Mutational Analysis of the Binding Pockets of the Diketo Acid Inhibitor L-742,001 in the Influenza Virus PA Endonuclease

The influenza virus PA endonuclease, which cleaves capped host pre-mRNAs to initiate synthesis of viral mRNA, is a prime target for antiviral therapy. The diketo acid compound L-742,001 was previously identified as a potent inhibitor of the influenza virus endonuclease reaction, but information on its precise binding mode to PA or potential resistance profile is limited. Computer- assisted docking of L-742,001 into the crystal structure of inhibitor-free N-terminal PA (PA-Nter) indicated a binding orientation distinct from that seen in a recent crystallographic study with L-742,001-bound PA-Nter (R. M. DuBois et al., PLoS Pathog. 8:e1002830, 2012). A comprehensive mutational analysis was performed to determine which amino acid changes within the catalytic center of PA or its surrounding hydrophobic pockets alter the antiviral sensitivity to L-742,001 in cell culture. Marked (up to 20-fold) resistance to L-742,001 was observed for the H41A, I120T, and G81F/V/T mutant forms of PA. Two- to 3-fold resistance was seen for the T20A, L42T, and V122T mutants, and the R124Q and Y130A mutants were 3-fold more sensitive to L-742,001. Several mutations situated at noncatalytic sites in PA had no or only marginal impact on the enzymatic functionality of viral ribonucleoprotein complexes reconstituted in cell culture, consistent with the less conserved nature of these PA residues. Our data provide relevant insights into the binding mode of L-742,001 in the PA endonuclease active site. In addition, we predict some potential resistance sites that should be taken into account during optimization of PA endonuclease inhibitors toward tight binding in any of the hydrophobic pockets surrounding the catalytic center of the enzyme

A versatile salicyl hydrazonic ligand and its metal complexes as antiviral agents

Acylhydrazones are very versatile ligands and their coordination properties can be easily tuned, giving rise to metal complexeswith different nuclearities. In the last fewyears, we have been looking for newpharmacophores able to coordinate simultaneously two metal ions, because many enzymes have two metal ions in the active site and their coordination can be a successful strategy to inhibit the activity of the metalloenzyme. As a part of this ongoing research, we synthesized the acylhydrazone H2L and its complexes with Mg(II), Mn(II), Co(II), Ni(II), Cu(II) and Zn(II). Their characterization, both in solution – also by means of potentiometric studies – and in the solid state, evidenced the ability of the o-vanillin hydrazone scaffold to give rise to different types of metal complexes, depending on the metal and the reaction conditions. Furthermore, we evaluated both the free ligand and its metal complexes in in vitro studies against a panel of diverse DNA- and RNA-viruses. In particular, the Mg(II), Mn(II), Ni(II) and Zn(II) complexes had EC50 values in the low micromolar range, with a pronounced activity against vaccinia viru

A versatile salicyl hydrazonic ligand and its metal complexes as antiviral agents

Acylhydrazones are very versatile ligands and their coordination properties can be easily tuned, giving rise to metal complexes with different nuclearities. In the last few years, we have been looking for new pharmacophores able to coordinate simultaneously two metal ions, because many enzymes have two metal ions in the active site and their coordination can be a successful strategy to inhibit the activity of the metalloenzyme. As a part of this ongoing research, we synthesized the acylhydrazone H2L and its complexes with Mg(II), Mn(II), Co(II), Ni(II), Cu(II) and Zn(II). Their characterization, both in solution - also by means of potentiometric studies - and in the solid state, evidenced the ability of the o-vanillin hydrazone scaffold to give rise to different types of metal complexes, depending on the metal and the reaction conditions. Furthermore, we evaluated both the free ligand and its metal complexes in in vitro studies against a panel of diverse DNA- and RNA-viruses. In particular, the Mg(II), Mn(II), Ni(II) and Zn(II) complexes had EC50 values in the low micromolar range, with a pronounced activity against vaccinia virus.publisher: Elsevier articletitle: A versatile salicyl hydrazonic ligand and its metal complexes as antiviral agents journaltitle: Journal of Inorganic Biochemistry articlelink: http://dx.doi.org/10.1016/j.jinorgbio.2015.05.013 content_type: article copyright: Copyright © 2015 Elsevier Inc. All rights reserved.status: publishe

Metal-chelating properties and antiviral activity of some 2-hydroxyphenyl amides

Influenza virus is an hot topic in medicinal chemistry and great efforts are ongoing for the discover of new antivirals able to overcome problems related to resistant strains and adverse side effects of current drugs. Influenza virus endonuclease is an attractive target for antiviral drug development and in particular the strategy to chelate the metal ion(s) within the active site proved to be an efficient mode to inhibit enzymatic activity. Our previous findings revealed that 2-hydroxyamide derivatives are able to chelate Mg2+ ions, forming complexes with different stoichiometric ratios. Here we report on the activity of the three ligands N-(4-fluorobenzyl)-2-hydroxybenzamide, N-(4-fluorobenzyl)-2,3-dihydroxybenzamide, and N1,N3-bis(4-fluorobenzyl)-2-hydroxyisophthalamide, containing the salicylic group, and their Mg2+ complexes (7)–(9), evaluated by means of virus yield assay in influenza virus-infected MDCK cells and vRNP reconstitution assay in HEK293T cells. In some cases, promising anti-influenza activities with EC50 values in the low micromolar range were found. As a contribute to clarify the activity in cells of the ligands, here we also present a study on the their coordinating properties towards the other essential metal ion Cu(II), carried out by potentiometric and calorimetric measurements

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

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 Nterminal 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 Mg2+) in the catalytic site of PA. By using HL1, H2L2, and HL3 as model ligands with Mg2+ ions, we isolated and fully characterized a series of complexes and tested them for inhibitory activity toward PA-Nter endonuclease. H2L2 and the corresponding Mg2+ complex showed an interesting inhibition of the endonuclease activity. The crystal structures of the uncomplexed HL1 and H2L2 and of the isolated magnesium complex [Mg(L3)2(MeOH)2]·2MeOH were solved by X-ray diffraction analysis. Furthermore, the speciation models for HL1, H2L2, and HL3 with Mg2+ 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 PANter 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

Novel indole-flutimide heterocycles with activity against influenza PA endonuclease and hepatitis C virus

Influenza viruses cause considerable morbidity and mortality, whether in the context of annual epidemics, sporadic pandemics, or outbreaks of avian influenza virus. For hepatitis C virus (HCV), an estimated 170 million people are chronically infected worldwide. These individuals are at high risk of developing progressive liver injury or hepatocellular carcinoma. Since the efficacy of currently approved antiviral drugs is threatened by emerging viral resistance and the cost remains high, new antiviral drugs are still required. By utilizing a structure-based approach, novel substituted indole-flutimide heterocyclic derivatives (1,2-annulated indolediketopiperazines) were rationally designed, synthesized and evaluated as influenza PA endonuclease inhibitors. The compounds were also tested for their antiviral effect against HCV. All N-hydroxyimides were potent PA endonuclease inhibitors while displaying low cytotoxicity. Compound 6 proved to be the most active analogue, while the most favorable indole substitution was fluorine at position 8 (compound 18). The chloro-derivative 24 showed additional potent anti-HCV activity and exhibited remarkable selectivity (>19). In accordance with the SAR data, removal of the hydroxyl group from the imidic nitrogen (compound 26) caused a complete loss of activity against influenza PA endonuclease as well as HCV. © The Royal Society of Chemistry 2016

An Integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus pa endonuclease

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