Annelies Stevaert

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

ABSTRACT 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.

The influenza virus polymerase complex: an update on its structure, functions, and significance for antiviral drug design

Influenza viruses cause seasonal epidemics and pandemic outbreaks associated with significant morbidity and mortality, and a huge cost. Since resistance to the existing anti‐influenza drugs is rising, innovative inhibitors with a different mode of action are urgently needed. The influenza polymerase complex is widely recognized as a key drug target, given its critical role in virus replication and high degree of conservation among influenza A (of human or zoonotic origin) and B viruses. We here review the major progress that has been made in recent years in unravelling the structure and functions of this protein complex, enabling structure‐aided drug design toward the core regions of the PA endonuclease, PB1 polymerase, or cap‐binding PB2 subunit. Alternatively, inhibitors may target a protein–protein interaction site, a cellular factor involved in viral RNA synthesis, the viral RNA itself, or the nucleoprotein component of the viral ribonucleoprotein. The latest advances made for these diverse pharmacological targets have yielded agents in advanced (i.e., favipiravir and VX‐787) or early clinical testing, besides several experimental inhibitors in various stages of development, which are all covered here.

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.

Antiviral therapies on the horizon for influenza.

Adequate response to severe influenza infections or pandemic outbreaks requires two complementary strategies: preventive vaccination and antiviral therapy. The existing influenza drugs, M2 blockers and neuraminidase inhibitors, show modest clinical efficacy and established or potential resistance. In the past three years, several new agents have entered the clinical pipeline and already yielded some promising data from Phase 2 trials. For two main categories, that is, the broadly neutralizing anti-hemagglutinin antibodies and small-molecule inhibitors of the viral polymerase complex, crystallography was instrumental to guide drug development. These structural insights also aid to expand the activity spectrum towards influenza A plus B viruses, or conceive nucleoprotein or polymerase assembly inhibitors. The practice of influenza therapy should radically change in the next decade.

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.

N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes

Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics. This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity. Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity. Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones. Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.

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.

Chlorogenic Compounds from Coffee Beans Exert Activity against Respiratory Viruses

Chlorogenic acids are secondary metabolites in diverse plants. Some chlorogenic acids extracted from traditional medicinal plants are known for their healing properties, e.g., against viral infections. Also, green coffee beans are a rich source of chlorogenic acids, with 5-O-caffeoylquinic acid being the most abundant chlorogenic acid in coffee. We previously reported the synthesis of the regioisomers of lactones, bearing different substituents on the quinidic core. Here, 3,4-O-dicaffeoyl-1,5-γ-quinide and three dimethoxycinnamoyl-γ-quinides were investigated for in vitro antiviral activities against a panel of 14 human viruses. Whereas the dimethoxycinnamoyl-γ-quinides did not show any antiviral potency in cytopathogenic effect reduction assays, 3,4-O-dicaffeoyl-1,5-γ-quinide exerted mild antiviral activity against herpes simplex viruses, adenovirus, and influenza virus. Interestingly, when the compounds were evaluated against respiratory syncytial virus, a potent antiviral effect of 3,4-O-dicaffeoyl-1,5-γ-quinide was observed against both subtypes of respiratory syncytial virus, with EC50 values in the submicromolar range. Time-of-addition experiments revealed that this compound acts on an intracellular post-entry replication step. Our data show that 3,4-O-dicaffeoyl-1,5-γ-quinide is a relevant candidate for lead optimization and further mechanistic studies, and warrants clinical development as a potential anti-respiratory syncytial virus drug.