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Research ArticleArticle

An Integrated Biological Approach to Guide the Development of Metal-Chelating Inhibitors of Influenza Virus PA Endonuclease

Annelies Stevaert, Salvatore Nurra, Nicolino Pala, Mauro Carcelli, Dominga Rogolino, Caitlin Shepard, Robert A. Domaoal, Baek Kim, Mercedes Alfonso-Prieto, Salvatore A. E. Marras, Mario Sechi and Lieve Naesens
Molecular Pharmacology February 2015, 87 (2) 323-337; DOI: https://doi.org/10.1124/mol.114.095588
Annelies Stevaert
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Salvatore Nurra
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Nicolino Pala
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Mauro Carcelli
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Dominga Rogolino
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Caitlin Shepard
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Robert A. Domaoal
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Baek Kim
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Mercedes Alfonso-Prieto
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Salvatore A. E. Marras
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Mario Sechi
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Lieve Naesens
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy (M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S., R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
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Abstract

The influenza virus PA endonuclease, which cleaves capped cellular pre-mRNAs to prime viral mRNA synthesis, is a promising target for novel anti–influenza virus therapeutics. The catalytic center of this enzyme resides in the N-terminal part of PA (PA-Nter) and contains two (or possibly one or three) Mg2+ or Mn2+ ions, which are critical for its catalytic function. There is great interest in PA inhibitors that are optimally designed to occupy the active site and chelate the metal ions. We focused here on a series of β-diketo acid (DKA) and DKA-bioisosteric compounds containing different scaffolds, and determined their structure-activity relationship in an enzymatic assay with PA-Nter, in order to build a three-dimensional pharmacophore model. In addition, we developed a molecular beacon (MB)–based PA-Nter assay that enabled us to compare the inhibition of Mn2+ versus Mg2+, the latter probably being the biologically relevant cofactor. This real-time MB assay allowed us to measure the enzyme kinetics of PA-Nter or perform high-throughput screening. Several DKA derivatives were found to cause strong inhibition of PA-Nter, with IC50 values comparable to that of the prototype L-742,001 (i.e., below 2 μM). Among the different compounds tested, L-742,001 appeared unique in having equal activity against either Mg2+ or Mn2+. Three compounds (10, with a pyrrole scaffold, and 40 and 41, with an indole scaffold) exhibited moderate antiviral activity in cell culture (EC99 values 64–95 μM) and were proven to affect viral RNA synthesis. Our approach of integrating complementary enzymatic, cellular, and mechanistic assays should guide ongoing development of improved influenza virus PA inhibitors.

Footnotes

    • Received September 1, 2014.
    • Accepted December 4, 2014.
  • A. Stevaert is holder of a PhD grant from the Flemish Agency for Innovation by Science and Technology (IWT). The authors acknowledge financial support by the Geconcerteerde Onderzoeksacties [GOA/15/019/TBA] from the KU Leuven; the Emory Pediatric Center for Drug Discovery fund; the Fondazione Banco di Sardegna; and the Italian Ministero dell’Istruzione, dell’Università e della Ricerca [PRIN 2010, 2010W2KM5L_003]. S.A.E. Marras is among a group of inventors who earn royalties for molecular beacon usage.

  • dx.doi.org/10.1124/mol.114.095588.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

  • Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 87 (2)
Molecular Pharmacology
Vol. 87, Issue 2
1 Feb 2015
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Research ArticleArticle

Metal-Chelating Inhibitors of Influenza Virus Endonuclease

Annelies Stevaert, Salvatore Nurra, Nicolino Pala, Mauro Carcelli, Dominga Rogolino, Caitlin Shepard, Robert A. Domaoal, Baek Kim, Mercedes Alfonso-Prieto, Salvatore A. E. Marras, Mario Sechi and Lieve Naesens
Molecular Pharmacology February 1, 2015, 87 (2) 323-337; DOI: https://doi.org/10.1124/mol.114.095588

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Research ArticleArticle

Metal-Chelating Inhibitors of Influenza Virus Endonuclease

Annelies Stevaert, Salvatore Nurra, Nicolino Pala, Mauro Carcelli, Dominga Rogolino, Caitlin Shepard, Robert A. Domaoal, Baek Kim, Mercedes Alfonso-Prieto, Salvatore A. E. Marras, Mario Sechi and Lieve Naesens
Molecular Pharmacology February 1, 2015, 87 (2) 323-337; DOI: https://doi.org/10.1124/mol.114.095588
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