tetano
Editor, Senior Moderator
ACS Chem Biol. 2012 Jan 2. [Epub ahead of print]
Identification of Influenza Endonuclease Inhibitors via a Novel Fluorescence Polarization Assay.
Baughman BM, Slavish PJ, Dubois RM, Boyd VA, White SW, Webb TR.
Abstract
Influenza viruses have been responsible for the largest pandemics in the previous century. Although vaccination and prophylactic antiviral therapeutics are the primary defense against influenza virus, there is a pressing need to develop new antiviral agents to circumvent the limitations of current therapies. The endonuclease activity of the influenza virus PAN protein is essential for virus replication and is a promising target for novel anti-influenza drugs. To facilitate the discovery of endonuclease inhibitors, we have developed a high-throughput fluorescence polarization (FP) assay, utilizing a novel fluorescein-labeled compound (Kd = 0.378 μM) and a PAN construct, to identify small molecules that bind to the PAN endonuclease active site. Several known 4-substituted 2,4-dioxobutanoic acid inhibitors with high and low affinities have been evaluated in this FP-based competitive binding assay, and there was a general correlation between binding and the reported inhibition of endonuclease activity. Additionally, we have demonstrated the utility of this assay for identifying endonuclease inhibitors in a small diverse targeted fragment library. These fragment hits were used to build a follow up library that that led to new active compounds which demonstrate FP binding and anti-influenza activities in plaque inhibition assays. The assay offers significant advantages over previously reported assays, and is suitable for high-throughput and fragment-based screening studies. Additionally the demonstration of the applicability of a mechanism-based 'targeted fragment' library supports the general potential of this novel approach for other enzyme targets. These results serve as a sound foundation for the development of new therapeutic leads targeting influenza endonuclease.
PMID:
22211528
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22211528
Identification of Influenza Endonuclease Inhibitors via a Novel Fluorescence Polarization Assay.
Baughman BM, Slavish PJ, Dubois RM, Boyd VA, White SW, Webb TR.
Abstract
Influenza viruses have been responsible for the largest pandemics in the previous century. Although vaccination and prophylactic antiviral therapeutics are the primary defense against influenza virus, there is a pressing need to develop new antiviral agents to circumvent the limitations of current therapies. The endonuclease activity of the influenza virus PAN protein is essential for virus replication and is a promising target for novel anti-influenza drugs. To facilitate the discovery of endonuclease inhibitors, we have developed a high-throughput fluorescence polarization (FP) assay, utilizing a novel fluorescein-labeled compound (Kd = 0.378 μM) and a PAN construct, to identify small molecules that bind to the PAN endonuclease active site. Several known 4-substituted 2,4-dioxobutanoic acid inhibitors with high and low affinities have been evaluated in this FP-based competitive binding assay, and there was a general correlation between binding and the reported inhibition of endonuclease activity. Additionally, we have demonstrated the utility of this assay for identifying endonuclease inhibitors in a small diverse targeted fragment library. These fragment hits were used to build a follow up library that that led to new active compounds which demonstrate FP binding and anti-influenza activities in plaque inhibition assays. The assay offers significant advantages over previously reported assays, and is suitable for high-throughput and fragment-based screening studies. Additionally the demonstration of the applicability of a mechanism-based 'targeted fragment' library supports the general potential of this novel approach for other enzyme targets. These results serve as a sound foundation for the development of new therapeutic leads targeting influenza endonuclease.
PMID:
22211528
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22211528