Access and Support for HTS Experiments
The cost of an HTS experiment is substantial. For approved projects, the Harold C. Simmons Cancer Center and UT Southwestern Medical Center provide considerable support for screening projects by underwriting fixed costs in the Core (e.g. salaries, infrastructure, equipment, etc.). This makes high-throughput screening affordable for young investigators as well as established researchers in the Simmons Cancer Center and the University at large. Principal investigators are only responsible for the cost of screening supplies and a scientist who will be dedicated to the project. The Core will provide the following without charge:
- Compounds or siRNAs
- Access to and technical expertise for equipment needed for HTS
- Scientific guidance and support for assay development
- Aid in conducting HTS experiments
- Data storage, processing, and analysis
- Cheminformatics and bioinformatics support
For HTS projects with a focus on cancer, the Simmons Cancer Center will cover 20 percent of the screening supplies costs for small screens (e.g. 8,000 compound pilot screens, miRNA screens, etc.) and 50 percent of larger screens (full library compound screens and genome-wide RNAi screens). Approval for Cancer Center funds for small screens is obtained through Bruce Posner, Ph.D., the Core Director. For larger screens, the HTS Oversight Committee decides if Cancer Center funding can be awarded. The maximum financial award for a screen is $12,500.
Tissue culture can be conducted in the investigator's laboratory or the HTS laboratory. If long-term culture work is to be conducted in the HTS lab, a daily fee of $5 will be assessed for use of HTS pipets, tubes, gloves, paper towels, and alcohol. The investigator's laboratory should supply their own plates or dishes, media, bottles, and any filter units needed.
For all RNAi screens, a supply fee of $425 will be assessed to cover the cost of tips ($250), plate seals ($120), and alcohol ($55). siRNA oligos are supplied free to investigators whose projects have been approved. For all microRNA screens, a supply fee of $100 will be assessed to cover the cost of tips, ethanol, tubes, pipettes, and plate sealing film.
Investigators using the high-content BD Pathway 855 screening microscope must pay $10/hour to use the microscope. This charge is to cover the cost of the microscope service contract. Data analysis can be conducted on the Attovision Workstation free of charge.
To gain access to the HTS Core facility and scientific staff for assay development and small screening projects, principal investigator(s) must first meet with the HTS Core Director, Bruce Posner, Ph.D., to discuss the assay and the goals for their screening project. In this discussion, they will describe the goals for the project, the assay development that has been completed to date, and downstream studies that will help advance the hits identified in their screen.
Access to Core is at the discretion of Dr. Posner and based largely on the originality of the screen, feasibility of the assay and screening strategy, and the therapeutic potential of the approach.
Once access is granted, the principal investigator will work collaboratively with the HTS Core to develop, optimize, and validate their assay for a small screen. The details of assay development are described in greater detail under HTS Screening of Chemical Compounds or siRNA Libraries.
Small projects defined as screens that interrogate small chemical or RNAi libraries in the Core or supplied by the principal investigator. The Core has several chemical sub libraries that include the Prestwick Collection (~1100 approved drugs), the MacMillan Natural Products Fraction Collection (~7,100 fractions representing ~30,000 natural products), the NIH Clinical Collection (~450 experimental drugs), and the 2K subset (~1800 natural product-like commercial compounds).
In order to gain access to the HTS Core and the institutional libraries for large screening projects, principal investigators must submit a short, four-page proposal that includes the following elements:
- Abstract describing the aims of the experiment and the assay to be used
- One page background describing relevance of the target of the experiment to cancer
- Description of the assay to be used
- Estimate of reagent costs
- Name of scientist dedicated to the experiment
- Description of secondary assays for prioritizing the compounds
- If applicable, a stated request for Simmons Cancer Center funding for screening supplies
Both the practicality and the scientific and therapeutic value of targets for screening are assessed by the Cancer Center HTS Oversight Committee, which is headed by Michael Roth, Ph.D., in the Biochemistry Department. Dr. Posner, the Core Director, and the HTS Core will help evaluate the cost and practicality of the proposed assay for HTS such as the number of liquid handling and wash steps, timing, reagent volumes, and necessary controls. If Cancer Center funding is requested, the HTS Oversight Committee will review the request and decide whether funds can be awarded. The maximal financial award is $12,500 and funds must be spent on screening supplies.
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To submit a proposal to the HTS Oversight Committee, send the proposal as a MS Word document or a PDF by email to Michael Roth, Ph.D., at email@example.com. Dr. Roth will submit the proposal to the HTS Oversight Committee for review and provide feedback to the principal investigator.
Assay development is conceptually similar for RNAi and compound screens. In both cases, the primary assay must reflect an appropriate biological context and be sufficiently robust to detect desired changes in the biological response when exposed to a genetic or chemical perturbagen.
Dr. Posner and the HTS staff will comment on the practicality of the assay, offer suggestions for counter and secondary assays, and, if needed, provide alternative assay approaches that have proven effective in previous projects. The Core brings more than 30 years of experience in assay development and screen execution and hence, is a valued collaborator in these aspects of HTS.
Both Core and project scientists work collaboratively to develop, refine, and validate parameters and controls (positive, negative, and neutral) for the primary assay such that it is robust (Z values > 0.45 over many assays and experimental days), tolerant of effects from DMSO, free from systematic effects (e.g. plating artifacts, liquid handling errors, etc.), simple (most assays have less than three liquid additions and are endpoint assays), and efficient in use of reagents, HTS equipment, personnel, and resources. Secondary assays are developed in parallel to the primary assay and must meet similar criteria. RNAi screens are typically run in 96 well microtiter plates (3 replicates per siRNA pool) while compounds screens are run in a 384 well format (1 replicate per compound).
Once an assay has an acceptable signal-to-noise ratio in small-scale experiments for chemical or RNAi screen, it is tested in 3 to 10 plates treated under "mock" conditions, an experiment in which the HTS protocol is executed but no actual library samples are tested. In the case of compound screening, DMSO is substituted for the chemical library in the test wells (columns 3 to 22 of a 384 well plate) and for RNAi screening, transfection reagent plus a non targeting siRNA or transfection reagent alone is substituted for the library in columns 2 to 11 of a 96 well plates. Assays that are reproducible and largely free of plate or systematic effects are deemed optimized and ready for screening provided approval has been obtained from the Core Director (small screening projects) or the HTS Oversight Committee (large screening projects).
Chemical Pilot Screens
For compound screens, a pilot experiment can be done with an 8,000 compound diversity subset. This subset is an approximate representation of the chemical space in the larger 200,000+ UT Southwestern small molecule library.
Principal investigators are required to supply a scientist to work on the HTS screen. This person will work with by the HTS scientific staff, who will operate the robotic liquid handlers and perform all operations involving the dispensing of compounds from library plates to experimental plates. The HTS staff scientists will also read the plates for each experimental run, analyze the data, and provide a report to the principal investigator and his scientist leading the screening effort. Following the primary screen, the principal investigator can opt to cherry pick hits from the primary screen for confirmation studies with the primary assay (3 replicates per compound) and further characterization using secondary and counter screens.
Results from assay optimization, mock screens, and pilot screens (if applicable) can be used in the proposal for a large genome-wide siRNA screen or a full library chemical screen (See Access to the HTS Core for Large Screening Projects). Once the HTS Oversight Committee approves a large screening project, the HTS Core will schedule everal experimental runs (25–40 plates per day) for the screen over an 8–12 week period. The scientist from the principal investigator's lab will participate in screen execution while HTS scientists will dispense the library, monitor screen quality, analyze the data, and provide regular reports of screen progress.
At the conclusion of the screen, the HTS Core will provide a summary of the primary data for the screen. For chemical screens, principal investigators can select 1,280 compounds for cherry picking and follow up studies. The Core provides 4 uL of a 5 mM stock solution for each cherry-picked compound. This is more than sufficient to carry out confirmation studies with the primary assay and additional follow-up studies that can include secondary assays and counter screens.
For RNAi screens, the Core will provide the ordering information for commercial siRNA libraries and the investigator can order primary hits that are of interest. The Core can provide follow-up support (e.g. inventory management, assay execution, data analysis, etc.) for siRNA screens, if needed.
One of the keys to our success as a collaborative Core facility has been the tight integration of HTS Core expertise with that in medicinal chemistry, ADME (Absorption, Distribution, Metabolism, and Excretion), and in vivo efficacy. Seven faculty members in the Biochemistry Department enable lead optimization and development in pre-clinical drug discovery programs. Their efforts are supported and enhanced by the Pharmacology Core that carries out ADME, toxicity, and efficacy studies.
The HTS Core participates in early pre-clinical hit-to-lead optimization with these groups as outlined in Figure 1. In this capacity, the HTS Core provides inventory, assay, data analysis, cheminformatics, and computational chemistry support for dose-response studies to help establish structure-activity relationships (SAR) for attractive chemical series that have been identified in chemical library screens. All data captured in each iteration of the lead optimization cycle is uploaded into the UT Southwestern Laboratory Information Management System (LIMS) to ensure the integrity of the data and facilitate data mining, reporting, and decision-making as leads are optimized and characterized. Drs. Anwu Zhou, Prema Latha Malllipeddi, and Hong Chen provide bio- and cheminformatic support for screening and serve as LIMS administrators.
Figure 1. Hit-to-Lead Development Cycle. Roles of the HTS, Chemistry, and Pharmacology Cores in their interactions with project biologists and chemists. The order and priority of the primary, secondary, and cell culture assays may change if the primary HTS assay is cell-based. (Adapted from MacCoss, M. and T.A. Baillie. Organic Chemistry in Drug Discovery. Science, 2004. 303(5665): p. 1810–1813.