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There is an urgent need for new antibacterial drugs that are effective against infections caused by multidrug-resistant pathogens. Novel nonfluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) have the potential to become such drugs because they display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. Bacterial topoisomerase inhibitors that are built on a tetrahydropyran ring linked to a bicyclic aromatic moiety through a syn-diol linker show potent anti-Gram-positive activity, covering isolates with clinically relevant resistance phenotypes. For instance, analog 49c was found to be a dual DNA gyrase-topoisomerase IV inhibitor, with broad antibacterial activity and low propensity for spontaneous resistance development, but suffered from high hERG K(+) channel block. On the other hand, analog 49e displayed lower hERG K(+) channel block while retaining potent in vitro antibacterial activity and acceptable frequency for resistance development. Furthermore, analog 49e showed moderate clearance in rat and promising in vivo efficacy against Staphylococcus aureus in a murine infection model.

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10 ؊7 ). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.Antibiotic resistance is a major health concern, and the existing antibiotics target only a handful of molecules. Therefore, there is an urgent need for antibiotics with novel mechanisms of action. Peptide deformylase (PDF; EC 3.5.1.27) is essential in a variety of pathogenic bacteria but is not required for cytoplasmic protein synthesis in eukaryotes and is therefore an interesting potential target for antibacterial agents. Protein synthesis in eubacteria, under normal conditions, is initiated by formyl-methionyl-tRNA (19). Consequently, all nascent polypeptides are synthesized with N-formyl-methionine at the N terminus. The formyl group is removed by PDF during elongation of the polypeptide chain (1, 7). As methionine aminopeptidase (EC 3.4.11.18) cannot hydrolyze N-blocked polypeptides, deformylation is also a prerequisite for protein maturation (10,22,27). Both PDF and MAP, are essential for growth in Escherichia coli (10,19,21). pdf gene mutants can only be obtained in E. coli strains lacking the gene for formyltransferase, the enzyme that N-formylates the methionyl-tRNA f Met (EC.2.1.2.9) (20). In a recent publication, we described the identification, optimization, and biological characterization of novel PDF inhibitors (3). These compounds were potent inhibitors of the isolated enzyme but only moderately active as antibacterials. In the accompanying paper, we describe transcription-translation assays that allowed us to demonstrate that the inhibitors were active as inhibitors of PDF in cell homogenates as well as in intact cells (4a). The experimental evidence presented here demonstrates that (i) antibacteri...

Pseudomonas (Comamonas) testosteroni T-2 completely converted p-toluenesulphonic acid (TS) orp-sulphobenzoic acid (PSB) to cell material, C 0 2 and sulphate, with growth yields of about 5 g protein (mol C)-l. PSB and sulphite were excreted as transient intermediates during growth in TS-salts medium. All reactions of a catabolic pathway involving sidechain oxidation and cleavage of the sulphonate moiety as sulphite were measurable in the soluble portion of cell extracts. Degradation of TS and PSB was inducible and apparently involved at least two regulons. TS was converted to p-sulphobenzyl alcohol in a reaction requiring NAD(P)H and 1 mol O2 (mol TS)-l. This alcohol was in an equilibrium (in the presence of NAD+) with p-sulphobenzaldehyde, which was converted to PSB in an NAD(P)+-dependent reaction. PSB was desulphonated to protocatechuic acid in a reaction requiring NAD(P)H and 1 mol O2 (mol PSB)-l. Experiments with '*02 confirmed involvement of a dioxygenase, because both atoms of this molecular oxygen were recovered in protocatechuate. Protocatechuate was converted to 2-hydroxy-4-carboxymuconate semialdehyde by a 4,Sdioxygenase. I N T R O D U C T I O NSeveral aliphatic, glyco and aryl sulphonates occur naturally (Laskin & Lechevalier, 1984), and so they might be expected to be biodegradable. However, some synthetic detergents (Cain, 198 l), sulphonated dyestuffs (Meyer, 198 1) and their precursors (Bretscher, 198 1) are generally poorly degraded. The appearance of these chemicals in rivers (Malle, 1978) indicates a need for a better understanding of their metabolism.Proof of quantitative degradation of individual aromatic sulphonates has accumulated in recent years (Cain & Farr, 1968;Brilon et al., 1981 a;Thurnheer et al., 1986;Nortemann et al., 1986; Ziirrer et al., 1987; Wittich et al., 1988;Locher et al., 1989) and desulphonation of aryl sulphonates serving as carbon sources for growth involves liberation of the sulphonate moiety as sulphite (Johnston et al., 1975). There have been very few observations of high rates of desulphonation by cell-free systems cf. Heyman & Molof, 1968; Willetts & Cain, 1972; Endo et al., 1977;Locher et al., 1989). In consequence, Brilon et al. (1981b) suggested a mechanism of dioxygenolytic desulphonation based on results of experiments with whole cells. Known degradative pathways for aromatic sulphonates with short sidechains seem to involve initial desulphonation, followed by standard pathways for the resultant aromatic ring structures (Cain & Farr, 1968; Brilon et al., 1981 b;Swisher, 1987). In at least one case, failure to desulphonate an intermediate caused serious growth inhibition (Kulla et al., 1983). p-Toluenesulphonate (TS), which is mainly used as a hydrotropic agent in detergent formulations, has served as a model compound in studies of the biodegradation of alkylated benzenesulphonates and was found to be degraded via 4-methylcatechol (Cain & Farr, 1968;Focht & Williams, 1970 We now present a complete mass balance for the metabolism of TS by Pseudomonas (Coma...

Low-molecular-weight beta-sulfonyl- and beta-sulfinylhydroxamic acid derivatives have been synthesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most of the compounds synthesized and tested displayed antibacterial activities that cover several pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these compounds as antibacterial agents is discussed with respect to selectivity, intracellular concentrations in bacteria, and potential for resistance development.

[structure: see text] Nostocarboline and seven derivatives were prepared and displayed minimal inhibitory concentration (MIC) values >or=100 nM against the growth of Microcystis aeruginosa PCC 7806, Synechococcus PCC 6911, and Kirchneriella contorta SAG 11.81, probably via the inhibition of photosynthesis. The natural product hybrid nostocarboline/ciprofloxacin displayed additional antibacterial activity against several Gram-negative bacteria (MICs >or=0.7 microM). Nostocarboline can thus be considered a potent, selective, readily available, natural algicide.