Structural Analysis of Specific Metal Chelating Inhibitor Binding to the Endonuclease Domain of Influenza pH1N1 (2009) Polymerase

Abstract

It is generally recognised that novel antiviral drugs, less prone to resistance, would be a desirable alternative to current drug options in order to be able to treat potentially serious influenza infections. The viral polymerase, which performs transcription and replication of the RNA genome, is an attractive target for antiviral drugs since potent polymerase inhibitors could directly stop viral replication at an early stage. Recent structural studies on functional domains of the heterotrimeric polymerase, which comprises subunits PA, PB1 and PB2, open the way to a structure based approach to optimise inhibitors of viral replication. In particular, the unique cap-snatching mechanism of viral transcription can be inhibited by targeting either the PB2 cap-binding or PA endonuclease domains. Here we describe high resolution X-ray co-crystal structures of the 2009 pandemic H1N1 (pH1N1) PA endonuclease domain with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme. Comparison of the binding mode of the different compounds and that of a mononucleotide phosphate highlights, firstly, how different substituent groups on the basic metal binding scaffold can be orientated to bind in distinct sub-pockets within the active site cavity, and secondly, the plasticity of certain structural elements of the active site cavity, which result in induced fit binding. These results will be important in optimising the design of more potent inhibitors targeting the cap-snatching endonuclease activity of influenza virus polymerase.

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

“…Structural studies have now revealed new opportunities for drug discovery that target the essential endonuclease activity of the virus (24)(25)(26). In the current study, we had two primary goals; to confirm that the known endonuclease inhibitor L-742,001 targets the enzyme within the intact virus by generating resistant mutants, and to survey the resistance potential of the endonuclease by characterizing the sites of mutation.…”

“…The mutation appears to adjust the two-metal coordination geometry and weaken the binding of the distal Mn 2+ . The distal and proximal Mn 2+ ions are coordinated by two and four side chains, respectively, which explains their different binding affinities (24). As regards the I79L mutation, this residue is adjacent to the two-metal locale and can potentially impact its conformation.…”

“…engages the centrally bound nucleotide of the substrate (24). Recent studies revealed how inhibitory compounds engage the active site (24)(25)(26).…”

“…Studies by others (24) have shown that the endonuclease domain from pH1N1 yields superior crystal structures of inhibitor complexes than the A/Vietnam/1203/2004 (H5N1) strain that we previously studied (26). Having demonstrated that the selected resistance mutations are also effective in the pH1N1, we switched to this strain for our kinetic, binding and structural analyses of the resistance mutants.…”

Structure and Assembly of the Influenza A Virus Ribonucleoprotein Complex

“…For instance, a recent structural study showed that several known endonuclease inhibitors, including four diketo compounds and a green tea catechin, bind to the endonuclease active site of the PA protein [44]. All these inhibitors chelate the two critical manganese ions in the active site of the enzyme, although some differences are noted in the overall ligand ordination of these compounds.…”