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.

Isosteres of hydroxypyridinethione as drug-like pharmacophores for metalloenzyme inhibition

“…[46] As the Mn 1 is relatively more labile, the influenza endonuclease assay also contained excess Mn 2+ (and Mg 2+ ), to be consistent with previous reports of PA N activity assays and for similar reasons as described in the NDM-1 assay. 31 However, the effect of excess Mn 2+ metal ions on the overall activity of metal binding inhibitors has been found to be insignificant so long as the total concentration of metal ions is above the K d of the more labile metal binding site. 47 Overall 5 , 7 , 21 , and 22 had the best activity against endonuclease with greater than 90% inhibition.…”

“…hCAII was prepared as previously reported, [30] MMP-12 was purchased from Enzo Life Sciences (Farmingdale, NY, USA), NDM-1 was supplied as a gift from Dr. David Tierney (U. Miami, Ohio), and influenza endonuclease was expressed and purified as previously reported. [31] …”

“…PA N was prepared as previously reported, [31] and the fluorescent ssDNA-oligo substrate was purchased from Sigma-Aldrich. The assay was performed in a Costar black 96-well plate.…”

Aryl and Arylalkyl Substituted 3‐Hydroxypyridin‐2(1H)‐ones: Synthesis and Evaluation as Inhibitors of Influenza A Endonuclease

“…In addition to Xofluza and related compounds, several different classes of influenza endonuclease inhibitors have been described. These include 2,4‐dioxobutanoic acid derivatives, 5‐hydroxy‐1,6‐dihydropyrimidine‐4‐carboxylic acid derivatives, flutimide and its derivatives, 2‐hydroxyphenyl amide derivatives, salicylaldehyde thiosemicabazones, various types of catechins, pyromeconic acid and pyridinone derivatives, N ‐acylhydrazone derivatives, 5‐hydroxy‐4‐pyridone‐3‐carboxylic acid derivatives, 4,5‐dihydroxypyrimidine‐6‐carboxamide derivatives, as well as tetramic acid derivatives …”

“…[24] In addition to Xofluza and related compounds, several different classes of influenza endonucleasei nhibitors have been described. These include 2,4-dioxobutanoic acid derivatives, [19,20,25,26] 5-hydroxy-1,6-dihydropyrimidine-4-carboxylic acid derivatives, [20] flutimide and its derivatives, [27] 2-hydroxyphenyla mide derivatives, [28] salicylaldehyde thiosemicabazones, [29] varioust ypes of catechins, [30,31] pyromeconic acid and pyridinone derivatives, [32] N-acylhydrazone derivatives, [33] 5-hydroxy-4-pyridone-3-carboxylic acid derivatives, [34] 4,5-dihydroxypyrimidine-6-carboxamide derivatives, [35] as well as tetramic acid derivatives. [36] From an X-ray crystallographic screening campaign of afragment library targeting the IAVe ndonuclease, we identified 5chloro-3-hydroxypyridin-2(1H)-one as ab imetalc helating ligand at the active site of the enzyme.…”