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.

The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase and retain potency against drug-resistance mutations. Our discovery that water molecules play an essential role in the drug binding is reported. Femtosecond experiments and theory expose molecular level dynamics of rilpivirine bound to HIV-1 reverse transcriptase. The two nitrile substituents (-CN), one on each arm of the drug, have vibrational spectra consistent with their protein environments being similar in crystals and in solutions. Two-dimensional vibrational-echo spectroscopy reveals a dry environment for one nitrile while unexpectedly the other is hydrogen-bonded to a mobile water molecule, not identified in earlier X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure indeed reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond, despite the enlargement and structural variation of the binding pocket, may help retain the potency of rilpivirine against the pocket mutations.

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X-ray crystallography has been an under-appreciated screening tool for fragment-based drug discovery due to the perception of low throughput and technical difficulty. Investigators in industry and academia have overcome these challenges by taking advantage of key factors that contribute to a successful crystallographic screening campaign. Efficient cocktail design and soaking methodologies have evolved to maximize throughput while minimizing false positives/negatives. In addition, technical improvements at synchrotron beamlines have dramatically increased data collection rates thus enabling screening on a timescale comparable to other techniques. The combination of available resources and efficient experimental design has resulted in many successful crystallographic screening campaigns. The three-dimensional crystal structure of the bound fragment complexed to its target, a direct result of the screening effort, enables structure-based drug design while revealing insights regarding protein dynamics and function not readily obtained through other experimental approaches. Furthermore, this “chemical interrogation” of the target protein crystals can lead to the identification of useful reagents for improving diffraction resolution or compound solubility.

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(1H)-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(1H)-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(1H)-one, the 5-aza analogue (5-hydroxy-2-(4-fluorophenyl)pyrimidin-4(3H)-one) did exhibit significant activity as an endonuclease inhibitor. The 6-aza analogue of 5-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one (6-(4-fluorophenyl)-4-hydroxypyridazin-3(2H)-one) also retained modest activity as an inhibitor. Several varied 6-phenyl-4-hydroxypyridazin-3(2H)-ones and 2-phenyl-5-hydroxypyrimidin-4(3H)-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(1H)-ones.

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

BackgroundThe recently approved anti-AIDS drug rilpivirine (TMC278, Edurant) is a nonnucleoside inhibitor (NNRTI) that binds to reverse transcriptase (RT) and allosterically blocks the chemical step of DNA synthesis. In contrast to earlier NNRTIs, rilpivirine retains potency against well-characterized, clinically relevant RT mutants. Many structural analogues of rilpivirine are described in the patent literature, but detailed analyses of their antiviral activities have not been published. This work addresses the ability of several of these analogues to inhibit the replication of wild-type (WT) and drug-resistant HIV-1.ResultsWe used a combination of structure activity relationships and X-ray crystallography to examine NNRTIs that are structurally related to rilpivirine to determine their ability to inhibit WT RT and several clinically relevant RT mutants. Several analogues showed broad activity with only modest losses of potency when challenged with drug-resistant viruses. Structural analyses (crystallography or modeling) of several analogues whose potencies were reduced by RT mutations provide insight into why these compounds were less effective.ConclusionsSubtle variations between compounds can lead to profound differences in their activities and resistance profiles. Compounds with larger substitutions replacing the pyrimidine and benzonitrile groups of rilpivirine, which reorient pocket residues, tend to lose more activity against the mutants we tested. These results provide a deeper understanding of how rilpivirine and related compounds interact with the NNRTI binding pocket and should facilitate development of novel inhibitors.

Edited by Norma Allewell HIV-1 integrase (IN) is essential for virus replication andCollectively, our findings provide a plausible path for structurebased development of second-generation ALLINIs.