An integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus PA endonuclease

Abstract

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) Mg(2+) or Mn(2+) 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 Mn(2+) versus Mg(2+), 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 Mg(2+) or Mn(2+). 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.

Fig. 1.

Illustration of the activity of DKAs in the plasmid-based PA-Nter assay. Recombinant PA-Nter (1 μg) was incubated with 1 μg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 in the presence of the test compounds. The endonucleolytic digestion of the plasmid was visualized by gel electrophoresis, and the amount of remaining intact plasmid was quantified. For L-742,001 and 10 (L-731,988), the IC₅₀ values were 0.5 μM and 0.8 μM, respectively. Wild-type (WT) enzyme showed complete cleavage with Mn²⁺ but was inactive when Mn²⁺ was omitted from the reaction mixture or replaced by Mg²⁺. The endonuclease activity was also completely abolished by the active site substitution K134A.

Fig. 2.

Three-dimensional arrangement of five pharmacophore features in the β-diketo acid motif. (A) Twelve active compounds 1–6, 10, 16, 34, 39–41 (i.e., β-diketo acids or esters with IC₅₀ values ≤ 2 μM) were aligned and mapped to generate a five-point pharmacophore. (B) The pharmacophore features are depicted as metal ligators (ML1, ML2, and ML3) in cyan, aromatic (Aro), and hydrophobic or aromatic (Hyd-Aro) features in pink. (C) Interfeature distances are given in angstroms (Å). (D) These distances are in agreement with both proposed models for interaction between the β-diketo acid motif and the metal ions. Left: in one model [which is based on the cocrystal structure of PA-Nter in complex with L-742,001 (DuBois et al., 2012)], the carboxylate and α-hydroxyl functionalities of the DKA motif coordinate one ion, and the third coplanar oxygen at the γ-position chelates the second metal ion together with the α-hydroxyl group. Middle: in an alternative model [previously proposed by us on the basis of L-742,001 docking within the holo form of PA-Nter, and assuming that L-742,001 mainly binds in its monodeprotonated form (Stevaert et al., 2013)], the γ-oxygen of the DKA motif is not involved. Instead, one metal is coordinated by the lone oxygen pair of the α-hydroxyl group and one oxygen atom of the carboxylate group, whereas the second metal ion interacts with both oxygens of the carboxylate moiety. Right: overlay of both models showing the intermetal distances.

Fig. 3.

Utility of the MB assay for determination of PA-Nter enzyme properties and activity of PA inhibitors. (A) Schematic representation of the MB assay. MB cleavage by recombinant PA-Nter (which can occur at different sites) separates the fluorophore (F) from the quencher (Q). The evolving fluorescence, indicating enzymatic activity, can be monitored in real-time. (B) Michaelis-Menten curve for MB-U₆A₂U₇-DFO. This MB was incubated with PA-Nter and the evolving fluorescence was recorded as a function of time and MB substrate concentration. The initial cleavage rate (V₀) was obtained from the slope (fluorescence units per minute) of the best-fit line derived from the 5–15-minute interval of the reaction. Values shown are the averages ± S.E.M. of three independent experiments. (C) Gel electrophoretic analysis of the MB cleavage pattern. The amount of intact MB-U₆A₂U₇-DFO substrate decreased over time when incubated with PA-Nter. The first-appearing cleavage products migrated faster than the intact MB substrate, whereas the late-cleavage products showed slower migration through the gel. When using Mn²⁺ as a cofactor, PA-Nter appears able to cleave shorter oligonucleotides than in the presence of Mg²⁺ or without addition of a bivalent metal (i.e., in the presence of the copurified metal). Addition of 100 mM EDTA completely abrogated the endonuclease activity. (D) Influence of the metal ion on the initial cleavage rate of MB-U₆A₂U₇-DFO and MB-het2-DFO. The initial cleavage rate (V₀; on the basis of the real-time fluorographs) for MB-U₆A₂U₇-DFO (left graph) was 1.4-fold and significantly higher with Mg²⁺ than with Mn²⁺, and still increased when no metal ion was added. An opposite effect was seen with MB-het2-DFO (right graph): In this case, the reaction rate was 2.2-fold higher in the presence of Mn²⁺ compared with Mg²⁺. The statistical significance (****P < 0.0001; ***P < 0.001; ns, not significant) was calculated by one-way analysis of variance followed by a Tukey’s multiple comparison test using GraphPad Prism. (E) Dose-dependent inhibition of MB cleavage by L-742,001. MB-U₆A₂U₇-DFO (at 20 nM) was incubated with 1 μg PA-Nter and increasing concentrations of L-742,001. The initial cleavage rate (V₀) was obtained from the slope (fluorescence units per minute) of the best-fit line derived from the 5–15-minute interval of the reaction. These V₀-values were used to calculate the percentage of inhibition. The inset shows the resulting dose-response curve.

Fig. 4.

Compounds 10 (L-731,988), 40, and 41 inhibit viral RNA synthesis besides affecting the virus entry process. Light gray bars: the test compounds L-742,001 (20 μM), 10 (200 μM), 40 (150 μM), 41 (150 μM), or chloroquine (80 μM) were added to MDCK cells, and after 30 minutes incubation at 35°C, influenza virus A/PR/8/34 was added. Dark gray bars: virus was added first and allowed to enter during 1 hour incubation, after which the compounds were added at the same concentrations as above. In both conditions, total cellular RNA was extracted at 10 hours postinfection (VC, untreated virus control). The number of vRNA copies was quantified by two-step real-time RT-PCR. On the y-axis, the fold increase in vRNA copies is shown relative to the viral copy number added at time zero. L-742,001 remains fully effective when added after virus entry. In contrast, the reported entry inhibitor chloroquine (Vanderlinden et al., 2012) is inactive when added at 1 hour postinfection. Compounds 10, 40, and 41 have a dual effect by acting both upon virus entry and viral RNA synthesis. Data shown are the mean ± S.E.M. of two independent tests.