HIGH THROUGHPUT SCREENING

INTRODUCTION

Over the past decade, several scientific advances and economic pressures have driven the need for improved drug discovery screening technology

Rapid progress in genomics has greatly expanded the number of potential therapeutic targets.

Combinatorial chemical synthesis has generated large numbers of compounds with the potential for pharmacological activity. H

High Throughput Screening (HTS) emerged as a way to screen these large chemical libraries against multiple targets while simultaneously reducing development and operating costs.

In terms of definition, HTS can be considered the process in which batches of compounds are tested for binding or biological activity against target molecules.

Today, many pharmaceutical companies are screening 10

Today, many pharmaceutical companies are screening 100,000-300,000 or more compounds perscreen to produce approximately 100-300 hits.

This is achieved using automated, robust miniaturized assays. The use of 384-well and higher density plates and commercially available plate-handling robotics has made HTS a reality

Several recent developments have led to new opportunities for the use of high throughput technologies at numerous stages of the drug discovery process.

• A typical HTS system has 7 essential components: targets, assays, compound libraries, automation, information systems, facilities, and scientists

TARGET

A target (or group of targets) is a substance that appears to be pivotal in a biological process, such that inhibition of its activity would produce a benefit

Inhibition of this receptor appears to slow tumor growth, so PGE2 is a good potential target

ASSAYS

An assay (or group of assays) displays whether a test compound (or antibody, etc.) has biological activity with the target.

This typically uses fluorescence or radioactivity as the indicator

Sometimes entirely new assays must be carefully designed utilizing new indicator technologies or reaction chemistries.

COMPOUND LIBRARIES

A library of compounds (or antibodies, etc.) ideally displays a wide range of structural diversity.

Sources of diversity can be compound collections from several outside vendors, natural compounds, and in-house combinatorial chemistry resources.

Library size, which can be in the millions, isn’t as important as library diversity, quality, and management.

AUTOMATION

Automation of the screening process is a necessity to complete a large number of assays in a reasonable amount of time.

This usually includes a miniaturization scheme, liquid handling robotics and vessels, a detection system, and raw data creation.

Information systems, facilities, scientists

Information systems are necessary for management of the process and data handling. This includes resource scheduling, assay tracking, compound management, and data collection.

Suitable facilities for the system are also an absolute necessity. This includes adequate lab space and environmental control

Finally, Scientists are needed to design the experiments, run and manage the system, and interpret the results

Cell based HTS

Cell-based assays use intact cells to measure the biological response stimulated by compounds.

Cell-based assays can provide pharmacological information on a compound’s penetration through the cell membrane, the effect of a compound on a cell’s overall metabolic pathway, and potentially a compound’s toxicity and ADME characteristics

For poorly defined or difficult to isolate targets, cell-based assays are becoming a practical alternative to biochemical approaches for screening compounds.

Cell-based assays have traditionally been used for secondary screening after hit compounds are identified in the first round of (primary) screening.

With recent advances in screening technology and automation, the number of cellbased HTS assays is increasing.

The cell line needs to be physiologically relevant and able to be produced in large quantities for HTS. Generally, cells are transformed to express the target of interest.

The second factor is the maintenance of consistent cell behavior to generate reproducible and reliable signal.

The behavior of cell lines in a HTS assay is dependent on many characteristics of the cell culture, including the media components such as antibiotics, as well as characteristics of the compound library, such as organic solvents introduced with the compounds

The third factor in running a successful cellbased screen is careful control of experimental conditions such as temperature, humidity, etc.

Variation in the conditions affecting cell growth can generate signal drift.

Other experimental condition considerations include compound concentration and incubation time.

In addition, compound cytotoxicity, which is compound concentration dependent, can result in false negatives or positives

AUTOMATION OF HIGH THROUGHPUT SCREENING

To start a basic automated cell-based HTS lab, some standard equipment is required.

At the minimum, a working HTS lab should have some sort of automation for the most repetitive tasks such as pipetting

A robotic liquid handler can be 96 or 384 channel, large or small volume, and its use adds to the speed and accuracy of assays.

The small volume pipettor can be used for compound transfer to assay plate, plate replication, pre-plating, and dilution.

The large volume pipettor is primarily used for cell addition and substrate addition to assay plates but can also be used for the same applications as the small volume pipettor if greater volumes are needed.

A pintool is retrofitted into an automated pipettor and usually has 384 pins with slots ground out of them.

The groove size and depth determines the amount of liquid it can pick up and transfer.

Using a pintool is a cost effective way to transfer 384 samples at a time. It is mostly used for compound transfer to assay plates

Another useful addition to an automated HTS arsenal is a 4-8 channel independent pipettor

4-8 channel pipettor allows for independent movement of individual tips.

This can be used for automating very labor intensive tasks such as hit-picking (selecting individual compounds for further testing out of the tens of thousands originally screened), serial dilutions for dose response testing, and even reagent addition to individual tubes to solubilize compounds for testing

Another extremely valuable addition to an HTS lab (which yields a greater walk away time) is some form of plate mover.

The track or linear non-robotic transport moves plates within a workstation while the robotic arm transport allows plates to be moved from one workstation to another.

Some robotic arm plate movers even have pager functions to let you know that they have finished and all is well.

Finally, a plate reader is essential because you need to be able to read your results.

Plate readers can read results in fluorescence, luminescence, or absorbance depending on the output needed for the assay