Metal-Chelating 2-Hydroxyphenyl Amide Pharmacophore for Inhibition of Influenza Virus Endonuclease

Abstract

The influenza virus PA endonuclease is an attractive target for development of novel anti-influenza virus therapeutics. Reported PA inhibitors chelate the divalent metal ion(s) in the enzyme's catalytic site, which is located in the N-terminal part of PA (PA-Nter). In this work, a series of 2-hydroxybenzamide-based compounds have been synthesized and biologically evaluated in order to identify the essential pharmacophoric motif, which could be involved in functional sequestration of the metal ions (probably Mg(2+)) in the catalytic site of PA. By using HL(1), H2L(2), and HL(3) as model ligands with Mg(2+) ions, we isolated and fully characterized a series of complexes and tested them for inhibitory activity toward PA-Nter endonuclease. H2L(2) and the corresponding Mg(2+) complex showed an interesting inhibition of the endonuclease activity. The crystal structures of the uncomplexed HL(1) and H2L(2) and of the isolated magnesium complex [Mg(L(3))2(MeOH)2]·2MeOH were solved by X-ray diffraction analysis. Furthermore, the speciation models for HL(1), H2L(2), and HL(3) with Mg(2+) were obtained, and the formation constants of the complexes were measured. Preliminary docking calculations were conducted to investigate the interactions of the title compounds with essential amino acids in the PA-Nter active site. These findings supported the "two-metal" coordination of divalent ions by a donor triad atoms chemotype as a powerful strategy to develop more potent PA endonuclease inhibitors.

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

“…However, with the availability of effective endonuclease assays and high resolution crystal structures, there have been numerous reports of novel chemotypes that inhibit this enzyme and an upsurge in accompanying medicinal chemistry (43)(44)(45)(46). Our studies have revealed some of the structural origins of how resistance can emerge, and this information can now guide medicinal chemists in their optimization of new inhibitor scaffolds and prioritization of early lead compounds.…”

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

“…This enzymatic endonuclease activity assay was performed according to a previously published method (Carcelli et al, 2014) with minor modifications. One microgram of recombinant PA-Nter was incubated with 1 mg (16.7 nM) of singlestranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 ml.…”

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

“…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. In many of these studies, a library of compound derivatives was screened with a limited number of influenza viruses and/or assays, making it difficult to compare the reported efficacies accurately (42).…”