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1.  MScreen: An Integrated Compound Management and High Throughput Screening (HTS) Data Storage and Analysis System 
Journal of biomolecular screening  2012;17(8):1080-1087.
High-throughput screening (HTS) has historically been used by the pharmaceutical industry to rapidly test hundreds of thousands of compounds to identify potential drug candidates. More recently, academic groups have used HTS to identify new chemical probes or small interfering RNA (siRNA) that can serve as experimental tools to examine the biology or physiology of novel proteins, processes, or interactions. HTS presents a significant challenge with the vast and complex nature of data generated. This report describes MScreen, a web-based, open-source cheminformatics application for chemical library and siRNA plate management, primary HTS and dose-response data handling, structure search, and administrative functions. Each project in MScreen can be secured with passwords or shared in an open information environment which enables collaborators to easily compare data from many screens, providing a useful means to identify compounds with desired selectivity. Unique features include compound, substance, mixture, and siRNA plate creation and formatting; automated dose-response fitting and quality control (QC); and user, target, and assay method administration. MScreen provides an effective means to facilitate HTS information handling and analysis in the academic setting so that users can efficiently view their screening data and evaluate results for follow-up.
doi:10.1177/1087057112450186
PMCID: PMC3600606  PMID: 22706349
chemoinformatics; data analysis software; open source; high-throughput screening
2.  Complementary Cell-Based High Throughput Screens Identify Novel Modulators of the Unfolded Protein Response 
Journal of Biomolecular Screening  2011;16(8):825-835.
Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.
doi:10.1177/1087057111414893
PMCID: PMC3374590  PMID: 21844328
unfolded protein response; endoplasmic reticulum stress; cell-based assay; luciferase reporter; natural products
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.
doi:10.1177/1087057110380571
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.
doi:10.1177/1087057110362101
PMCID: PMC2882805  PMID: 20237205
VirF; Shigella flexneri; AraC family; HTS; transcriptional activators
5.  High-Throughput Screening for Small Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay 
Journal of biomolecular screening  2009;14(2):161-172.
Guanine nucleotide-exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytokskeletal changes, motility, growth, survival, and gene transcription. The RhoGEF Leukemia-Associated RhoGEF (LARG) is a member of the Regulators of G-protein Signaling Homology Domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide binding assay utilizing BODIPY-Texas Red-GTPγS (BODIPY-TR-GTPγS), we performed a ten-thousand compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a non-fluorescent radioactive guanine nucleotide binding assay measuring LARG-stimulated [35S] GTPγS binding to RhoA, thus ruling out non-specific fluorescent effects. All five compounds selectively inhibited LARG-stimulated RhoA [35S] GTPγS binding, but had little to no effect upon RhoA or Gαo [35S] GTPγS binding. Therefore, these five compounds should serve as promising starting points for the development of small molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.
doi:10.1177/1087057108328761
PMCID: PMC2698131  PMID: 19196702
high-throughput screening; fluorescence polarization; RhoGEF; RhoA; LARG; Drug Discovery

Results 1-5 (5)