Identification of Influenza Endonuclease Inhibitors Using a Novel Fluorescence Polarization Assay

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• 作者：Brandi M. Baughman ; P. Jake Slavish ; Rebecca M. DuBois ; Vincent A. Boyd ; Stephen W. White ; Thomas R. Webb
• 刊名：ACS Chemical Biology
• 出版年：2012
• 出版时间：March 16, 2012
• 年：2012
• 卷：7
• 期：3
• 页码：526-534
• 全文大小：357K
• 年卷期：v.7,no.3(March 16, 2012)
• ISSN：1554-8937

文摘

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 PA_N 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 (K_d = 0.378 渭M) and a PA_N construct, to identify small molecules that bind to the PA_N 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 that 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 鈥渢argeted fragment鈥?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.