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.

Understanding how compound penetration occurs across the complex cell walls of Gram-negative bacteria is one of the greatest challenges in discovering new drugs to treat the infections they cause. A combination of next-generation transposon sequencing, computational metadynamics simulations (CMDS), and medicinal chemistry was used to define genetic and structural elements involved in facilitated carbapenem entry into Pseudomonas aeruginosa. Here we show for the first time that these compounds are taken up not only by the major outer membrane channel OccD1 (also called OprD or PA0958) but also by a closely related channel OccD3 (OpdP or PA4501). Transport-mediating molecular interactions predicted by CMDS for these channels were first confirmed genetically, then used to guide the design of carbapenem analogs with altered uptake properties. These results bring us closer to the rational design of channel transmissibility and may ultimately lead to improved permeability of compounds across bacterial outer membranes.

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Prior research has suggested that family businesses may have a higher proclivity to behave as good corporate citizens, as compared with nonfamily firms, which may impact financial performance. However, while this idea is intuitively appealing, the actual antecedents of corporate citizenship behaviour in the family firm context have not yet been assessed systematically. In addition, empirical evidence regarding the impact of citizenship behaviour on firm performance remains inconclusive. Drawing from the literature on family business goals and corporate citizenship, our conceptual framework proposes that family-centred and business-centred goals act as determinants of Family business goals 35family firm citizenship, but each with a different impact. Furthermore, we argue that family firm reputation partially mediates the relationship between family firm citizenship behaviour and family firm performance. We discuss implications for family business research and practice and offer suggestions for future studies.

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An NMR-based approach marries the two traditional screening technologies (phenotypic and target-based screening) to find compounds inhibiting a specific enzymatic reaction in bacterial cells. Building on a previous study in which it was demonstrated that hydrolytic decomposition of meropenem in living Escherichia coli cells carrying New Delhi metallo-β-lactamase subclass 1 (NDM-1) can be monitored in real time by NMR spectroscopy, we designed a cell-based NMR screening platform. A strong NDM-1 inhibitor was identified with cellular IC50 of 0.51 μm, which is over 300-fold more potent than captopril, a known NDM-1 inhibitor. This new screening approach has great potential to be applied to targets in other cell types, such as mammalian cells, and to targets that are only stable or functionally competent in the cellular environment.

An NMR-based approachm arries the two traditional screening technologies (phenotypic and target-based screening) to find compounds inhibiting as pecific enzymatic reaction in bacterial cells.B uilding on ap revious study in which it was demonstrated that hydrolytic decomposition of meropenem in living Escherichia coli cells carrying New Delhi metallo-b-lactamase subclass 1(NDM-1) can be monitored in real time by NMR spectroscopy, we designed acell-based NMR screening platform. As trong NDM-1 inhibitor was identified with cellular IC 50 of 0.51 mm,w hich is over 300-fold more potent than captopril, ak nownN DM-1 inhibitor.T his new screening approach has great potential to be applied to targets in other cell types,such as mammalian cells,and to targets that are only stable or functionally competent in the cellular environment.