High-Throughput Screening Assays for Accelerated Drug Discovery

This article is sponsored by BellBrook Labs and INTEGRA BIOSCIENCES.

High-throughput screening emerged in the late 1980s, driven by advances in chemistry and genomics. With recent breakthroughs in automation and robotics, high-throughput screening is now a mainstay in early-stage drug development, allowing researchers to evaluate many thousands of drug candidates in relatively short time frames. In this article, discover what high-throughput assays are, some of the key types of high-throughput assays, the technological advances that led to their development, and nascent methods in this field.

A laboratory robotics platform pipetting compounds onto a 96-well plate. — High-throughput screening relies on robotics and automation, reducing experimental costs and hands-on time, and increasing speed.
©iStock, Hris

What Are High-Throughput Screening Assays?

High-throughput screening assays are methods that harness the power of automation to test thousands of chemical compounds or biological structures simultaneously.¹ Scientists commonly use these assays in early-stage drug discovery to identify potential targets, validate leads, and test downstream toxicology.

For example, pharmaceutical companies can perform high-throughput screening to narrow down a library of thousands of different chemical compounds against a human cancer cell line in the search for an active target. High-throughput screening assays accelerate target identification and validation, reducing costs and minimizing risks in drug development.

High-Throughput Screening Assays: Types and Methods

High-throughput assays can be broadly categorized into target-based or phenotypic screens. In target-based assays, researchers use cells expressing a known molecular target, such as a pathogenic protein or a cell surface receptor, and screen a library of compounds to identify a drug candidate that acts on the desired protein. This type of high-throughput screening typically uses purified target proteins and in vitro biochemical methods to measure the specific activity of chemical compounds in a library.²

Target-based versus phenotypic drug discovery: Target-based drug discovery begins with targeted assays that identify leads for a known drug target. Phenotypic drug discovery begins with phenotypic assays performed on a disease model to identify a lead before identifying a target. In both cases, lead molecules proceed through optimization and, if successful, pre-clinical and clinical trials. — High-throughput assays are often categorized as target-based or phenotypic screens.
Modified from © istock.com, Pikovit44, grafikazpazurem, Yevheniia Bunha, Irfan Setiawan; designed by erin lemieux

In contrast, scientists use phenotypic screening assays when they do not have a known molecular target. Cell-based assays for high-throughput screening help researchers select chemical compounds based on their effects on the cell phenotype or function. For this type of screening, researchers grow cells in a monolayer within multi-well plates, apply compounds to each well, and assess biological effects such as cell death, or reduced production of a pathological protein. Functional readouts from phenotypic, cell-based assays provide scientists with detailed information that they cannot gain from biochemical assays.³

Förster/fluorescence resonance energy transfer

Förster resonance energy transfer assays, otherwise known as fluorescence resonance energy transfer (FRET) assays, are biochemical screening methods that measure fluorescence as a readout. They use two fluorescent molecules, a donor and an acceptor. When the donor fluorophore absorbs light within the correct range, the molecule becomes excited and emits fluorescent light.⁴ If the acceptor fluorophore is in close proximity (<10 nanometers), energy transfer from the donor will cause the acceptor to fluoresce as well.⁴

Scientists often use this type of high-throughput screening method to study processes and interactions within cells that change the molecular proximity of the donor and acceptor, such as a chemical ligand binding to its receptor, the localization of proteins within cells, or protein-protein interactions. For example, they might engineer a cell line to express a fluorescently labeled disease-associated surface receptor and use a FRET assay to rapidly assess if any labeled compounds from a large library bind to that receptor. While versatile, FRET assays are only suitable when the targets are within 10 nanometres, and the use of an external light source to excite the donor means they have a low signal-to-background ratio.

Luminescence

Luminescence assays, including chemiluminescence and bioluminescence, measure the light produced during chemical and enzymatic reactions.⁵ Because they do not require excitation by an external light source, luminescence assays lack several of the disadvantages of FRET, such as high background noise and low sensitivity with increasing molecular distance

Scientists can use luminescence in a range of different high-throughput screening applications, including several cell-based assays. For example, by measuring luminescence in an ATP-dependent reaction within cancer cells, scientists can determine cell viability after drug exposure and identify which drug or compound kills the cancer cells.³ Researchers can also use luminescence in reporter gene assays by inserting a gene encoding a luciferase enzyme downstream of a gene of interest, measuring the luminescent signal as an indicator of gene expression.⁵

High-Throughput Assay Development and Technological Advances

High-throughput screening assays rely heavily on advanced robotics and automation. Scientists can employ a range of multi-well plate formats, from 96 to 1536 wells per plate, using microplate readers to measure the fluorescence, luminescence, or other readouts across all wells of the plate simultaneously. This allows researchers to screen many thousands of compounds per day.²

In recent years, high-content screening has gained popularity. Unlike high-throughput screening methods, which measure a single readout per well of a multi-well plate, high-content screening is an image-based, phenotypic screening method that gathers multi-parametric data from each well.⁶ By combining automated high-throughput microscopy, multiplexed fluorescence, and quantitative data analysis, high-content screening allows researchers to measure complex phenotypic changes, such as cell viability, morphology, and structure, in parallel.⁷ High-content screening assays have also been developed for the study of stem-cell-derived organoids and other three-dimensional cell cultures.⁸

Using next-generation sequencing technology, researchers can incorporate multiomics analysis into high-throughput screens, for example, to explore the links between genotype and phenotype.⁹ Advances in microfluidics technology have also improved the efficiency of high-throughput screening assays; by reducing reaction volumes and increasing speed, microfluidics methods enable scientists to perform more multiplexed, ultra-high-throughput screening.¹⁰

Scientists have further accelerated drug development by integrating artificial intelligence and machine learning into high-throughput screening methods. Using machine learning to guide high-throughput screening by predicting outcomes, or simplifying downstream analysis from high-content screens, researchers can reduce experimental costs and improve efficiency.¹¹

Future High-Throughput Assay Advances

As technology continues to advance, researchers will be able to increase multiplexing capabilities and gather more detailed data from both target-based and phenotypic high-throughput screening assays, improving the efficiency and speed of drug development.

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