High Throughput Screening (HTS)

Cancer Center Support for HTS experiments

The cost of an HTS experiment is substantial.  For approved projects, The Simmons Cancer Center HTS core laboratory will provide access to the UTSW compound collection and/or to genomic siRNA resources. The core will provide the following without charge:

• compounds or siRNAs
• access to equipment needed for HTS
• aid in conducting HTS experiments
• data processing and analysis

Laboratories conducting HTS experiments must dedicate at least one scientist to conduct the experiments and must provide all reagents needed. The HTS Core assists investigators in identifying reagent sources.  The Simmons Cancer Center will cover one half the reagent costs for approved projects to be conducted by Cancer Center Members.

Project Approval

Cancer Center members propose screening projects to the HTS Core to be evaluated by the Cancer Center HTS Oversight Committee for feasibility and relevance to cancer. The Oversight Committee decides which projects receive Cancer Center support and provides advice on the design of the primary assay as well as for secondary assays. Approval from the Oversight Committee allows funding for assay development and validation. Once the Committee receives data from the HTS Core Laboratory demonstrating good screening statistics, the project will be approved for support for HTS from the Comprehensive Cancer Center.  Instructions on how to apply for project support can be found here.

The Cancer Center HTS Oversight Committee

Steven McKnight, Ph.D., Professor and Chairman of Biochemistry

John Minna, M.D., Professor of Internal Medicine, Director, Hamon Center for Therapeutic Oncology Research

Lawrence Lum, Ph.D., Assistant Professor of Cell Biology

Michael Roth, Ph.D., Professor and Vice Chairman of Biochemistry

Michael White, Ph.D., Associate Professor of Cell Biology

Experiments are judged by the committee for:

• relevant to cancer
• importance and scientific interest of the project goals
• probability that proposed assays can identify compounds or genes of interest

Target assessment and assay evaluation

Both the practicality and the scientific and therapeutic value of targets for screening are assessed by the Cancer Center HTS Oversight Committee. The HTS Core staff will then assess the cost and practicality of the proposed assay for HTS. This includes aspects such as the number of liquid handling and wash steps, timing, reagent volumes, and necessary controls. For advice about these aspects in preparation for applying for Cancer Center Support, contact Michael Roth, Ph.D.

HTS assay development and testing

HTS staff work with the initiating laboratory to develop the proposed assay into a form suitable for HTS. Compound screens are run in 384 well plates without replicates. siRNA experiments are typically run in triplicate in 96-well plates.

For reasons of cost and reliability, assays must be end-point assays and simplified to minimize liquid handling steps.

The assay signal to noise ratio must be optimized to produce statistics indicating that a compound or siRNA with the desired activity can be reliably distinguished from compounds or siRNAs lacking activity in the assay. No experiment will have access to compound or genomic siRNA resources unless the assay reproducibly achieves the desired signal to noise statistics.

HTS experiments require controls on each plate both for quality control and for data normalization. Both positive and negative controls must be optimized.

The HTS laboratory works to minimize the cost of the experiment, which often requires changing reagents and sometimes completely redesigning the assay.

HTS screening of chemical compounds or siRNA libraries

Once an assay has an acceptable signal to noise ratio in small scale experiments, it is tested in 10 to 20 plates treated with DMSO to determine reproducibility under conditions similar to HTS screening.

Assays that are reproducible are then screened against a test library of 8,000 compounds at 5 µM concentration to determine the “hit” rate.

Assays that pass these evaluations are then screened at rates between 8,000 to 19,000 compounds per day, depending upon the assay type. Cancer Center members are required to supply a scientist to conduct the HTS screen. This person will be aided the HTS staff, who operate the robotic liquid handlers and perform all operations involving the dispensing of compounds from library plates to experimental plates.

siRNA experiments are developed in a similar manner, except that they are run in 96 well format in replicate plates and no test library is used before the genomic screen is begun. Just as with compounds, siRNA experimental controls must achieve a reproducible signal to background ratio in runs of multiple plates before the project is allowed to screen the genomic library.

Only complete genomic screens of the siRNA library are allowed.

Data analysis and storage

HTS experiments generate a large data set in which the link between well location, content, and experimental value must be rigorously maintained. HTS staff are responsible for this, as well as for monitoring daily quality control and flagging plates with unacceptable control statistics that must be repeated. The HTS Core processes screening data and adds it to the Laboratory’s screening database, which connects compound or siRNA identity to activity in each assay run by the lab.

The HTS laboratory provides screening data in Excel files to the initiating investigators and, at the conclusion of the primary HTS experiment, aids in defining compounds or siRNAs of interest.

“Hit” validation

The HTS laboratory assembles a “hit collection” of compounds or siRNAs that had activity in the assay exceeding a predetermined value, typically 3 standard deviations or more from the mean of the experimental population.

The “hit collection” is screened again in the primary assay and compounds or siRNAs that show repeated activity are then subjected to additional secondary assays that can be run in multi-well format with the limited amount of compound or siRNA available.

Based upon the results of secondary screens, lack of activity in other HTS experiments run by the Core (screening history) and other criteria such as compound structure, a small number of compounds or siRNA oligonucleotides are purchased for continued investigation in the initiating laboratory. This is a critically important step in the HTS process. Because subsequent experiments are time consuming and expensive, very few compounds or siRNAs can be subjected to more intense analysis. The typical compound set chosen for follow up is 10 to 20 from an initial hit collection of several thousand.

For siRNA experiments, one can choose to select siRNAs of interest out of a second genomic library at a cost of $5000 for each experiment (see library descriptions).

Once compounds of interest are purchased for additional experimentation, the purity and identity of the compounds can be determined by the Medicinal Chemistry Core in the Biochemistry Department on a fee for service basis (contact Douglas Frantz, Ph.D.). In cases where compounds are no longer available commercially, commercial suppliers will usually contract to synthesize the compounds. The Medicinal Chemistry core has the capability to synthesize a limited number of compounds each year.

Compound Libraries

The HTS Core laboratory has multiple copies of 200,000 small, drug-like compounds dissolved in DMSO and arrayed in 384 well plates. The compounds were purchased from major suppliers to the pharmaceutical industry with a small but growing number of compounds synthesized at UT Southwestern. Compounds were selected for being able to pass 48 structure-based filters that identified undesirable characteristics as well as for satisfying a relaxed set of Lipinski’s rules for good bioavailability¹. Those that passed and were purchased represented the desirable structural diversity available from the companies shown below as of November 2004 (for Comgenex, 2005).

Genomic siRNA Libraries

The HTS core manages a human genomic library from Dharmacon and Drosophila genomic RNAi library from Ambion that were purchased by UT Southwestern and are available free of charge to UT Southwestern investigators who propose experiments acceptable to an Oversight Committee (contact Michael Roth, Ph.D., for information). The human genomic library is composed of pools of 4 double-stranded siRNA oligos specific for each of 21,125 human genes arrayed in 96 well plates. The HTS Core laboratory also manages smaller quantities of a similar human genomic library purchased from QIAGEN by the NCI program project led by Steve McKnight, Ph.D. This second library is used to assemble smaller sets of siRNAs to validate hits from primary screens of the Dharmacon library. It is available to Cancer Center investigators outside the PPG at a cost of $5000 per experiment. In addition to being composed of pools of siRNA sequences for each gene distinct from those comprising the Dharmacon library, the QIAGEN library is the only cost effective way to validate a collection of several hundred potential hits resulting from a genomic screen. Currently individual pools of siRNAs targeting a gene cost ~$100 per pool from either Dharmacon or QIAGEN, making validation of the entire set of “hits” from a genomic screen prohibitively expensive if they are purchased individually. Use this link for information on ordering oligos from Dharmacon. All libraries are kept in multiple copies in -80 °C or -20°C freezers within the 1900 sq ft HTS laboratory.

HTS Laboratory

Location:  L4.182 & L4.184

Equipment

The HTS laboratory has all of the equipment required for HTS. The laboratory operates two Beckman FX liquid handlers, a Beckman SPAN-8 liquid handler for cherry picking, 8 Multidrop instruments for dispensing reagents into plates, two plate washers, two plate sealers, a CLIPR luminescence plate reader and an EnVison multimode plate reader, a Coulter Cell Counter and all necessary cell culture equipment. The laboratory has access to a Beckman IC-100 Image Cytometer for microscope based screening of individual plates.

Personnel:

Shugang Wei, Ph.D.

Olga Jeter, M.S.

Mridula Vishwanath, M.S.

Weihua Hao, Ph.D.

Screening Capacity

The HTS Laboratory has conducted two compound screens and two genomic siRNA screens simultaneously, representing the current maximum daily screening capacity of the laboratory. Individual experiments differ considerably in throughput, depending upon assay complexity. Typical screening rates are 25 to 40 plates per day for cell based compound screens (8,000 to 12,800 compounds) and 10 siRNA source plates containing 800 gene specific sequences for RNAi. The latter experiments usually require comparing two conditions in triplicate (60 plates per day). At these rates HTS of either compounds or siRNAs requires 5 weeks for primary screening and one to two weeks for repeating failed plates, cherry picking, and hit validation.

In 2006 the HTS lab completed 5 compound screens and 7 genomic siRNA screens, with two additional compound screens in progress at year’s end. Typically, 2 to 4 assays are being developed for HTS at any time, with some of these not proceeding to full HTS for lack of funding. The laboratory currently could conduct as many as 20 compound or siRNA experiments per year. Lack of funds for HTS experiments rather than limitations of infrastructure is the major factor restraining the growth of such experiments at UT Southwestern.

Screening Costs

A major activity of the HTS lab during assay development is to minimize screening costs by limiting the cost of reagents. The most common compound or siRNA assay run by the laboratory is cell-based with a luciferase reporter. Reagent costs for HTS experiments conducted in 2005-2006 ranged between $0.25 and $0.15/well with the lower figure the target for assay development. A screen of the full compound library usually requires 700 plates, including those for repeating failed plates and assay development. Plate and liquid handler tips are approximately $7,000-$10,000/screen and regent costs at $0.15 per well are approximately $40,000. Screens of the human genomic library typically require $25,000 in reagent costs. The major costs for the experiments, the expensive equipment, the cost of acquiring and formatting the compound and siRNA libraries and staff salaries are shared by the NCI program project, the Cancer Center and UT Southwestern.

Project Approval Application

Projects proposed for funding through the Simmons Comprehensive Cancer Center must provide the following information.

• 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

Send this information as a word document or PDF by email to michael.roth@utsouthwestern.edu for distribution to the Oversight Committee.

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