Technologies and Instrumentation in the HTS Resource
The High-Throughput Screening Shared Resource has three Biomek FX robotic liquid handlers with exchangeable 96- and 384-pipette heads for standard HTS liquid handling, a Biomek Span-8, and an Echo 555 for selecting compounds or siRNAs of interest from screening plates (cherry-picking).
The Echo is also used to dispense compounds for both single- and multi-dose experiments to support HTS confirmation screening and SAR studies.
Three PerkinElmer multi-mode EnVision readers are available for quantitating light-based endpoint assays (fluorescence, luminescence, absorbance, and AlphaScreen) in 96- and 384-well plate formats.
A RapidFire 300 reader is also available and uses mass spectroscopy to monitor the progress of enzyme-catalyzed reactions.
The HTS Shared Resource operates a BD Pathway 855 automated microscope for high-content, image-based screening of 96- and 384-well plates. To date, the HTS resource has implemented assays to measure calcium kinetics, mitochondrial health, mitotic index, cell proliferation, DNA damage and repair, cytoplasmic and nuclear translocation, neurite outgrowth, and cell migration.
The laboratory has a BioTek EL406 plate washer/dispenser, eight Titertek Multidrop liquid handlers, ABgene and Velocity11 plate sealers, two barcode printers, and two Apricot (96/384 well) pipettors. The lab has standard cell culture equipment (four tissue culture hoods, two Olympus microscopes, a Bio-Rad cell counter, three Eppendorf 5810 centrifuges) and sufficient incubator space (18 CO2 incubators) to support about seven concurrent cell-based screens and the associated maintenance cultures. Two monitored, cryogenic storage units (Thermo Fisher) house mammalian cell banks.
A BioMicroLab XL100 is used to handle 1-dram vials submitted by chemists for testing or for incorporation in the HTS chemical library. This instrument transfers compound samples from these vials to 2D-barcoded Matrix tubes for storage and cherry-picking. It has an onboard decapping tool, a four-place Satorius balance, and a 2D barcode reader. 2D and 1D barcode readers are used to read entire racks of Matrix storage tubes (96 tubes per rack) and the rack 1D barcodes, respectively.
The HTS resource uses several software programs and database systems to track chemical and RNAi inventory and to analyze, store, maintain, retrieve, and visualize data and information accumulated by screening projects. Current platforms include Core Informatics LIMS (v 4.2), Genedata Screener suite (v. 11) for data analysis, Accelrys' Pipeline Pilot (v. 9) for data aggregation and summarization, MOE (Chemical Computing Group), Glide (Schrodinger Inc.), HPCR (high performance computing via TACC), and TIBCO's Spotfire (v. 6) for data visualization.
Investigators have access to the UT Southwestern chemical library, which consists of 200,000 small molecules from commercial sources, 2,500 compounds from UT Southwestern chemists, and about 7,100 partially purified natural product fractions (each natural product fraction contains 3-10 natural products). The HTS laboratory has multiple copies of the institutional library, which is dissolved in DMSO and arrayed in 384-well plates.
The commercial compounds were purchased from major suppliers to the pharmaceutical industry. These compounds passed 48 structure-based filters that identified undesirable characteristics as well as satisfied a relaxed set of Lipinski's rules for good bioavailability. Those that were purchased represented the desirable structural diversity available from the following companies: ChemDiv (100,000 compounds), ChemBridge (75,500), ComGenex (22,000), Prestwick Chemical, (1,100) and TimTec (500), (ComGenex, 2005).
Associate Professor John MacMillan, Ph.D., a member of the UT Southwestern Biochemistry Department and the Simmons Comprehensive Cancer Center, provides the marine natural product fraction collection. As a principal investigator with expertise in natural product chemistry and isolation methods, Dr. MacMillan has established a "preparative pipeline" for a natural product screening collection that currently contains approximately 6,500 natural product fractions from a diverse group of marine-derived actinomycetes, Firmicutes, and Proteobacteria, the majority of which come from the Gulf of Mexico (Texas, Florida, and Louisiana) and the rich estuary system of South Carolina.
He has cultured these bacteria using a specialized "toolbox" of small-molecule signaling compounds (e.g., N-acyl homoserine lactones, siderophores, surfactins, etc.) that, in appropriate combination and concentration, can mimic the natural environment of each bacterial strain. These strains represent an affordable and renewable resupply of any natural product of interest from these sources.
The diversity of the bacterial collection and the associated natural products is considerable, with the former containing representatives from the order of Actinomycetales (10 different families) as well as strains from genera Gordonia (Corynebacteriaceae), Frankia, the alphaproteobacteria Erythrobacter (non-actinomycete strains), and bacilli.
The chemistry of the natural products isolated from these bacteria has proved diverse with respect to structural classes (e.g., polyketides, alkaloids, terpenes, and hybrid-structure). As would be expected, the natural products library contains novel compounds as well as those that have been previously characterized.
The HTS resource manages a human genomic library from Dharmacon as well as C. elegans and Drosophila genomic RNAi libraries from Ambion that were purchased by UT Southwestern. These libraries are available free of charge to UT Southwestern investigators who propose experiments acceptable to an oversight committee (contact Bruce Posner, Ph.D., for details - Bruce.Posner@UTSouthwestern.edu).
The human genomic library is composed of pools of four double-stranded siRNA oligos specific for each of 21,125 human genes arrayed in 96-well plates. All libraries are kept in multiple copies in -80°C or -20°C freezers within the 3,900-square-foot HTS laboratory.