Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease

Abstract

Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or “snatched” from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.

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

“…This construct is similar to that previously studied (24) apart from a nonessential flexible loop (residues 51-72) that was deleted in our previous studies (26). Crystals of this construct routinely produced apo and complex structures at 2.3 Å resolution or better (SI Appendix, Tables S3-S5).…”

“…We used the inhibitor L-742,001, a 4-substituted 2,4-dioxobutanoic acid that effectively inhibits viral growth in both cell culture and mouse models (18,28,29). We recently confirmed that L-742,001 inhibits the activity of the isolated endonuclease domain and structurally characterized its binding at the active site (26). Here, we demonstrate that resistant mutations do not emerge by simply passaging the virus repeatedly in the presence of the compound.…”

“…L-742,001 has a trefoillike structure and is therefore an ideal molecule to probe the resistance landscape of the rather large endonuclease active site locale. L-742,001 (26) and related compounds (24) engage the central two-metal core and interact with many of the surrounding residues. We initially attempted to obtain resistant variants by passaging wild-type A/Puerto Rico/8/34 (henceforth PR8) virus in the presence of L-742,001 with a gradual increase in inhibitor concentration (10 μM, 20 μM, and 40 μM).…”

“…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.…”

“…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.…”