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PLoS Pathogens. Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease
[Source: PLoS Pathogens, full page: (LINK). Abstract, edited.]
Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease
Rebecca M. DuBois 1,2, P. Jake Slavish 3, Brandi M. Baughman 3,4, Mi-Kyung Yun 1, Ju Bao 1, Richard J. Webby 2, Thomas R. Webb 3,4, Stephen W. White 1,4*
1 Department of Structural Biology, St. Jude Children?s Research Hospital, Memphis, Tennessee, United States of America, 2 Department of Infectious Diseases, St. Jude Children?s Research Hospital, Memphis, Tennessee, United States of America, 3 Department of Chemical Biology and Therapeutics, St. Jude Children?s Research Hospital, Memphis, Tennessee, United States of America, 4 Integrated Program in Biomedical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America
Abstract
Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or ??snatched?? from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.
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Citation: DuBois RM, Slavish PJ, Baughman BM, Yun M-K, Bao J, et al. (2012) Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease. PLoS Pathog 8(8): e1002830. doi:10.1371/journal.ppat.1002830
Editor: Andrew Pekosz, Johns Hopkins University - Bloomberg School of Public Health, United States of America
Received March 26, 2012; Accepted June 13, 2012; Published August 2, 2012
Copyright: ? 2012 DuBois et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH/NIAID grant AI098757 (to TRW), Cancer Center core grant CA21765, the Children?s Infection Defense Center, the Hartwell Center, and the American Lebanese Syrian Associated Charities (ALSAC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract No. W-31-109-Eng-38. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Stephen.White@stjude.org
________
Author Summary
Seasonal and pandemic influenza have enormous impacts on global public health. The rapid emergence of influenza virus strains that are resistant to current antiviral therapies highlights the urgent need to develop new therapeutic options. A promising target for drug discovery is the influenza virus PA protein, whose endonuclease enzymatic activity is essential for the ??cap-snatching?? step of viral mRNA transcription that allows transcripts to be processed by the host ribosome. Here, we describe a structure-based analysis of the mechanism of inhibition of the influenza virus PA endonuclease by small molecules. Our X-ray crystallographic studies have resolved the modes of binding of known and predicted inhibitors, and revealed that they directly block the PA endonuclease active site. We also report a number of molecular interactions that contribute to binding affinity and specificity. Our structural results are supported by biochemical analyses of the inhibition of enzymatic activity and computational docking experiments. Overall, our data reveal exciting strategies for the design and optimization of novel influenza virus inhibitors that target the PA protein.
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Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease
Rebecca M. DuBois 1,2, P. Jake Slavish 3, Brandi M. Baughman 3,4, Mi-Kyung Yun 1, Ju Bao 1, Richard J. Webby 2, Thomas R. Webb 3,4, Stephen W. White 1,4*
1 Department of Structural Biology, St. Jude Children?s Research Hospital, Memphis, Tennessee, United States of America, 2 Department of Infectious Diseases, St. Jude Children?s Research Hospital, Memphis, Tennessee, United States of America, 3 Department of Chemical Biology and Therapeutics, St. Jude Children?s Research Hospital, Memphis, Tennessee, United States of America, 4 Integrated Program in Biomedical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America
Abstract
Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or ??snatched?? from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.
________
Citation: DuBois RM, Slavish PJ, Baughman BM, Yun M-K, Bao J, et al. (2012) Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease. PLoS Pathog 8(8): e1002830. doi:10.1371/journal.ppat.1002830
Editor: Andrew Pekosz, Johns Hopkins University - Bloomberg School of Public Health, United States of America
Received March 26, 2012; Accepted June 13, 2012; Published August 2, 2012
Copyright: ? 2012 DuBois et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH/NIAID grant AI098757 (to TRW), Cancer Center core grant CA21765, the Children?s Infection Defense Center, the Hartwell Center, and the American Lebanese Syrian Associated Charities (ALSAC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract No. W-31-109-Eng-38. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Stephen.White@stjude.org
________
Author Summary
Seasonal and pandemic influenza have enormous impacts on global public health. The rapid emergence of influenza virus strains that are resistant to current antiviral therapies highlights the urgent need to develop new therapeutic options. A promising target for drug discovery is the influenza virus PA protein, whose endonuclease enzymatic activity is essential for the ??cap-snatching?? step of viral mRNA transcription that allows transcripts to be processed by the host ribosome. Here, we describe a structure-based analysis of the mechanism of inhibition of the influenza virus PA endonuclease by small molecules. Our X-ray crystallographic studies have resolved the modes of binding of known and predicted inhibitors, and revealed that they directly block the PA endonuclease active site. We also report a number of molecular interactions that contribute to binding affinity and specificity. Our structural results are supported by biochemical analyses of the inhibition of enzymatic activity and computational docking experiments. Overall, our data reveal exciting strategies for the design and optimization of novel influenza virus inhibitors that target the PA protein.
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