Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics, based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against gram-negative Pseudomonas sp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homologue of the ß-barrel protein LptD (Imp/OstA), which functions in outer membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications. A synthesized antibiotic targets a protein involved in outer membrane biogenesis to selectively kill Pseudomonas pathogens. 1 Peptidomimetic Antibiotics Target Outer Membrane Biogenesis in Pseudomonas aeruginosa AbstractAntibiotics with new mechanisms of action are urgently required to combat the growing health

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OxyB is a cytochrome P450 enzyme that catalyzes the first phenol coupling reaction during the biosynthesis of vancomycin-like glycopeptide antibiotics. The phenol coupling reaction occurs on a linear peptide intermediate linked as a C-terminal thioester to a peptide carrier protein (PCP) domain within the multidomain glycopeptide nonribosomal peptide synthetase (NRPS). Using model peptides with the sequence (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP and (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-(S)Dpg-S-PCP (where Hpg = 4-hydroxyphenylglycine, and Dpg = 3,5-dihydroxyphenylglycine), or containing (R)Leu instead of (R)(NMe)Leu, attached to recombinant PCPs derived from modules-6 and -7 in the vancomycin NRPS, we show that cross-linking of Hpg4 and Tyr6 by OxyB can occur in both hexapeptide- and heptapeptide-PCP conjugates. Thus, whereas OxyB may act preferentially on a hexapeptide still linked to the PCP-6 in NRPS subunit-2, it is possible that a linear heptapeptide intermediate linked to PCP-7 in NRPS subunit-3 may also be transformed into monocyclic product. For turnover, OxyB requires electrons, which in vitro can be supplied by spinach ferredoxin and E. coli flavodoxin reductase. Turnover is also dependent upon the presence of molecular oxygen. The model substrate (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP is transformed into cross-linked product by OxyB with a kcat of 0.1 s-1 and Km in the range 4-13 muM. Equilibrium binding of this substrate to OxyB, monitored by UV-vis, is accompanied by a typical low-to-high spin state change in the heme, characterized with a Kd of 17 +/- 5 muM.

Thanatin targets protein-protein interactions between LptA and LptD in the periplasmic bridge required for LPS transport.

Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-Å resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys 347 being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel ␤-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The discovery of novel antibiotics with new mechanisms of action is an important goal in antibiotic research to combat infections caused by multidrug-resistant bacteria, in particular Gram-negative microorganisms with their unique asymmetric outer membrane (OM) 3 (1). Naturally occurring cationic host defense peptides that form part of the innate immunity in many organisms have recently attracted great interest in the search for new clinically useful antibiotics (2). We explore here an approach to antibiotic discovery based upon host defense peptide-inspired macrocyclic peptidomimetics as a potential source of antibiotics displaying target selectivity and potency not seen with naturally occurring host defense peptides. In previous work (3), we described a family of macrocyclic ␤-hairpin peptidomimetics with potent and selective antimicrobial activity against Pseudomonas spp., which were shown to have a novel mechanism of action targeting the ␤-barrel outer membrane protein LptD in Pseudomonas aeruginosa and inhibiting its key lipopolysaccharide (LPS) transport function in OM biogenesis (4). We report here the discovery of a new conformationally constrained ␤-hairpin peptidomimetic (called JB-95) having potent antimicrobial activity against a panel of Grampositive and Gram-negative bacteria and, in particular, against Escherichia coli. JB-95 shows minimal inhibitory concentrations (MICs) of ϳ0.25 g/ml against E. coli, including many multidrug-resistant clinical strains. We report the solution structure of JB-95 and investigations into its mechanism of action against E. coli. Experimental ProceduresPeptide Synthesis-The methods for synthesis and characterization of all peptides have been described previously (5). JB-95 was of Ͼ95% purity by analytical reverse phase HPLC ( Antibacterial Assays-MICs were determined in microtiter plates in Mueller-Hinton-I (MH-I) medium using the broth

During the biosynthesis of glycopeptide antibiotics of the vancomycin family, several oxidative phenol coupling reactions take place. An oxygenase (OxyB) from the vancomycin producer catalyzes the first of these coupling reactions to a significant extent only when the putative hexapeptide substrate is linked as a thioester to a peptide carrier domain (PCD) derived from the non‐ribosomal peptide synthetase (see picture).

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