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1.  Application of a High-throughput Fluorescent Acetyltransferase Assay to Identify Inhibitors of Homocitrate Synthase 
Analytical biochemistry  2010;410(1):133-140.
Homocitrate synthase (HCS) catalyzes the first step of L-lysine biosynthesis in fungi by condensing acetyl-Coenzyme A and 2-oxoglutarate to form 3R-homocitrate and Coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust, fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of ~41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized over 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases and enzymes involved in lipid metabolism.
PMCID: PMC3115995  PMID: 21073853
homocitrate synthase; antifungal; high-throughput screen; fluorescent assay; MMBC; ThioGlo 1; acetyltransferase
2.  Chemical Screens Against A Reconstituted Multi-Protein Complex: Myricetin Blocks DnaJ Regulation of DnaK through an Allosteric Mechanism 
Chemistry & biology  2011;18(2):210-221.
DnaK is a molecular chaperone responsible for multiple aspects of proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors, we screened plant-derived extracts against a re-constituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK’s intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaJ. Together, these results highlight a “gray box” screening approach, which approximates a limited amount of the complexity expected in physiological, multi-protein systems.
PMCID: PMC3057461  PMID: 21338918
3.  High Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume By Exploiting Energy Transfer 
Journal of biomolecular screening  2010;15(10):1211-1219.
Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate-detection reagents, such as quinaldine red (QR). While such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70’s weak enzymatic activity have combined to create significant challenges in high throughput screening. To overcome these difficulties, we have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal. Biochem. 2005, 342:254–259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, we tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~ 0.6, CV ~8%) and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70-family members.
PMCID: PMC3052282  PMID: 20926844
phosphate; malachite green; ATPase; molecular chaperone; fluorescence assay
4.  HTS of the Virulence Regulator VirF: A Novel Antibacterial Target for Shigellosis 
Journal of biomolecular screening  2010;15(4):379-387.
Shigella flexneri is a human enteropathogen that infects ca. 165 million people and claims more than 1 million lives per year worldwide. Although shigellosis has been considered a disease of the “Third World,” like many other contagious diseases, it does occur in developed countries. The emergence of drug and multi-drug-resistant strains of Shigella emphasize the need for novel antibiotic development. VirF, an AraC-type transcriptional regulator, is responsible for the expression of all downstream virulence factors that control intracellular invasion and cell-to-cell spread of Shigella. Gene knockout studies have validated that inhibition of VirF expression is sufficient to block the normal life cycle of Shigella in the host and thereby increase susceptibility to the host immune system. The authors have developed a high-throughput, cell-based assay to monitor inhibition of VirF using β-galactosidase as a reporter protein. Using an avirulent strain of Shigella, they have screened libraries containing ~42,000 small molecules. Following confirmation and dose-response analysis, they have identified 25 compounds that demonstrate VirF inhibition in vivo ≥55% in comparison to the controls and little general antibacterial activity (measured by cell growth, OD600). The authors are in the process of confirming these “hits” in several secondary assays to assess the mechanism of action.
PMCID: PMC2882805  PMID: 20237205
VirF; Shigella flexneri; AraC family; HTS; transcriptional activators

Results 1-4 (4)