A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy. IMPORTANCEThe prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

No abstract

Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z′ of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.

BACKGROUND AND PURPOSE11β-hydroxysteroid dehydrogenase type I (11β-HSD1), a target for Type 2 diabetes mellitus, converts inactive glucocorticoids into bioactive forms, increasing tissue concentrations. We have compared the pharmacokinetic-pharmacodynamic (PK/PD) relationship of target inhibition after acute and repeat administration of inhibitors of 11β-HSD1 activity in human, rat and mouse adipose tissue (AT). EXPERIMENTAL APPROACHStudies included abdominally obese human volunteers, rats and mice. Two specific 11β-HSD1 inhibitors (AZD8329 and COMPOUND-20) were administered as single oral doses or repeat daily doses for 7-9 days. 11β-HSD1 activity in AT was measured ex vivo by conversion of 3 H-cortisone to 3 H-cortisol. KEY RESULTSIn human and rat AT, inhibition of 11β-HSD1 activity was lost after repeat dosing of AZD8329, compared with acute administration. Similarly, in rat AT, there was loss of inhibition of 11β-HSD1 activity after repeat dosing with COMPOUND-20 with continuous drug cover, but effects were substantially reduced if a 'drug holiday' period was maintained daily. Inhibition of 11β-HSD1 activity was not lost in mouse AT after continuous cover with COMPOUND-20 for 7 days. CONCLUSIONS AND IMPLICATIONSHuman and rat AT, but not mouse AT, exhibited tachyphylaxis for inhibition of 11β-HSD1 activity after repeat dosing. Translation of observed efficacy in murine disease models to human for 11β-HSD1 inhibitors may be misleading. Investigators of the effects of 11β-HSD1 inhibitors should confirm that desired levels of enzyme inhibition in AT can be maintained over time after repeat dosing and not rely on results following a single dose.

Nicotinic acetylcholine receptors (nAChRs) are neurotransmitter-gated ion channels in which five subunits coassemble to form either a homomeric or heteromeric complex. In vertebrates, nAChRs are expressed at the neuromuscular junction (NMJ) and also within the central and peripheral nervous system (the "neuronal" nAChRs). In addition to the five nAChR subunits expressed at the NMJ ( a l , PI, y, 6, E), eleven neuronal nAChR subunits have been identified ( a 2 -1~9 and P2-p4). We are interested in examining the properties of recombinant neuronal nAChRs by expressing defined combinations of cloned rat nAChR subunit cDNAs in a variety of cultured mammalian cell lines. We have examined the influence of different host cell types and of nAChR subunit domains upon the efficiency of folding, subunit co-assembly and cell-surface expression of neuronal nAChRs.Nicotinic AChRs are also expressed abundantly within the invertebrate nervous system and are important targets for insecticides. It has, however, proved to be particularly difficult to obtain functional recombinant insect nAChRs by heterologous expression of cloned subunit cDNAs. We have obtained evidence to suggest that insect cell lines provide an environment better suited to the efficient folding and assembly of insect nAChRs. In particular, we have found Drosophila S2 cells to be a useful expression system for characterisation of nAChRs and other ion charhels and also of G-protein coupled receptors cloned from DrosophifaThe success in expressing recombinant proteins using heterologous expression systems is highly variable. Expression systems which employ prokaryote and lower eukaryote organisms (e.g. bacteria and yeast), frequently yield high levels of recombinant protein. This is largely due to the ability of these organisms to grow to high biomass in liquid culture. However, proteins which require complex folding or post-translational modification are often inadequately or incorrectly processed in these organisms.Higher eukaryote expression systems are more suited to processing complex proteins but are often 'ill-equipped' for high level expression of proteins. The expression of seven transmembrane helix (STH) receptors presents a particular challenge since folding, insertion in the cell membrane and in some cases coupling via G-proteins are all required for expression of functional receptors.We have expressed a wide range of STH receptors using the LCR/MEL expression system. This system utilizes the globin gene locus control region (LCR) which promotes the expression of genes in a copy numberdependent, position-independent manner in murine erythro-leukaemia (MEL) cells. The LCR/MEL system enables the rapid identification of recombinant MEL cell clones which are capable of high level expression of functional STH receptors.Despite observing high levels of recombinant mRNA we were unable to detect functional chemokine receptors which could bind the appropriate ligand using the LCR/MEL system o r using the baculovirus expression system. This may be explained by the...