Bacterial resistance to known therapeutics has led to an urgent need for new chemical classes of antibacterial agents. To address this we have applied a Staphylococcus aureus fitness test strategy to natural products screening. Here we report the discovery of kibdelomycin, a novel class of antibiotics produced by a new member of the genus Kibdelosporangium. Kibdelomycin exhibits broad-spectrum, gram-positive antibacterial activity and is a potent inhibitor of DNA synthesis. We demonstrate through chemical genetic fitness test profiling and biochemical enzyme assays that kibdelomycin is a structurally new class of bacterial type II topoisomerase inhibitor preferentially inhibiting the ATPase activity of DNA gyrase and topoisomerase IV. Kibdelomycin is thus the first truly novel bacterial type II topoisomerase inhibitor with potent antibacterial activity discovered from natural product sources in more than six decades.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial and community-acquired pathogen for which few existing antibiotics are efficacious. Here we describe two structurally related synthetic compounds that potentiate beta-lactam activity against MRSA. Genetic studies indicate that these agents target SAV1754 based on the following observations: (i) it has a unique chemical hypersensitivity profile, (ii) overexpression or point mutations are sufficient to confer resistance, and (iii) genetic inactivation phenocopies the potentiating effect of these agents in combination with beta-lactams. Further, we demonstrate these agents inhibit peptidoglycan synthesis. Because SAV1754 is essential for growth and structurally related to the recently reported peptidoglycan flippase of Escherichia coli, we speculate it performs an analogous function in S. aureus. These results suggest that SAV1754 inhibitors might possess therapeutic potential alone, or in combination with beta-lactams to restore MRSA efficacy.

The emergence of drug-resistant bacteria coupled with the limited discovery of novel chemical scaffolds and druggable targets inspires new approaches to antibiotic development. Here we describe a chemical genomics strategy based on 245 Staphylococcus aureus antisense RNA strains, each engineered for reduced expression of target genes essential for S. aureus growth. Attenuation of gene expression can sensitize cells to compounds that inhibit the activity of a gene product or associated process. Pools of strains grown competitively in the presence of bioactive compounds generate characteristic profiles of strain sensitivities reflecting compound mechanism of action. Here, we validate this approach with a structurally and mechanistically diverse set of reference antibiotics and, in the accompanying paper in this issue of Chemistry & Biology (Huber et al., 2009), demonstrate its use in the discovery of new cell wall inhibitors.

1 This study compares the activity of BMS-180560 (2-butyl-4-chloro-1-[[1-[2-(2H-tetrazol-5-yl)phenyl]-1H-indol-4-yl]methyl]-lH-imidazole-5-carboxylic acid), an insurmountable angiotensin II (All) receptor antagonist, with that of losartan and EXP3174 in functional and biochemical models of AII-receptor activation.2 BMS-180560 selectively inhibited ['25I]-Sar'Ile8AII (['25I]SI-AII) binding to rat aortic smooth muscle (RASM) cell and rat adrenal cortical AT, receptors (Ki = 7.6 ± 1.2 and 18.4 ± 3.9 nM respectively) compared to adrenal cortical AT2 receptors (Ki = 37.6 ± 1.3 JiM). The K, values of BMS-180560 and EXP3174, but not losartan, varied as a function of the BSA concentration used in the assays, indicating that the diacid drugs bound to albumin.3 BMS-180560 (3-300 nM) increased the KD of SI-AII for RASM cell AT, receptors. Only at high concentrations of BMS-180560 (300nM) were Bmax values decreased.4 BMS-1 80560 inhibited AII-stimulated contraction of rabbit aorta with a calculated KB = 0.068 ± 0.048 nM and decreased maximal AlI-stimulated contraction at 1 nM BMS-180560 by 75%. In the presence of 0.1% BSA, a higher KB value (5.2 ± 0.92 nM) was obtained. Losartan behaved as a competitive antagonist with a KB = 2.6 ± 0.13 nM. Contraction stimulated by endothelin-1, noradrenaline, KCl, or the TXA2 receptor agonist U-46619 were unaffected by BMS-180560 (1 nM).5 All stimulated the acidification rates of RASM cells as measured by a Cytosensor microphysiometer with an EC50 of 18 nM. Losartan (30 nM) shifted the All concentration-effect curves in a competitive manner whereas BMS-180560 (0.01 and 0.1 nM) decreased the maximum responses by 60 and 75% respectively. Inhibition by losartan and BMS-180560 could be reversed following washout although recovery took longer for BMS-180560. (17-55%). BMS-180560 (3 and 10 nM) increased the EC50 for All and decreased the maximum response by 30 and 80% respectively. The inhibition by EXP3174 and BMS-180560 could be reversed by inclusion of losartan (200 nM) indicating that the inhibition was not irreversible. 7 In conclusion, BMS-180560 is a potent, specific, predominantly competitive, reversible All receptor antagonist, which displays insurmountable receptor antagonism. At concentrations of BMS-180560 which have no effect on receptor number, BMS-180560 produced insurmountable antagonism of AII-stimulated second messenger formation, extracellular acidification, and smooth muscle contraction.

This report describes the integration of laser-scanning f luorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated f luorescence associated with a receptorcontaining particle. To illustrate the various binding formats accommodated by this technique, saturation-and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G proteincoupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laserscanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step f luorescent immunoassays.The surge in identification of disease-related genes (1) and the combinatorial expansion of compound collections used in ''drug discovery'' (2) have compelled both the biotechnology and pharmaceutical industries to seek more efficient bioassays and screening methods to search for and develop new medicines. The measurement of binding interactions forms the basis of many assays and typically requires the physical separation of free ligand, L [e.g., [