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 21 or Mn 21 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 b-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 21 versus Mg 21, 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 highthroughput screening. Several DKA derivatives were found to cause strong inhibition of PA-Nter, with IC 50 values comparable to that of the prototype L-742,001 (i.e., below 2 mM). Among the different compounds tested, L-742,001 appeared unique in having equal activity against either Mg 21 or Mn 21 . Three compounds (10, with a pyrrole scaffold, and 40 and 41, with an indole scaffold) exhibited moderate antiviral activity in cell culture (EC 99 values 64-95 mM) 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.

A Novel Endonuclease Inhibitor Exhibits Broad-Spectrum Anti-Influenza Virus Activity In Vitro

“…The mean number of plaques in each well was calculated, and the EC 50 s were determined by using the log (inhibitor) versus response logistic nonlinear regression equation in GraphPad Prism 6.0 software. In each case, the selective index (SI) was determined by calculating the ratio of the CC 50 to the EC 50 .…”

“…Endonuclease inhibition assay. Endonuclease activity assays were performed as described previously (33,34,42,50). Briefly, the 209-residue N-terminal domain of the influenza A virus PA protein (PA N ) was cloned and His tag purified (42).…”

“…6D). The CC 50 and nanomolar EC 50 s of RO-7 resulted in favorable selective indices (SI ϭ CC 50 /EC 50 ) as determined in the plaque number reduction (SI ϭ 1,053.1), virus yield reduction (SI ϭ 743.8), and cell viability (SI ϭ 3718.8) assays. Thus, RO-7 exhibits activity against influenza A virus in cellbased assays, with minimal cytotoxicity.…”

“…To evaluate the activity of RO-7 against influenza viruses in vitro, we initially performed 3 assays in MDCK cells, namely, a plaque number reduction assay, a virus yield reduction (TCID 50 ) assay, and a cell viability assay. Using the A/California/04/2009 (H1N1)pdm09 virus, we observed mean EC 50 s of 11.3 nM and 16.0 nM in the plaque number and virus yield reduction assays, respectively ( Fig.…”

“…This process is essential to completing the replication cycle and is widely recognized as a prime antiviral target. PA endonuclease inhibitors, including the 4-substituted 2,4-dioxobutanoic acid derivatives (29,30) and the substituted 2,6-diketopiperazine natural product flutimide (31,32), were characterized 2 decades ago in enzymatic, cell-based, and limited in vivo assays (29), where they demonstrated potent (micromolar 50% inhibitory concentrations [IC 50 s]) and cap-dependent inhibition of influenza A and B viruses. Subsequently, marchantins, catechins (33,34), hydroxamic acid and N-hydroxyimides (35,36), 3-hydroxypyridin-2(1H)-one derivatives (37, 38), 3-hydroxyquinolin-2(1H)-one derivatives (39), fullerene derivatives (40), and carboxamide derivatives (41) were identified as PA protein inhibitors.…”