Crystallographic fragment screening and structure-based optimization yields a new class of influenza endonuclease inhibitors

Abstract

Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, we report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase. A high-resolution crystal form of pandemic 2009 H1N1 influenza polymerase acidic protein N-terminal endonuclease domain (PAN) was engineered and used for fragment screening leading to the identification of new chemical scaffolds binding to the PAN active site cleft. During the course of screening, binding of a third metal ion that is potentially relevant to endonuclease activity was detected in the active site cleft of PAN in the presence of a fragment. Using structure-based optimization, we developed a highly potent hydroxypyridinone series of compounds from a fragment hit that defines a new mode of chelation to the active site metal ions. A compound from the series demonstrating promising enzymatic inhibition in a fluorescence-based enzyme assay with an IC50 value of 11 nM was found to have an antiviral activity (EC50) of 11 μM against PR8 H1N1 influenza A in MDCK cells.

Figure 1

Description of PA_N structure. (A) APBS calculated electrostatic potential surface of the active site cleft of PA_N. The binding of a ligand described in this study (7) is shown with yellow carbon atoms and a carboxamide derivative with three chelating groups crystallized by Dubois et al. is shown in gray (PDB code: 4E5I). The subpockets are labeled as previously described. (B) Superposition of the pH1N1 2009 PA_N structures from this study and one previously described (PDB code: 4AVQ).

Figure 2

Discovery of a third active site metal. Metal-coordinating bonds are depicted as black dashed lines and hydrogen bonds as blue dashed lines. Residues with significant structural changes upon binding of a ligand are displayed with the apo structure shown in orange. (A) Structure of 1 bound to subpockets 2 and 3. Compound 1 coordinates to a third metal (M3), which coordinates to Glu80. The electron density (dark blue mesh) is calculated from a F_o-F_c omit map and contoured at 5σ. Electron density calculated from anomalous data (cyan) is contoured at 3σ. (B) Structure of 1 calculated electrostatic potential surface (APBS) and subpocket labels. (C) Stereoview of the active site cleft showing electron density for the metal ions and coordinating waters in the apo PA_N structure from a 100 mM CaCl₂ soak. The locations of the metal ions from the soak of 1 are shown in green whereas the locations of the calcium cations from a 100 mM soak are shown in yellow. The electron density for the metal and coordinating waters is shown (omit map contour of 2.5σ).

Figure 3

Structure of 2 bound to PA_N. Metal-coordinating bonds are depicted as black dashed lines, hydrophobic and cation-π interactions as grey, and hydrogen bonds as blue dashed lines. Residues with significant structural changes upon binding of a ligand are depicted with the apo structure shown in orange. Electron density calculated from anomalous scattering data (cyan) is contoured at 3σ. (A) Compound 2 bound at subpockets 2 and 3, and the active site (omit map contoured at 4σ). (B) Side view of 2 bound at the active site superposed with 2,4-dioxo-4-phenylbutanoic acid (PDB code: 4E5G).

Figure 4

Development of the pyridinone series of compounds. Metal coordinating bonds are depicted as black dashes, hydrophobic and cation-π interactions are grey, and hydrogen bonds as blue dashes. Residues with significant structural changes upon binding of a ligand are shown with the apo structure colored orange. (A) 3 bound at the active site (omit map contoured at 3σ). (B) Compound 4 bound at subpockets 2 and 3, and the active site (omit map 4σ contours). (C) 5 bound at the active site (omit map contour of 4.5σ). (D) 6 bound at the active site (omit map with 4σ contours). (E) 7 bound at the active site (omit map contoured at 4σ).

Figure 5

Viral yield inhibition assay and cytotoxicity assay. Virus yield assay using MDCK cells and the A/Puerto Rico/8/1934 (H1N1) influenza strain was used to determine the antiviral activity of 7; oseltamivir and 0.1% (v/v) DMSO (only) were used as the positive and negative controls. Cytotoxicity for 7 (shown in red) was determined from MDCK cells after 24 hours using the MTT assay.