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.

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

“…Four resistance mutations were identified, T20A, I79L, F105S, and E119D, and all map to the endonuclease active site thereby confirming it as the target of L-742,001. These mutations partially overlap with those identified in an earlier L-742,001 study (35), and elicited resistance in both of the influenza strains tested.…”

“…T20A may not be a bona fide resistant mutation because over 98% of deposited sequences harbor this variation (SI Appendix, Table S6). Nonetheless, alanine provides a selective advantage when L-742,001 is present and T20A was also identified in the earlier studies (35). This residue is located on the opposite side of the active site to Phe105, and they may act in concert to influence the positioning of the p-chlorobenzene and phenyl arms of L-742,001 (Fig.…”

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

“…The PA protein is highly conserved among all influenza A viruses (79), with critical enzymatic domains retaining 99% homology (42); it is less likely to tolerate resistant mutations (25,80,81). PA inhibitor-resistant mutants have been generated by cell culture passage for L-742,001 (30,66), a 2-substituted-4,5-dihydroxypyrimidine derivative (42). This L-742,001-resistant mutant strain (PA T20A) was significantly more impaired in replication capacity (3-fold EC 50 change) than the zanamivir-resistant mutant (NA Q199G) that arose through passages in MDCK cells within the same study (ϳ30,000-fold EC 50 change), suggesting a profile of lower resistance for this endonuclease inhibitor than for the NAI (66).…”

“…This process is essential to completing the replication cycle and is widely recognized as a prime antiviral target. 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.…”

Virtual Screening and Biological Validation of Novel Influenza Virus PA Endonuclease Inhibitors

“…The relevance of the pocket delimited by Val122, Arg124, and Tyr130 was previously proposed in our mutational analysis of the binding pockets of L-742,001 (see Figure 4b). 19 Likewise, this pocket also proved to be of critical importance for the binding of three recently identified PAIs with strong inhibitory activity, as demonstrated in PA-Nter cocrystallization experiments. 18,20 Taken together, our docking results suggest that the superior PA-Nter inhibitory activity of 10 (IC 50 = 0.94 μM) is related to its optimal orientation for metal chelation, combined with its engagement into the Val122−Arg124−Tyr130 cavity.…”