Phenyl Substituted 4‑Hydroxypyridazin-3(2<i>H</i>)‑ones and 5‑Hydroxypyrimidin-4(3<i>H</i>)‑ones: Inhibitors of Influenza A Endonuclease

Seasonal and pandemic influenza outbreaks remain a major human health problem. Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is attractive for the development of new agents for the treatment of influenza infection. Our earlier studies identified a series of 5- and 6-phenyl substituted 3-hydroxypyridin-2­(1<i>H</i>)-ones that were effective inhibitors of influenza endonuclease. These agents identified as bimetal chelating ligands binding to the active site of the enzyme. In the present study, several aza analogues of these phenyl substituted 3-hydroxypyridin-2­(1<i>H</i>)-one compounds were synthesized and evaluated for their ability to inhibit the endonuclease activity. In contrast to the 4-aza analogue of 6-(4-fluorophenyl)-3-hydroxypyridin-2­(1<i>H</i>)-one, the 5-aza analogue (5-hydroxy-2-(4-fluorophenyl)­pyrimidin-4­(3<i>H</i>)-one) did exhibit significant activity as an endonuclease inhibitor. The 6-aza analogue of 5-(4-fluorophenyl)-3-hydroxypyridin-2­(1<i>H</i>)-one (6-(4-fluorophenyl)-4-hydroxypyridazin-3­(2<i>H</i>)-one) also retained modest activity as an inhibitor. Several varied 6-phenyl-4-hydroxypyridazin-3­(2<i>H</i>)-ones and 2-phenyl-5-hydroxypyrimidin-4­(3<i>H</i>)-ones were synthesized and evaluated as endonuclease inhibitors. The SAR observed for these aza analogues are consistent with those previously observed with various phenyl substituted 3-hydroxypyridin-2­(1<i>H</i>)-ones

Differential Isotopic Enrichment To Facilitate Characterization of Asymmetric Multimeric Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein–ligand and protein–protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched (¹⁵N-labeled) p51 and unlabeled p66. To enable detection of ¹⁵N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% ¹⁵N model. Our results demonstrated that ¹⁵N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but ¹⁵N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure

3‑Hydroxyquinolin-2(1<i>H</i>)‑ones As Inhibitors of Influenza A Endonuclease

Several 3-hydroxyquinolin-2­(1<i>H</i>)-ones derivatives were synthesized and evaluated as inhibitors of 2009 pandemic H1N1 influenza A endonuclease. All five of the monobrominated 3-hydroxyquinolin­(1<i>H</i>)-2-ones derivatives were synthesized. Suzuki-coupling of <i>p</i>-fluorophenylboronic acid with each of these brominated derivatives provided the respective <i>p</i>-fluorophenyl 3-hydroxyquinolin­(1<i>H</i>)-2-ones. In addition to 3-hydroxyquinolin-2­(1<i>H</i>)-one, its 4-methyl, 4-phenyl, 4-methyl-7-(<i>p</i>-fluorophenyl), and 4-phenyl-7-(<i>p</i>-fluorophenyl) derivatives were also synthesized. Comparative studies on their relative activity revealed that both 6- and 7-(<i>p</i>-fluorophenyl)-3-hydroxyquinolin-2­(1<i>H</i>)-one are among the more potent inhibitors of H1N1 influenza A endonuclease. An X-ray crystal structure of 7-(<i>p</i>-fluorophenyl)-3-hydroxyquinolin-2­(1<i>H</i>)-one complexed to the influenza endonuclease revealed that this molecule chelates to two metal ions at the active site of the enzyme

Crystallographic Fragment Screening and Structure-Based Optimization Yields a New Class of Influenza Endonuclease Inhibitors

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 (PA_N) was engineered and used for fragment screening leading to the identification of new chemical scaffolds binding to the PA_N 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 PA_N 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

Detecting Allosteric Sites of HIV‑1 Reverse Transcriptase by X‑ray Crystallographic Fragment Screening

HIV-1 reverse transcriptase (RT) undergoes a series of conformational changes during viral replication and is a central target for antiretroviral therapy. The intrinsic flexibility of RT can provide novel allosteric sites for inhibition. Crystals of RT that diffract X-rays to better than 2 Å resolution facilitated the probing of RT for new druggable sites using fragment screening by X-ray crystallography. A total of 775 fragments were grouped into 143 cocktails, which were soaked into crystals of RT in complex with the non-nucleoside drug rilpivirine (TMC278). Seven new sites were discovered, including the Incoming Nucleotide Binding, Knuckles, NNRTI Adjacent, and 399 sites, located in the polymerase region of RT, and the 428, RNase H Primer Grip Adjacent, and 507 sites, located in the RNase H region. Three of these sites (Knuckles, NNRTI Adjacent, and Incoming Nucleotide Binding) are inhibitory and provide opportunities for discovery of new anti-AIDS drugs