Novel broad-spectrum inhibitors of bacterial methionine aminopeptidase.

With increasing emergence of multi-drug resistant infections, there is a dire need for new classes of compounds that act through unique mechanisms. In this work, we describe the discovery and optimization of a novel series of inhibitors of bacterial methionine aminopeptidase (MAP). Through a high-throughput screening campaign, one azepinone amide hit was found that resembled the native peptide substrate and possessed moderate biochemical potency against three bacterial isozymes. X-ray crystallography was used in combination with substrate-based design to direct the rational optimization of analogs with sub-micromolar potency. The novel compounds presented here represent potent broad-spectrum biochemical inhibitors of bacterial MAP and have the potential to lead to the development of new medicines to combat serious multi-drug resistant infections.

X-ray crystal structures of Escherichia coli RNA polymerase with switch region binding inhibitors enable rational design of squaramides with an improved fraction unbound to human plasma protein.

Squaramides constitute a novel class of RNA polymerase inhibitors of which genetic evidence and computational modeling previously have suggested an inhibitory mechanism mediated by binding to the RNA polymerase switch region. An iterative chemistry program increased the fraction unbound to human plasma protein from below minimum detection levels, i.e., <1% to 4-6%, while retaining biochemical potency. Since in vitro antimicrobial activity against an efflux-negative strain of Haemophilus influenzae was 4- to 8-fold higher, the combined improvement was at least 20- to 60-fold. Cocrystal structures of Escherichia coli RNA polymerase with two key squaramides showed displacement of the switch 2, predicted to interfere with the conformational change of the clamp domain and/or with binding of template DNA, a mechanism akin to that of natural product myxopyronin. Furthermore, the structures confirmed the chemical features required for biochemical potency. The terminal isoxazole and benzyl rings bind into distinct relatively narrow, hydrophobic pockets, and both are required for biochemical potency. In contrast, the linker composed of squarate and piperidine accesses different conformations in their respective cocrystal structures with RNA polymerase, reflecting its main role of proper orientation of the aforementioned terminal rings. These observations further explain the tolerance of hydrophilic substitutions in the linker region that was exploited to improve the fraction unbound to human plasma protein while retaining biochemical potency.

Small-molecule inhibitors of gram-negative lipoprotein trafficking discovered by phenotypic screening.

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.

Understanding the effects of decompaction maintenance on the infill state and play performance of third-generation artificial grass pitches.

Third generation artificial grass pitches have been observed to get harder over time. The maintenance technique of rubber infill decompaction is intended to help slow, or reverse, this process. At present, little is understood about either the science of the infill compaction process or the efficacy of decompaction maintenance. The objective of this study was to measure the changes in rubber infill net bulk density, force reduction (impact absorption) and vertical ball rebound under various levels of compactive effort in controlled laboratory-based testing. The assessments were repeated after the systems had been raked to simulate the decompaction maintenance techniques. These tests defined the limits of compaction (loose to maximally compacted) in terms of the change in rubber infill net bulk density, force reduction and vertical ball rebound. Site testing was also undertaken at four third generation pitches immediately pre and post decompaction, to determine the measurable effects in the less well controlled field environment. Rubber infill net bulk density was found to increase as compactive effort increased, resulting in increased hardness. Decompacting the surface was found to approximately fully reverse these effects. In comparison, the site measurements demonstrated similar but notably smaller magnitudes of change following the decompaction process suggesting that the field state pre and post decompaction did not reach the extremes obtained in the laboratory. The findings suggest that rubber infill net bulk density is an important parameter influencing the hardness of artificial grass and that decompactions can be an effective method to reverse compaction related hardness changes.

Dancer perceptions of the force reduction of dance floors used by a professional touring ballet company.

The mechanical properties of dance floors have the potential to influence dancers' performance and injury risk. Little information is available that describes dancers' preferences for dance floor mechanical properties. Investigation of dancers' perceptions of varied dance floors can serve to enlighten governing bodies, floor manufacturers, and the dance community. The aim of this study was to assess the perceptions of dancers from a touring professional ballet company regarding four floors with varied force reduction (FR) that were created to replicate those used by the company in normal dance training and performance. A specialized questionnaire was developed that incorporated a series of qualitative and quantitative measures that could be used by participants to express their perceptions of the custom built dance floors. Floor FR was quantified with reference to the protocols specified by European standards. Dancer perceptions were in general agreement with floor FR values; however, some discrepancies were observed. Dancers expressed a preference for floor FR within the mid to upper limits (57% to 72%) of the European standards, although a minority preferred low FR (approximately 36%) floors. A limited ability to perceive inconsistencies in FR across test floors was observed, which may have implications for injury risk. Investigation of the perceptions of dancers from more diverse backgrounds, on floors that provide a closer representation of typical dance studio and stage sizes, over longer periods of time, would provide further insight into the perceptual and adaptive responses of dancers to varied floor mechanical properties.

A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria.

Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.

The role of a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase druggability.

The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor.

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.

Novel rapidly diversifiable antimicrobial RNA polymerase switch region inhibitors with confirmed mode of action in Haemophilus influenzae.

A series of inhibitors with a squaramide core was synthesized following its discovery in a high-throughput screen for novel inhibitors of a transcription-coupled translation assay using Escherichia coli S30 extracts. The inhibitors were inactive when the plasmid substrate was replaced with mRNA, suggesting they interfered with transcription. This was confirmed by their inhibition of purified E. coli RNA polymerase. The series had antimicrobial activity against efflux-negative strains of E. coli and Haemophilus influenzae. Like rifampin, the squaramides preferentially inhibited synthesis of RNA and protein over fatty acids, peptidoglycan, and DNA. However, squaramide-resistant mutants were not cross-resistant to rifampin. Nine different mutations were found in parts of rpoB or rpoC that together encode the so-called switch region of RNA polymerase. This is the binding site of the natural antibiotics myxopyronin, corallopyronin, and ripostatin and the drug fidaxomicin. Computational modeling using the X-ray crystal structure of the myxopyronin-bound RNA polymerase of Thermus thermophilus suggests a binding mode of these inhibitors that is consistent with the resistance mutations. The squaramides are the first reported non-natural-product-related, rapidly diversifiable antibacterial inhibitors acting via the switch region of RNA polymerase.

The synthesis and selective IL-2 inhibitory activity of bis piperazine-phenol Mannich adducts.

Novel phenol bis-Mannich adducts were identified as IL-2 expression inhibitors in a T cell proliferation screening assay. Analogues of the lead compound were prepared through parallel synthesis and a highly selective IL-2 inhibitor was discovered that provided a suitable compound for further optimization.