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Lipopolysaccharide from Escherichiu coli 01 11, its gulE derivative when grown in galactose, E. coli 086, and Sulmonella typhimuvium LT2 all contain antigenic side chains and separate into more than 40 components by electrophoresis in gradients of polyacrylaniide containing sodium dodecylsulfate. These components from E. coli 0111 are not interconvertible and show a heterogeneous size distribution when fractionated with Sephadex (3-200. Isoelectric focusing of this mixture in pH 3.5 -10 ampholines reveals a single component, ruling out extensive charge heterogeneity.The relative antigenic side chain lengths for the components, estimated using ratios of galactose in antigenic side chain to phosphate in the lipid-A-core oligosaccharide region, show that the size heterogeneity is due to differences in the number of antigenic side chain units per molecule and ranges from none to over 40. Preference for molecules of specific chain lengths, especially short ones, was observed.In contrast, the galE mutant grown without galactose does not synthesize antigenic side chains, and more than 90 % of its lipopolysaccharide migrates as a single band at a position corresponding to the lowest-molecular-weight component from the above preparations. Lipopolysaccharide from E. coli PL2, a K12 strain lacking antigenic side chain, separates into two low-molecular-weight components on electrophoresis. These results confirm that the heterogeneity which we observe in lipopolysaccharide containing antigenic side chains, is due to the side chain rather than the lipid-A -core oligosaccharide region.Lipopolysaccharide is found uniquely in the outer membrane of gram-negative bacteria and is important in the structure [ 1,2] and function [3,4] of this membrane. Variations in its composition also influence host responses to invasion by gram-negative organisms [5].Lipopolysaccharide consists of lipid A, core oligosaccharide, and antigenic side chain. The linkage of sugar residues [6] and the chemical structure within the lipid A moiety [7,8] is known for lipopolysaccharide from several organisms. Biosynthesis of lipid A has also been investigated [9].

SUMMARYThere is an urgent need for the discovery and development of new antitubercular agents that target novel biochemical pathways and treat drug-resistant forms of the disease. One approach to addressing this need is through high-throughput screening of drug-like small molecule libraries against the whole bacterium in order to identify a variety of new, active scaffolds that will stimulate additional biological research and drug discovery. Through the Molecular Libraries Screening Center Network, the NIAID Tuberculosis Antimicrobial Acquisition and Coordinating Facility tested a 215,110-compound library against M. tuberculosis strain H37Rv. A medicinal chemistry survey of the results from the screening campaign is reported herein. CONFLICT OF INTEREST STATEMENTCompeting interests: Dr. Goldman is a NIAID staff member who either in the past or currently provides oversight for the project that generated the data used as the basis for this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public AccessAuthor Manuscript Tuberculosis (Edinb) The MLSCN was established in 2005 as a pilot program to assemble a large library of biologically relevant small molecules and make them available through a network of HTS laboratories to researchers worldwide through a competitive assay submission process. Acceptance of the TAACF assay into the MLSCN program made available the unique resources of the NIH Small Molecule Repository (SMR), significantly expanding the spectrum of molecules tested for activity against TB. For this screen, a 215,110-compound library from the SMR was examined for anti-TB activity using the assay described previously, 7 with the only change to the screening protocol being the elimination of the polyethylene incubator bags, resulting in the identification of a number of novel chemical scaffolds. Moreover, even for classes of compounds identified earlier during testing of the NIAID ChemBridge library, 7 additional examples emerged that further clarified the structure-activity picture. Since the compounds in the SMR have been examined in scores of diverse assays undertaken by the MLSCN, and the results published on the NIH PubChem website, 8 another motivation for conducting the MLSCN campaign is the ability to correlate antituberculosis activity of the hits with other biological activities that these compounds may possess, potentially providing information about possible mechanisms of action or toxicity. The raw screening results upon which the structural analysis below is based are now publicly available on PubChem (assay AIDs 1332 and 1626). MATERIALS ...

A whole-cell C. albicans screen was designed to identify novel inhibitors interacting with the synthesis, assembly and regulation of the fungal cell wall. C. albicans was grown in a paired broth assay in 96-well plates with natural product extracts or pure chemical compoundsin the presence and absence of the osmotic stabilizer, sorbitol. Growthwas visually examinedover a 7-day period and scored into different growth categories. Positives from the sorbitol rescue were then examined under the microscope for morphological alterations and grouped into several morphological classes. Sorbitol protection and cell morphology were indicators of novel antifungal agents from natural product extracts and pure compounds.

Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Peptidoglycan synthesis and degradation is a process in bacteria that involves multiple enzymes under strict temporal and spatial regulation. We used a set of small molecules that inhibit the transglycosylation step of peptidoglycan synthesis to discover genes that help to regulate this process. We identified a gene responsible for the susceptibility of Escherichia coli cells to killing by glycolipid derivatives of vancomycin, thus establishing a genetic basis for activity differences between these compounds and vancomycin.

Ramoplanin (Figure 1) is a cyclic glycolipodepsipeptide antibiotic that kills gram positive bacteria by inhibiting cell wall biosynthesis. Ramoplanin was shown to block the conversion of Lipid I to Lipid II, 1 a reaction that is catalyzed by the intracellular GlcNAc transferase, MurG (Scheme 1). It was proposed that ramoplanin inhibits MurG by complexing Lipid I, which prevents it from being utilized as a substrate. Below we show that ramoplanin also inhibits the polymerization of Lipid II; therefore, we propose that another mechanism by which ramoplanin can kill bacterial cells is through inhibition of the transglycosylation step of peptidoglycan synthesis. Using a synthetic analogue of Lipid II, we present evidence that enzyme inhibition by ramoplanin involves substrate binding. Ramoplanin undergoes a conformational change upon substrate binding, and the resulting complexes self-associate to form fibrils. The significance of fibril formation is discussed.The mechanism of action of ramoplanin has been investigated in permeabilized bacterial cells and membrane preparations by following the incorporation of radiolabel from a precursor into various intermediates along the pathway to peptidoglycan. 1-3 A limitation of these assays is that if one enzymatic step is blocked, then no information can be obtained about subsequent steps. Thus, because ramoplanin prevents the formation of Lipid II, it is not possible to determine whether it also inhibits the polymerization of Lipid II. We reinvestigated the ability of ramoplanin to block Lipid II polymerization using a modified membrane assay 4 in which the transglycosylases are selectively inhibited to permit the buildup of radiolabeled Lipid II. Following removal of the inhibitor, peptidoglycan synthesis commences. The effect of ramoplanin on Lipid II polymerization was evaluated by monitoring the amount of radioactive peptidoglycan formed in the presence of increasing concentrations of ramoplanin. Ramoplanin blocks the polymerization of Lipid II and thus is an inhibitor of the transglycosylation step of peptidoglycan synthesis (Figure 2).Ramoplanin was proposed to act by complexing substrates required for peptidoglycan synthesis. 1 Unfortunately, difficulties in isolating Lipid intermediates from bacterial cells have hindered studies of their interactions with ramoplanin. 5,6 Moreover, the natural Lipid intermediates contain a 55 carbon polyprenol chain that renders them insoluble in water, and thus difficult to use in biophysical studies of complex formation. We recently developed a synthetic route to a soluble Lipid I analogue (1) to use in studying MurG, 7 the GlcNAc transferase that converts Lipid I to Lipid II. Using purified MurG, we have now made the corresponding Lipid II analogue 2 from 1, as shown (Scheme 2). 8,9 Compound 2 is identical to natural Lipid II except that the 55 carbon chain has been replaced with a 10 carbon unit so that the compound is freely water soluble. 10 The ability of ramoplanin to interact with 2 was investigated by NMR (Figure 3). Ti...

Formation of branched glucan, glucan-glucan cross links, and glucan-chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor p-1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor /3-1,3 glucan, yielding a linear glucan containing a p-1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429-4411, An apparent K,, of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent K, of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H,O competing for reaction at the activated donor site, thus resulting in hydrolysis.However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for p-1,3 glucan as acceptor, in as much as no transfer was detected when either hexa-N-acetyl-chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of p-1,3 glucan into the previously existing cell wall structure.Keywords. Glucosyltransferase ; fungal wall ; BGL2 protein ; wall assembly ; Saccharomyces.Synthesis and assembly of the fungal cell wall is a complex process about which we know much less compared to the analogous events occurring in bacteria. We do know that linear polymers of chitin, p-1,4 N-acetylglucosamine, and glucan, p-1,3 glucose, are synthesized by microsomal fractions prepared from fungal cells when iincubated in the presence of their appropriate UDP-sugars [l, 21. The process appears to be vectorial in that UDP-sugar presenf in the cytoplasm is polymerized, with concurrent translocation through the membrane of the growing polymeric chain. However, little if anything is known about the following aspects of p-1,3 polymer synthesis: (a) initiation of chain synthesis, (b) regulation of chain initiation, (c) translocation through the membrane, (d) mechanism and regulation of termination of chain synthesis, and (e) localization of chain synthesis during different phases of wall expansion. Furthermore, glucan found in the mature cell wall consists of highly branched molecules containing both p-1,3 and p-1,6 linkages.

The continuing spectre of resistance to antimicrobial agents has driven a sustained search for new agents that possess activity on drug resistant bacteria. Although several paths are available to reach this goal, the most generalized would be the discovery and clinical development of an agent that acts on a new target which has not yet experienced selective pressure in the clinical setting. Such a target should be essential to the growth and survival of bacteria, and sufficiently different from, or better still non-existent in, the human host. The transglycosylation reaction that polymerizes biochemical intermediates into peptidoglycan qualifies as such a target. This biochemical system accepts the basic unit N-acetylglucosamine-beta-1, 4-N-acetyl-muramyl-pentapeptide-pyrophosphoryl-undecaprenol (lipid II), and leads to polymerization of the N-acetylglucosamine -beta-1, 4-N-acetyl-muramyl-pentapeptide segment into peptidoglycan. Approaches to targeting this reaction include modification of known glycolipid and glycopeptide natural product antibiotics. The synthesis and antibacterial activity of synthetic analogs of moenomycin having novel antibacterial activities not present in the parent structure will be presented, together with the combinatorial chemistry and assay systems leading to their discovery. Likewise, we will discuss chemical modifications to specific glycopeptide antibiotics that have extended their spectrum to include vancomycin resistant enterococci that substitute D-alanyl-D-lactate for D-alanyl-D-alanine in their peptidoglycan. Two differing theories, one positing the generation of high affinity, specific binding to D-alanyl-D-lactate via glycopeptide dimerization and/or membrane anchoring, and the other supporting direct targeting of the modified glycopeptide to the transglycosylation complex, seek to explain the mechanism of action on vancomycin resistant enterococci. Biochemical evidence in support of these two theories will be discussed.