title: An integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus PA endonuclease.
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) Mg(2+) or Mn(2+) 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 Mn(2+) versus Mg(2+), 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 Mg(2+) or Mn(2+). 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.
authors.affiliation: 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.).
authors.forename: Annelies
authors.initials: A
authors.lastname: Stevaert
authors.affiliation: 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.).
authors.forename: Salvatore
authors.initials: S
authors.lastname: Nurra
authors.affiliation: 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.).
authors.forename: Nicolino
authors.identifier: http://orcid.org/0000-0002-9681-5233
authors.initials: N
authors.lastname: Pala
authors.affiliation: 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.).
authors.forename: Mauro
authors.initials: M
authors.lastname: Carcelli
authors.affiliation: 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.).
authors.forename: Dominga
authors.initials: D
authors.lastname: Rogolino
authors.affiliation: 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.).
authors.forename: Caitlin
authors.initials: C
authors.lastname: Shepard
authors.affiliation: 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.).
authors.forename: Robert A
authors.initials: RA
authors.lastname: Domaoal
authors.affiliation: 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.).
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
authors.affiliation: 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.).
authors.forename: Mercedes
authors.initials: M
authors.lastname: Alfonso-Prieto
authors.affiliation: 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.).
authors.forename: Salvatore A E
authors.initials: SA
authors.lastname: Marras
authors.affiliation: 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.).
authors.forename: Mario
authors.identifier: http://orcid.org/0000-0003-2983-6090
authors.initials: M
authors.lastname: Sechi
authors.affiliation: 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.) lieve.naesens@rega.kuleuven.be.
authors.forename: Lieve
authors.initials: L
authors.lastname: Naesens
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D002614:Chelating Agents / Q000737:chemistry / Q000494:pharmacology*; D004285:Dogs; D055808:Drug Discovery / Q000379:methods*; D004720:Endonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008968:Molecular Conformation; D009975:Orthomyxoviridae / Q000187:drug effects* / Q000201:enzymology*
chemical_list: D000998:Antiviral Agents; D002614:Chelating Agents; D004720:Endonucleases