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Cell-based Assay for High Throughput Screening of HIV Protease Inhibitors
Chen-Chen Kan The successful development of clinically effective inhibitors of the HIV protease is a major triumph for structure-based drug design. However, AIDS remains a disease continually needing new anti-viral agents because of the rapid emergence of drug-resistance. Dr. Kan's lab has developed a safe, convenient, and cost-effective E. coli-based assay system that involves co-expression of an engineered beta-galactosidase as an HIV protease substrate and the HIV protease precursor comprising the trans-frame region and the protease domain. The production of active HIV protease in this system results in a loss of beta-galactosidase activity, which can be detected in high-throughput screens. This E. coli-based system was used as a tool for primary screen of library compounds for inhibitors against HIV protease. The lab is currently working to develop and test a human CD4+ cell-based assay system to serve as a secondary screening platform for inhibitors identified using the E. coli-based assay system. Screening Methods in Antimicrobial Drug Discovery
Molly Schmid Because of the rapid development of microbial drug resistance there is a great need for inexpensive and efficient assays that allow discovery of novel antibacterial molecules with specific mechanisms of action. These molecules could provide the starting point for the development of new classes of antibiotics. In addition, because the mechanism of antibacterial activity in these molecules is known, information can be gained about microbial cell biology. Dr. Schmid's lab is developing a high throughput assay for antibacterial compounds with pathway selectivity for DNA replication, cell wall biosynthesis and protein secretion. S. aureus strains with well defined conditional mutations in essential genes related to these pathways are used in the assay to provide a screening system for antibacterial molecules that can penetrate whole cells. In this situation, a chemical compound, which is not lethal to wild-type bacteria, acts in concert with a mutation in a pathway that also is not lethal (unless the appropriate conditions are applied), to produce a lethal effect. Compounds identified in the screen that show promising antibacterial activity will be characterized further. Dr. Schmid's lab is using the relatively new technique of fragment-based screening to discover novel small molecule antibacterial compounds with activity against one or more bacteria in the group of moderately dangerous pathogens identified by the federal government as Category B priority agents. Fragment-based screening has the potential to allow identification of new chemical entities with antibacterial activity that might have been missed with high throughput screening techniques. Protein crystallization and high resolution X-ray diffraction of the target proteins will be used to identify and characterize the binding of small (MW 164-342) chemical fragments with highly resolved chemical structures and co-structures. Four bacterial target families (NAD synthetase, farnesyl diphosphate synthase, peptidyl-tRNA hydrolase and glucosamine-6-phosphate synthase) were selected for their essentiality in key gram negative species and the desirable attributes of the proteins for structural biology and fragment screening approaches. Once a fragment is shown to bind a target, it will then be optimized for potent antibacterial activity in a process that relies on the chemical structure of the fragment. The ultimate goal of this project is to discover optimized compounds with improved potency and drug-like properties that can serve as safe, orally effective antibacterial agents. Small Molecule Antibacterial Agents as Selectable Genetic Markers
Molly Schmid Another project being conducted in Dr. Schmid's lab is the development of small molecule antibacterial agents and corresponding resistance markers that can be used in genetic manipulations of various pathogens. Strain manipulations and identification of the resistance markers will be performed in Staphylococcus aureus (a gram-positive surrogate for the pathogens Bacillus anthracis, Clostridium botulinum, and Listeria monocytogenes), and/or Salmonella typhimurium (a gram-negative surrogate for the pathogens Yersinia pestis, Y. enterocolitica, Francisella tularensis, Burkholderia pseudomallei, B. mallei, Shigella sonnei, E. coli, and Campylobacter jejuni). The use of a surrogate bacterium to undertake identification and optimization of the resistance markers allows the work to proceed more safely and without the stringent containment facilities required by the federal government for these category A and B priority agents. The use of the surrogate bacteria will also provide the ability to identify resistance markers that are unable to recombine with the genomes of the pathogens mentioned above, which will be a benefit in future development of the selectable marker as a useful genetic tool.
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