HTS Paper Draft

7/31/2019 HTS Paper Draft

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UCSD Extension

Course: Pre-Clinical Drug Discovery and Development,BIOL-40125 Spring 2003, Section ID: 039644

Instructor: Mubarack Muthalif

Group Project:

HIGH THROUGHPUT SCREENING INDRUG DISCOVERY

Group Members:

Eric Allen

Candace Crogan-GrundyTatyana NisanJessica RiversShaoxian Sun


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HIGH THROUGHPUT SCREENING IN DRUG DISCOVERY

Eric Allen, Candace Crogan-Grundy, Jessica Rivers, Tatyana Nisan, Shaoxian Sun

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TABLE OF CONTENT

1. INTRODUCTION.....................................................................................................3

2. OVERVIEW OF HIGH THROUGHPUT SCREENING IN DRUG DISCOVERY........4

3. CELL BASED HIGH THROUGHPUT SCREENING................................................6

4. AUTOMATION OF HIGH THROUGHPUT SCREENING.........................................7

4.1 Overview.........................................................................................................7

4.2 Maxim Pharmaceuticals: A Case Study......................................................10

5. WHATS NEXT FOR HIGH THROUGHPUT IN DRUG DISCOVERY?..................12

6. BIBLIOGRAPHY...................................................................................................14


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1. INTRODUCTION

Over the past decade, several scientific advances and economic pressures have

driven the need for improved drug discovery screening technology [1, 2, 3]. Rapid

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

while combinatorial chemical synthesis has generated large numbers of compounds with

the potential for pharmacological activity. 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 100,000-300,000 or more compounds per

screen to produce approximately 100-300 hits [3]. 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.

In parallel with the drive towards higher throughput and miniaturization, there is

increasing emphasis on producing quality leads, not just hits, with the desirable drug-

like properties [3, 4]. Several recent developments have led to new opportunities for the

use of high throughput technologies at numerous stages of the drug discovery process [2,

4].

This paper will examine the current state of HTS in Drug Discovery, with an

emphasis on the cell-based assays and automation. It will also outline the emerging

applications of HTS in the drug discovery and development process.


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2. OVERVIEW OF HIGH THROUGHPUT SCREENING IN DRUG DISCOVERY

A typical HTS system has 7 essential components: targets, assays, compound libraries,

automation, information systems, facilities, and scientists. These are each described next.

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. For example, the

prostaglandin E2 receptor (PGE2) has been implicated in tumor-induced immune

suppression. Inhibition of this receptor appears to slow tumor growth, so PGE2 is a good

potential target [5].

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. While some assays are standard and relatively straightforward, others are

more complex and prone to error. Sometimes entirely new assays must be carefully

designed utilizing new indicator technologies or reaction chemistries. Assay kits may be

purchased from outside vendors also.

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, isnt as important as library diversity, quality,

and management.

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.


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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.

With all the necessary HTS components in place, screening can begin. A diagram of

the interplay between the HTS components is shown below in Figure 1.

Figure 1

Target

Assay

Library

Scientist

Automation

Scholarly

Publications

Financial

Rewards

Information

Systems


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Now that the basics of HTS have been covered, applications to entire cells will now be

discussed.

3. CELL BASED HIGH THROUGHPUT SCREENING

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

compounds. Unlike a cell-free biochemical assay, where only the isolated target protein

is being utilized, cell-based assays can provide pharmacological information on a

compounds penetration through the cell membrane, the effect of a compound on a cells

overall metabolic pathway, and potentially a compounds 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.

However, due to having more variables per assay, and being more complicated to

automate than most biochemical assays, 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 cell-

based HTS assays is increasing. To run a successful cell-based screen, a number of

factors need to be considered, and experiments need to be well-controlled [11]. The first

factor is the selection of the cell line. 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, and stable cell lines are most preferred. 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


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the compound library, such as organic solvents introduced with the compounds. Thus,

cell production requires close monitoring. The third factor in running a successful cell-

based 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. Cell-based screening requires particular

instrumentation and assay plates to meet its special needs. Liquid handling instruments

are required to deliver uniformly viable cells to 96- or 384-well plates. Poly-lysine or

other types of coated plates are preferred to improve the adherence of cells. Controls and

standard compounds are regularly run to monitor the consistency of the cells.

Cell-based assays can provide information that is not available through

biochemical assays. However, it has some drawbacks and data needs to be carefully

interpreted. It can generate false signal if compounds have problems penetrating the cell

membrane, if cytotoxicity effects occur, if the compounds hit off-target, or when

interference of signal readout occurs. The hit compounds from cell-based assays are

normally followed up with confirmatory biochemical assays.

4. AUTOMATION OF HIGH THROUGHPUT SCREENING

4.1 Overview

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 [9]. 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


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small volume pipettor can be used for compound transfer to assay plates, library re-

plating, plate replication, pre-plating (adding liquid to plates before the assay is run,

usually done in bulk), 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 new

technology on the market is a pintool. 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. While the 96/384 pipettor pipettes in one solid block, a 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. There are two main designs for plate movers in

an automated laboratory set up. 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 [10]. Either design allows for more reliable timing plate to plate,

and many plate movers have a built in timer function which means you can run assays


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over weekends, essentially creating a seven day work week. Some robotic arm plate

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

One of the drawbacks of using a track or linear transport system is that you must

have a large or long lab if you want to add peripherals. Stackers or liner systems do tend

to be faster and more reliable than robotic arms, but they can still drop plates. An

advantage to a robotic arm system versus a linear system is that the cluster design of

other instruments around an arm is space saving so you can add more peripherals with

less space needed.

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. The detection needed is based on the

detection reagents used in the assay. There are plate readers available that have all three

reading abilities and can read up to 1536 well plates.

Once the basic equipment is in place, other instruments can be integrated into the

setup, such as an automated incubator, barcode readers, plate sealers, and stackers.

Adding an automated incubator with the ability to interface with the plate movement

robotics is a fairly new concept in cell based HTS. It provides the ability to move plates

from the incubation environment to other devices then return them to the incubator [7].

This is important in cell based assay automation as cells typically require control of

temperature, pH, and humidity in order to maintain viability, and assays often have

required incubation periods [7]. This brings up another issue in cell-based automation,

which is contamination.


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With manual assays, protection from contaminants such as dust and other airborne

particles is often done by simply placing lids on the microplates. Lidding can be

maintained with automation as some robotic arms can pick up and move lidded plates as

well as remove, track, and replace lids during the assay steps. If this is too time

consuming or not practical, protection from contaminants can be accomplished by

enclosing the laboratory automation robotics and other devices within a controlled

environmental chamber such as a laminar flow hood or bio-safety cabinet [7].

These are just the basic instruments and equipment needed for an HTS lab, there

are, however, many different designs and layouts depending on the needs of the lab. Just

as there is no one way to accomplish a cell-based assay, there is no one way to automate

a cell-based assay. Depending on the structure of the experiment, as well as the available

time, space, and budget, it might be best to automate a single small portion, automate

several portions and link them together manually, or automate an entire assay [7].

Robotic systems that provide users with systems that are flexible, scalable, and which

dont go out of date when the users needs change are in high demand. Flexibility is

needed to add or remove components from the system very easily without redesigning the

entire system [8]. Also important in the ever-changing shift toward miniaturization is the

need for scalability.

4.2 Maxim Pharmaceuticals: A Case Study

Maxim Pharmaceuticals HTS laboratory operates a working whole-cell based

screening platform. Their purpose is to screen for inhibitors and inducers of apoptosis or

programmed cell death. The medical justification for inhibitors is their possible use in

preventing unwanted cell death often seen in heart attacks, strokes and degenerative


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diseases such as Alzheimers and Parkinsons Disease. The justification for inducers is

primarily to kill cancer cells or cells that do not have the normal cell death machinery.

Maxim Pharmaceuticals uses a proprietary substrate that is specific for activated caspase.

The screens are performed in 384-well format and are whole cell based. Activated

caspase indicates cell death through apoptosis specifically, not just cell death by necrosis.

They screen compound libraries against numerous cancer cell lines. The research flow at

Maxim is typical of cell based screening elsewhere. It starts with compound inventory

and solubilization, compound transfer and plate replication, cell addition, incubation,

plate reading, and finally data analysis. At Maxim the laboratory layout is a combination

of track and robotic arm systems. Plates usually get reagent added with a small volume

96 channel pipettor, compound is added with a 384 pintool head, cells are added with a

large volume 96 channel pipettor (all these steps are linked to stackers, hence this portion

is considered a linear setup). The plates are manually loaded into an automated

incubator. Once the incubation period is finished, the plates are moved with a robotic

arm from the incubator to a large volume 96 channel pipettor, substrate is added, and the

plates are moved to a fluorescent plate reader, stopping by a barcode reader on the way to

keep track of what data corresponds to what compound. The barcodes are then read and

the plates are moved back (by the robotic arm) to the incubator for further incubation.

Once done incubating a second time, the plates are moved by the arm from the incubator

to the scanner, then to the plate reader for a final read. Once they are done with the final

read, they are essentially trash. A plate sealer has been integrated with the arm so these

plates are sealed for splash-free disposal into a hazardous waste barrel. Once the assay

run is complete the robotic arm uses the pager function to call one of the employees to let


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them know the assay run has finished without problems. Data analysis is done for the

primary screens using a software package to handle the massive quantities of data

generated. The HTS laboratory at Maxim also handles all of the secondary screens such

as Dose Response and Growth Inhibition. The dilutions for these assays are done off-line

using 8 channel pipettors and then the integrated automation is used for the actual assay.

Data from these smaller assays is analyzed using templates in Excel.

5. WHATS NEXT FOR HIGH THROUGHPUT IN DRUG DISCOVERY?

HTS has revolutionized compound screening. However, it has also introduced

new challenges to the drug discovery and development process. Just a small fraction of

primary hits generated by the HTS can easily overwhelm traditional follow-up testing,

therefore creating new bottlenecks in the drug discovery process [4]. Thus, it is now time

to explore the application of the high throughout technologies to those new bottlenecks in

the areas of medicinal chemistry, structural biology, pharmacology, ADME studies, and

toxicology.

Identifying metabolic and pharmacokinetic properties earlier during drug

discovery provides a clear advantage for further drug development. In vitro ADME

studies, such as the use of isolated hepatocytes or liver microsomes to study drug

metabolism, are being adapted to assay formats similar to those currently running on high

throughput platforms with the aid of fluorogenic cytochrome-P450 substrates, and can be

readily configured as high throughput systems [2, 4].

Information-rich assays, such as those based on fluorescence microscopy, can add

significant value to each data point generated by high throughput screens. Sub cellular

responses to pharmacological agents can now be monitored using a combination of


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molecular sensors and automated high-speed scanning microscopes [2]. The latest

instruments operate at rates comparable to those of microtiter-plate readers, thus allowing

for broadening the application of HTS in pharmacological profiling and target validation.

High throughput enabled technologies will be fundamental to more efficient

clinical trials by providing the means to base the studies on well-characterized surrogate

disease-markers in genotype-selected populations. Traditionally, clinical trials enroll

thousand of patients to meet the fairly broad range of subject selection criteria. In

contrast, Genentech obtained FDA approval for herseptin (trastuzumab) a treatment for

metastatic breast cancer using the data from two Phase III studies with less than 700

patients with HER2 protein overexpression [2].

Overall, high throughput technologies have much potential in future drug

development and design: from their immediate promise to shorten the preclinical

development timeline to eventually paving the way to make drugs prescribed or tailor-

made according to the genetic makeup of an individual.


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6. BIBLIOGRAPHY

1. Sundberg, S. High-throughput and ultra-high-throughput screening: solution- and

cell-based approaches. Current Opinion in Biotechnology, 2000, 11, 1, 47-53.

2. Inglese, J.Expanding the HTS paradigm. Drug Discovery Today, 2002, 7, 18, S105-

S106

3. Rubenstein, K. Overcoming the Innovation Deficit. Market Analysis Report. Drug and

Market Development, 4 May 2000.

4. Kariv, I.; Rourick R.A.; Kassel, D.B.; Chung, T.D.Y. Improvement of Hit-to-Lead

Optimization of in Vitro HTS Experimental Models for Early Determination of

Pharmacokinetic Properties. Combinatorial Chemistry and High Throughput

Screening, 2002, 5, 459-472.

5. Yang, Li et al. Cancer-associated immunodeficiency and dendritic cell abnormalities

mediated by the prostaglandin EP2 receptor, The Journal of Clinical Investigation,

March 2003, 111, 5, 727.

6. Devlin, John P. High-Throughput Screening as a Discovery Resource. In Mei et al

(Ed.), Integrated Drug Discovery Technologies. (pp. 365 394). New York, NY:

Marcel Dekker, Inc.

7. Hudson Cell Based Assay Automation

http://www.hudsoncontrol.com/products/cell_based_assay_automation.htm

8. Dera, Skip. Modular Systems Gear Toward Automation. Drug Discovery &

Development, 2002, 5, 8, 59-65.

9. Lesney, Mark S. Juggling the Mix. Modern Drug Discovery, January 2002, 26-31.


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10.Hamilton, S.D., Kramer, G.W., and Russo, M.F. An Introduction to Laboratory

Automation, Lab Automation 2002.

11.Johnston P. A. & Johnston, P. A. Cellular platforms for HTS: three case studies.

Drug discovery today, 2002, 7, 353-363.

Documents

HTS Paper Draft