Fragment-Based Identification of Influenza Endonuclease Inhibitors

Abstract

The influenza virus is responsible for millions of cases of severe illness annually. Yearly variance in the effectiveness of vaccination, coupled with emerging drug resistance, necessitates the development of new drugs to treat influenza infections. One attractive target is the RNA-dependent RNA polymerase PA subunit. Herein we report the development of inhibitors of influenza PA endonuclease derived from lead compounds identified from a metal-binding pharmacophore (MBP) library screen. Pyromeconic acid and derivatives thereof were found to be potent inhibitors of endonuclease. Guided by modeling and previously reported structural data, several sublibraries of molecules were elaborated from the MBP hits. Structure-activity relationships were established, and more potent molecules were designed and synthesized using fragment growth and fragment merging strategies. This approach ultimately resulted in the development of a lead compound with an IC50 value of 14 nM, which displayed an EC50 value of 2.1 μM against H1N1 influenza virus in MDCK cells.

Figure 1

Structural model of the influenza RNA-dependent RNA polymerase PA subunit (PDB code 4M5Q). The endonuclease active site employs two divalent metal cations to facilitate the hydrolytic cleavage of the phosphodiester backbone of nucleic acids. Key active site residues and binding pockets are highlighted. Pocket 2 is obscured by residue Tyr24. Divalent metal cations are shown as orange spheres with the bridging hydroxide anion shown as a red sphere.

Figure 2

Proposed binding of pyromeconic acid (1) to the PA subunit active site. Ring positions 5 and 6 were deemed amenable to further derivatization for targeting hydrophobic pocket 2 and key residues Tyr24 and Arg84. Derivatization at the ring position 1 was proposed to probe interactions with pocket 4.

Figure 3

Docking analysis of 71 in influenza PA endonuclease (PDB code 4M4Q). (a) Docked structure of 71 bound to endonuclease. Hydrophobic interactions were found between the 6-position phenylaminomethyl moiety and hydrophobic pocket 2, as well as halogen bonding with Arg82. The N-phenyltetrazole moiety was found to hydrogen-bond simultaneously to Arg124 and Lys34. (b) Ligand interaction diagram detailing ligand/protein/solvent interactions rendered in two dimensions. Interactions between the ligand and protein active site are displayed as colored dotted lines: coordination bonds in purple, hydrogen bonds and π–π interactions in green, and halogen bonds in blue. Blue halos indicate a measure of ligand solvent-exposure, with larger halos indicating greater exposure.

Figure 4

Potency and cytotoxicity analysis of 71 and a reported diketo acid inhibitor 89 in MDCK cell lines. Cytotoxicity was determined by incubating cells in the presence of inhibitors for 48 h, followed by evaluating cell viability. Potency was determined by coadministration of inhibitor and a lethal challenge of virus particles, followed by a 48 h incubation and subsequent analysis of cell viability.

Figure 5

Chemical structure and inhibitory activity of several reported influenza endonuclease inhibitors that contain a phenyltetrazole moiety. Reported IC₅₀ values for each compound are provided. The MBP portion of each molecule is highlighted in blue. Compounds were initially reported by Parhi et al. and Sagong et al.

Figure 6

Structural comparison of several phenyltetrazole-containing influenza endonuclease inhibitors. Left: Crystal structure of Sagong-11 (PDB code 4W9S). Middle: Crystal structure of a derivative of Parhi-6 (PDB code 4M4Q). Right: Docking analysis of 71 (PDB code 4M4Q). Crystal structures of phenyltetrazole-containing inhibitors show the binding orientation of the phenyltetrazole moiety to be essentially conserved. Docking analysis of 71 predicts a very similar binding mode for this moiety to that observed in crystallographically validated inhibitors.

Scheme 1

Reagents and conditions: (a) triethyl orthoformate, H₂SO₄ (catalytic), rt, 24 h; (b) 4-nitrophenyl trifluoroacetate, pyridine, rt, 18 h; (c) ethyl or tert-butyl acetoacetate, NaH, reflux in dry THF, 4–6 h; (d) TFA, CH₂Cl₂, rt, 2–4 h; (e) HATU, triethylamine, DMF, 60 °C, o/n; (f) 1:1 HCOOH/H₂O, 80 °C, 2–6 h.

Scheme 2

Reagents and conditions: (a) benzyl bromide, K₂CO₃, DMF, 80 °C, 8–12 h; (b) thionyl chloride, CH₂Cl₂, rt, 8 h; (c) R₁R₂NH, triethylamine, DMF, 75 °C, o/n; (d) 5:5:1 HOAc/HCl/TFA, rt to 40 °C, 24–48 h; (e) sodium azide, DMF, rt, o/n; (f) triphenylphosphine, THF, rt, 30–60 min; (g) acid chloride or sulfonyl chloride, CH₂Cl₂, rt, o/n; (h) BCl₃, CH₂Cl₂, 0 °C, 30 min, rt, 30 min.

Scheme 3

Reagents and conditions: (a) thionyl chloride, CH₂Cl₂, rt, 4–6 h; (b) zinc dust, HCl, water, 70 °C, 4–6 h; (c) benzyl bromide, K₂CO₃, DMF, 80 °C, 8–12 h; (d) aryl- or alkylamine, HOAc, 3:1 EtOH/H₂O, microwave 125 °C, 90–120 min; (e) BCl₃, CH₂Cl₂, 0 °C, 30 min, rt, 30 min; (f) 5:5:1 HOAc/HCl/TFA, rt to 40 °C, 24–48 h.

Scheme 4

Reagents and conditions: (a) 1:1 HCOOH/H₂O, 80 °C, 2–6 h; (b) benzyl bromide, K₂CO₃, DMF, 80 °C, 8–12 h; (c) arylamine, HOAc, 3:1 EtOH/H₂O, microwave 130 °C, 3–5 h; (d) 5:5:1 HOAc/HCl/TFA, rt to 40 °C, 24–48 h; (e) 4% KOH, 1:2:1 THF/MeOH/water, rt, o/n; (f) RCH₃NH, triethylamine, DMF, 75 °C, o/n.