffer were added per well, the plates were centrifuged at 300xg for 30 seconds and incubated at RT for 60 minutes. The final volume was 8 ul. The plates were read using an Envision Microplate Reader (excite at 680nm and read emission at 570 nm).
Dose Response Assay Protocol: This assay was performed using the primary assay methodology with a dose-response, stacked-plate format. These compounds were screened in a 10-point dilution series ranging from 100 uM to 0.195 uM and a final DMSO concentration of 1%. The assay was performed as described for the primary screen except that the dose-response assay uses a stacked-plate method wherein each compound dilution is inter-plate rather than intra-plate. In this method, all of the compounds on a plate are at a single concentration. This design is driven by the efficiency of liquid handling, making it quick to generate simultaneous concentration curves for up to 1280 compounds in a 1536-well plate. The dose response testing confirmed the primary screen hits and provided the IC50 data necessary to determine the lead agents to be advanced in secondary screens and target localization studies.
In the primary screen a compound was deemed "Inactive" if it had a percent inhibition < 28.99%. Out of a total of 335,445 compounds screened, 1,902 compounds showed >/= 28.99% inhibition at the 12.5 uM compound for a hit rate of approximately 0.29%. Therefore, 1,902 compounds were ordered for follow up dose response experiments.
In the confirmatory assay, inhibition was determined at each of 10 tested concentrations ranging from 100 uM to 0.195 uM. Compounds that showed >30% inhibition at any test concentration were considered Active. IC50 values were calculated for compounds using a 4 parameter Levenburg-Marquardt algorithm, with the maximum and minimum locked at 0 and 100 respectively.
Compounds identified as Active in the primary screen that were not available for followup testing were labeled as Inconclusive.
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In the confirmatory (dose response) screen, active compounds were scored on a scale of 41-80. Based on the
IC50 results, compounds that did not show activity were given the score 0.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)