High Throughput Screening - Methods, Protocols [Methods In M.pdf - Biochemia - majsterek666

Methods in Molecular Biology TM

VOLUME 190

High

Throughput

Screening

Methods and Protocols

Edited by

William P. Janzen

HUMANA PRESS

Methods in Molecular Biology TM

High

Throughput

Screening

Methods and Protocols

Edited by

William P. Janzen

HUMANA PRESS

High Throughput Screening Assays

Design and Implementation

of High Throughput Screening Assays

Ricardo Macarrón and Robert P. Hertzberg

1. Introduction

In most pharmaceutical and biotechnology companies, high throughput

screening (HTS) is a central function in the drug-discovery process. This has

resulted from the fact that there are increasing numbers of validated therapeu-

tic targets being discovered through advances in human genomics, and increas-

ing numbers of chemical compounds being produced through high-throughput

chemistry initiatives. Many large companies study 100 targets or more each

year, and in order to progress these targets, lead compounds must be found.

Increasingly, pharmaceutical companies are relying on HTS as the primary

engine driving lead discovery.

The HTS process is a subset of the drug discovery process and can be described

as the phase from Target to Lead. This phase can be broken down in the follow-

ing steps:

Target Choice

⇓

Reagent Procurement

⇔

Assay Development and Validation

⇓

HTS Implementation

⇓

Data Capture, Storage and Analysis

⇓

Leads

It is critically important to align the target choice and assay method to ensure

that a biologically relevant and robust screen is configured. Every screening

Screening Collections

⇒

From: Methods in Molecular Biology, vol. 190: High Throughput Screening: Methods and Protocols

Edited by: W. P. Janzen © Humana Press Inc., Totowa, NJ

Macarrón and Hertzberg

laboratory can relate stories of assays being delivered that are incompatible

with modern robotic screening instruments and unacceptable in terms of signal

to background or variability. To avoid this problem, organizations must ensure

that communication between therapeutic departments, assay-development

groups, and screening scientists occurs early, as soon as the target is chosen,

and throughout the assay-development phase.

Reagent procurement is often a major bottleneck in the HTS process. This

can delay the early phases of assay development, e.g., when active protein

cannot be obtained, and also delay HTS implementation if scale-up of protein

or cells fails to produce sufficient reagent to run the full screen. For efficient

HTS operation, there must be sufficient reagent available to run the entire

screening campaign before production HTS can start. Otherwise, the campaign

will need to stop halfway through and the screening robots will have to be

reconfigured for other work. Careful scheduling between reagent procurement

departments and HTS functions is critical to ensure optimum use of robotics and

personnel. To improve scheduling, modern HTS laboratories are moving toward

a supply-chain model similar to that used in industrial factories.

Successful HTS implementation is multidisciplinary and requires close

alignment of personnel maintaining and distributing screening collections,

technology specialists responsible for setting up and supporting HTS automa-

tion, biologists and biochemists with knowledge of assay methodology, infor-

mation technology (IT) personnel capable of collecting and analyzing large

data sets, and chemists capable of examining screening hits to look for patterns

that define lead series. Through the marriage of these diverse specialties, thera-

peutic targets can be put through the lead discovery engine called HTS and

lead compounds will emerge.

2. Choice of Therapeutic Target

While disease relevance should be the main driver when choosing a thera-

peutic target, one should also consider factors important to the HTS process.

These factors are technical, i.e. whether a statistically robust and sufficiently

simple assay can be configured, as well as chemical. Chemical considerations

relate to the probability that compounds capable of producing the therapeuti-

cally relevant effect against a specific target are: 1) present in the screening

collection, 2) can be found through screening, and 3) have drug-like physico-

chemical properties.

Years of experience in HTS within the industry have suggested that certain

targets are more ‘chemically tractable’ than others. Recent studies of top-selling

prescription drugs have shown that G-protein coupled receptors (GPCRs), ion

channels, nuclear hormone receptors and proteases are among the most exploit-

High Throughput Screening Assays

able target classes, i.e., drugs against these targets produce the highest sales.

Among these targets, GPCRs are normally thought of as the most chemically

tractable, since there are more GPCR drugs on the market than drugs for any

other target class. Furthermore, evidence indicates that HTS campaigns against

GPCRs produce lead compounds at a higher rate than many other target classes

(1) . Kinases are another chemically tractable class that often affords lead com-

pounds from screening ( see Chapter 4); however, while many kinase inhibitors

are in clinical trials, none have yet reached the market.

On the other side of the spectrum, targets that work via protein-protein inter-

actions have a lower probability of being successful in HTS campaigns. One

reason for this is the fact that compound libraries often do not contain com-

pounds of sufficient size and complexity to disrupt the large surface of protein-

protein interaction that is encountered in these targets. Natural products are

one avenue that may be fruitful against protein-protein targets, since these com-

pounds are often larger and more complex than those in traditional chemical

libraries ( see Chapter 9). The challenge for these targets is finding compounds

that have the desired inhibitory effect and also contain drug-like properties

(e.g., are not too large in molecular weight). Recently, several groups have

begun to tackle this problem by screening for small fragments that inhibit the inter-

action and joining them together to produce moderate-sized potent inhibitors.

Certain subsets of protein-protein interaction targets have been successful

from an HTS point of view. For example, chemokines receptors are technically

a protein-protein interaction (within the GPCR class) and there are several

examples of successful lead compounds for targets in this class (2) . Similarly,

certain integrin receptors that rely on small epitopes (i.e., RGD sequences)

have also been successful at producing lead compounds (3) . There may be

other classes of tractable protein-protein interactions that remain undiscovered

due to limitations in compound libraries.

Based on the thinking that chemically tractable targets are easier to inhibit,

most pharmaceutical companies have concentrated much of their effort on these

targets and diminished work on more difficult targets. While this approach

makes sense from a cost-vs-benefit point of view, one should be careful not to

eliminate entirely target classes that would otherwise be extremely attractive

from a biological point of view. Otherwise, the prophecy of chemical tractabil-

ity will be self-fulfilled, since today’s compound collections will not expand

into new regions and we will never find leads for more difficult biologically

relevant targets. There is clearly an important need for enhancing collections

by filling holes that chemical history has left open. The challenge is filling

these holes with drug-like compounds that are different from the traditional

pharmacophores of the past.

Macarrón and Hertzberg

A second and equally important factor to consider when choosing targets is

the technical probability of developing a robust and high-quality screening assay.

The impact of new assay technologies has made this less important, since there

are now many good assay methods available for a wide variety of target types

( see Subheading 3. ). Nevertheless, some targets are more technically difficult

than others. Of the target types mentioned earlier, GPCRs, kinases, proteases,

nuclear hormone receptors, and protein-protein interactions are often relatively

easy to establish screens for. Ion channels are more difficult, although new tech-

nologies are being developed that make these more approachable from an HTS

point of view (4) . Enzymes other than kinases and proteases must be considered on

a case-by-case basis depending on the nature of the substrates involved.

Reagent procurement is also a factor to consider, obtaining sufficient reagents

for the screening campaign can sometimes be time-consuming, expensive, and

unpredictable. In the case of protein target, this depends on the ease with which

the particular protein(s) can be expressed and purified; the amount of protein

needed per screening test; and the commercial cost of any substrates, ligands,

or consumables.

All of these factors must be considered on a case-by-case basis and should

be evaluated at the beginning of a Target-to-Lead effort before making a choice

to go forward. Working on an expensive and technically difficult target must

be balanced against the degree of validation and biological relevance. While

the perfect target is chemically tractable, technically easy, inexpensive, fully

validated, and biologically relevant, such targets are rare. The goal is to work

on a portfolio that spreads the risk among these factors and balances the avail-

able resources.

3. Choice of Assay Method

There are usually several ways of looking for hits of any given target. The

first and major choice to make is between a biochemical or a cell-based assay

( see Chapter 6). By biochemical we understand an assay developed to look for

compounds that interact with an isolated target in an artificial environment.

This has been the most popular approach in the early 1990s, the decade in

which HTS became a mature and central area of drug discovery. This bias

toward biochemical assays for HTS is partly driven by the fact that cell-based

assays are often more difficult to run in high throughput. However, recent

advances in technology and instrumentation for cell-based assays have occurred

over the past few years. Among these is the emergence of HTS-compatible

technology to measure GPCR (5) and ion channel function (4) , confocal imag-

ing platforms for rapid cellular and subcellular imaging, and the continued

development of reporter-gene technology.

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