Stevaert Annelies, Nurra Salvatore, Pala Nicolino, Carcelli Mauro, Rogolino Dominga, Shepard Caitlin, Domaoal Robert A, Kim Baek, Alfonso-Prieto Mercedes, Marras Salvatore A E, Sechi Mario, Naesens Lieve
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.).
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.)
Mol Pharmacol. 2015 Feb;87(2):323-37. doi: 10.1124/mol.114.095588. Epub 2014 Dec 4.
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.
流感病毒PA内切酶可切割带帽的细胞前体mRNA以启动病毒mRNA合成，是新型抗流感病毒疗法的一个有前景的靶点。该酶的催化中心位于PA的N端部分（PA-Nter），含有两个（也可能是一个或三个）Mg(2+)或Mn(2+)离子，这些离子对其催化功能至关重要。人们对经过优化设计以占据活性位点并螯合金属离子的PA抑制剂非常感兴趣。我们在此聚焦于一系列含有不同支架的β-二酮酸（DKA）及其生物电子等排体化合物，并通过PA-Nter酶促试验确定它们的构效关系，以构建三维药效团模型。此外，我们开发了一种基于分子信标（MB）的PA-Nter试验，使我们能够比较对Mn(2+)和Mg(2+)的抑制作用，后者可能是生物学上相关的辅因子。这种实时MB试验使我们能够测量PA-Nter的酶动力学或进行高通量筛选。发现几种DKA衍生物对PA-Nter有强烈抑制作用，其IC50值与原型L-742,001相当（即低于2 μM）。在所测试的不同化合物中，L-742,001表现独特，对Mg(2+)或Mn(2+)具有同等活性。三种化合物（10：具有吡咯支架，40：和41：具有吲哚支架）在细胞培养中表现出中等抗病毒活性（EC99值为64 - 95 μM），并被证明会影响病毒RNA合成。我们整合互补的酶促、细胞和机制试验的方法应能指导改进的流感病毒PA抑制剂的持续开发。
