ࡱ> OQNg JbjbjVV .0r<r<TTTTThhh8,huDCCCCCCC$FHDTDTT/D111ZTTC1C11nP7l:pp5V3^X8 CED0uDx8gIgI@l:gITl:t 1DDauDgI : Study guide for research assistants Read "Discovery of a small molecule that blocks wall teichoic acid biosynthesis in Staphylococcus aureus" (J.G. Swoboda et al., ACS Chemical Biology 4: 875-83, 2009). The full text of this paper can be accessed online (from UW computers) by following the links from this web page: http://www.ncbi.nlm.nih.gov/pubmed/19689117. Use the study guide below to help you understand the paper. You are welcome to discuss the paper with Greg and/or other people at any time. When you are satisfied with your overall understanding of the paper, please answer the "Questions for lab notebook" in your notebook; these won't be given a letter grade but will be checked! General background Much of our work consists of preparing for and conducting target-based screens in which we seek inhibitors of specific, isolated proteins. Aside from the success of the Plasmodium DHODH project (see previous study guides), purely target-based screening has not often led to new drug candidates. Though almost all existing chemotherapeutics for infectious diseases have sprung from traditional cell-based (or phenotypic) assays, these have severe limitations as well. In particular, optimization of hit compounds can be difficult and costly without knowledge of the compounds mechanism of action. The assigned paper is innovative primarily in its screening strategy, which combines features of target-based and cell-based screening in a best-of-both-worlds approach lauded by M.N. Gwynn et al. (Ann N Y Acad Sci 1213: 5-19, 2010): Mechanism-based whole cell screens can combine a targeted approach with a biologically integrated context. The focus on a particular pathway as a target (the wall teichoic acid biosynthesis pathway, in this case) insures that initial hits can be scrutinized carefully with respect to mechanism of action and can be developed relatively quickly through medicinal chemistry and structural biology. The assays cellular context insures that compounds identified as hits can disrupt the target in vivo (avoiding compounds that are unsuitable as leads because they cannot permeate cells, for example) and permits searches for inhibitors of an entire pathway (rather than targeting a single protein whose druggability may be uncertain). Abstract Note the definition of conditionally essential: genes that cannot be deleted except in strains incapable of initiating polymer synthesis. The best hit compound was designated 1835F03. My guess is that it was in well F3 of plate 1835 of the high-throughput screen. An ABC transporter is an ATP-Binding Cassette transporter, a type of transmembrane protein that uses the energy of ATP hydrolysis to move molecules across membranes. Introduction The first paragraph defines virulence factors as bacterially expressed molecules that are non-essential for survival in vitro but are required for robust infection in a host. In other words, bacteria lacking these factors can grow well in test tubes or on agar plates but not inside a host. Third paragraph: As with peptidoglycan biosynthesis, these polysaccharides are all assembled intracellularly on a bactoprenol carrier lipid Bactoprenol thus serves as a lipid anchor for the polysaccharides before they are transferred to peptidoglycan. It is shown in Figure 1 as a rod-shaped thing sticking into the cell membrane. Results and Discussion Compound 1835F03 has no activity against other Gram-positive strains that contain WTAs. From a drug development perspective, this was probably disappointing to the researchers. A hit compound is most likely to be developed into a drug if it can kill diverse bacteria (and thus has a large potential market) rather than just one particular species. The top left of p. 879 refers to heterologous complementation. As you may know, complementation is the introduction of a gene (often on a plasmid) to replace the function of a deleted or mutated gene. (This is often an important step in showing that a certain gene is responsible for a certain phenotype.) Heterologous complementation is when the newly introduced gene comes from a different species (Bacillis subtilis, in this case). 1835F03 was found to be bacteriostatic i.e., the cells stop dividing but dont die. (The other option is that it could have been bacteriocidal.) This is not necessarily good news from a drug development standpoint, as bacteriocidal drugs are preferred over bacteriostatic ones. Questions for lab notebook 1. Several WTA biosynthesis genes are conditionally essential in S. aureus. Why do tarO mutations compensate for mutations in other enzymes in the pathway? 2. Why do many polysaccharide polymers function as virulence factors? In other words, why are they necessary for invasion of a host? 3. Could the authors screening strategy have led to the discovery of inhibitors of TarO or TarA? Why or why not? 4. Explain how Figure 5 suggests that 1835F03 acts upon TarGH. 5. Toward the end of page 879, the authors say, These results confirm that the mutations are within the target rather than simply suppressor mutations that allow for the transport of truncated WTAs (which would occur if a conditionally essential enzyme upstream of TarG were inhibited). Explain. 6. As you know, bacterial evolution of resistance to drugs remains a formidable challenge. If a new drug were developed to block the function of TarG, would resistance be likely to evolve quickly? Explain.     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