High Throughput Screening

Evolution of the drug discovery process

The discovery of penicillin by Sir Alexander Fleming in 1928 is widely considered to be one of mankind’s greatest medical advances. This antibiotic, produced by the fungal genus Penicillium, is effective against a wide range of Gram-positive bacteria, including the causative agents of pneumonia and septicemia. The outbreak of World War II was a driving force behind mass production of the drug, which is credited with saving the lives of thousands of soldiers who would otherwise have died from wound infections.
The need to produce penicillin on a large scale, and the subsequent evolution of drug resistant bacteria, led to rapid advancements in synthetic organic chemistry during the latter half of the twentieth century. Synthetic methods offered the potential to manufacture drugs for the treatment of specific conditions and, since effective therapeutics were unavailable for many diseases, received significant investment. The drug discovery process has since grown enormously in sophistication and, broadly speaking, is these days divided into a number of distinct phases.

 

The drug discovery process

The drug discovery process

High Throughput Screening (HTS)

The intended target of a drug discovery program could be any one of a vast number of biological entities including, but not limited to, enzymes, receptors, ion channels and nucleic acids. Following target identification and validation, compound screening assays are developed with the aim of identifying chemicals that will interact with the target and which therefore have the potential to influence the disease state. These molecules are referred to as “hits”.

Hit discovery often relies on HTS, the process of testing libraries of tens or hundreds of thousands of compounds for their activity against the target. HTS typically assumes no prior knowledge of which chemotypes might be effective, whereas methods such as focused screening utilize smaller subsets of molecules that are believed to have a high likelihood of activity. HTS assays are typically performed in 384- or 1536-well microtiter plates and rely on the use of automation such as liquid handlers, plate washers, plate stackers and plate readers. Factors which should be considered when running an HTS include:

• Inclusion of relevant positive and negative controls, plus standard curves
• Acceptable signal: background ratio (the assay window)
• Z’ factor (a statistical parameter which takes in to account the assay window, as well as the level of variance around the positive and negative control signals)
• The screening concentration of the test compounds
• Number of replicates
• The DMSO sensitivity of the assay
• False negative and false positive hit rates
• Assay cost

Protein kinases and protein phosphatases

Protein phosphorylation and dephosphorylation, mediated by kinases and phosphatases respectively, control a wide range of cellular functions, and as a result have been the focus of many drug discovery programs. Kinases transfer phosphate groups from ATP to amino acids which have free hydroxyl (-OH) groups (serine, threonine or tyrosine), whereas phosphatases remove phosphate groups from these same amino acids. Phosphorylation and dephosphorylation events produce changes in the function of a target protein, for example by altering its cellular location, its enzyme activity, or its association with other biomolecules. Chemical inhibition of kinases or phosphatases can therefore be used to modulate cellular activities, which in turn can impact on a disease state.

Chemical_inhibition_of_kinase_activity

Chemical inhibition of kinase activity. Grey = enzyme substrate, purple = chemical inhibitor.

HTS assay readouts

A variety of assay readouts can be used for HTS, including the measurement of fluorescent intensity, fluorescence polarization, Förster Resonance Energy Transfer (FRET), colorimetric detection, radiometry, and luminescent detection. Many of these rely on the use of labeled secondary antibodies for detection, however it is often beneficial to instead directly conjugate the primary antibody to the detection moiety. This can reduce background signal, improve data quality through assay simplification, and can save both time and money.

Lightning-Link® antibody labeling

Lightning-Link® from Expedeon is an innovative technology that enables direct labeling of antibodies or proteins. The product range includes kits for labeling antibodies with enzymes or with a wide range of fluorophores, including tandem dyes.

The benefits of Lightning-Link® include:

  • Quick and easy to use
  • Requires only 30 seconds hands-on time
  • No separation steps involved so 100% of the antibody or protein is recovered
  • Possibility to label from as little as 10ug to a gram or more of antibody

Lightning-Link® from Expedeon is an innovative technology that enables direct labeling of antibodies or proteins. The product range includes kits for labeling antibodies with enzymes or with a wide range of fluorophores, including tandem dyes.

Kinase assay using directly conjugated antibody

Use of a directly conjugated antibody to screen for kinase inhibitors. a. Kinase phosphorylation of the target protein, which is captured by a specific antibody bound to a microplate b. indirect detection of the phosphorylated target c. direct detection of the phosphorylated target d. chemical inhibition of the kinase, resulting in no assay signal. Use of a directly conjugated antibody for detection can save time and money, as well as improving data quality through assay simplification and reduction of background signal.

PiColorLock™ Phosphate Detection System

Dyes such as Malachite Green are often used to measure the inorganic phosphate (Pi) which is released by phosphatase activity, however the dye-Pi complexes are prone to precipitation and the reagents are very acidic which can lead to non-enzymatic hydrolysis of many phosphorylated substrates. The PiColorLock™ Phosphate Detection System from Expedeon is a superior product that is designed to measure the activity of any enzyme which generates Pi, including phosphatases, ATPases, GTPases and heat shock proteins. The reagent is compatible with almost any assay buffer, including DMSO, the solvent most commonly used in HTS. PiColorLock™ has a unique accelerator to speed up color development, and a stabilizer to suppress non-enzymatic backgrounds with acid-labile substrates such as ATP and GTP. Furthermore, the reagent has a wide linear range, and color development is not inhibited by high concentrations of protein.

PiColorLock process diagram with inhibition

Principle of the PiColorLock™ Phosphate Detection System. Grey = enzyme substrate, red = chemical inhibitor.

 

In addition to our Lightning-Link® antibody labeling kits and our PiColorLock™ Phosphate Detection System we offer a wide range of products to simplify your assay development and screening. Contact us to find out more!

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Dye-based detection of inorganic phosphate

August 25th, 2017

Application notes

Dyes such as Malachite Green are regularly used to detect inorganic phosphate (Pi), an important inorganic ion in a wide variety of biological functions. In the presence of Pi these reagents change color, and a simple absorbance reading can be employed to measure the amount of Pi which has been released from a substrate.  …

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A-Z Guide to Fluorochromes

August 23rd, 2017

Download a Free Copy   Lightning-Link® fluorescent antibody and protein labeling kits allow you to covalently label any antibody in under 20 minutes with only 30 seconds hands-on time. We currently have over 35 label in our range of fluorescent dyes. This guide provides a general overview of each fluorochrome in our range including their…

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Absorption-Emission Table

August 23rd, 2017

Lightning-Link® is an innovative technology that enables direct labeling of antibodies, proteins, peptides or other biomolecules with only 30 seconds hands-on time.

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Simplifying Antibody Conjugation Process

August 23rd, 2017

Antibodies are widely employed in the quantification of antigens in complex biological samples. Using techniques such as Western blotting, ELISA, and immunohistochemistry researchers are able to measure a single antigen, or perhaps a limited number of antigens, in each sample. In the post-genomics era, advances in multiplex immunoassay technologies now allow scores or even hundreds…

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Antibody Purification Guide

August 23rd, 2017

Expedeon specializes in easy to use bioconjugation kits which enable the direct labeling of antibodies or proteins with enzymes, fluorescent labels, biotin, streptavidin, gold nanoparticles, latex beads or oligonucleotides. Unfortunately, many antibodies are provided in buffers which contain additives that are incompatible with labeling technologies, making puri cation a key consideration prior to carrying out…

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Guide to labeling your primary antibody

August 23rd, 2017

Antibodies are used to detect and quantify antigens in techniques such as flow cytometry, ELISA, western blotting, immunohistochemistry and lateral flow. The antibody that binds to the antigen is called a ‘primary antibody’ and it confers specificity to the assay. Most types of immunoassays also incorporate a ‘label’ whose purpose is to provide measurability. Assays…

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