The biotechnology and pharmaceutical industries explore a wide range of compounds in hopes of finding new treatments and the next potential “wonder drug.” Given the breadth of conditions and diseases, combined with the range of possible biological targets and potential therapeutics, researchers need fast and efficient methods for finding the right targets. Doing so often involves high-throughput screening (HTS).

The bio-discovery process depends on testing as many candidate compounds and potential leads as possible.

A key to facilitating and maximizing workflow is to work with samples in multiwell plates, and automation can play a big part in the screening process.

Although not new, HTS is continually being improved upon. Therefore, many scientists still trust and rely on the technique. According to Chris Grimley, vice president of marketing at Labcyte, a company that makes acoustic liquid handlers, “HTS is still a key component of the drug discovery process, but in the past decade or so, researchers started using more focused approaches to screening based on in silico knowledge of protein targets or historical data.”

Here we discuss some of the HTS tools and applications used by researchers who are screening biotherapeutics and their targets.

Keys to using kinases

Many applications of HTS for therapeutics involve kinases. “Kinases have to be validated first, if indicated, as a true target that reflects the altered phenotype or plays a pivotal role in a certain pathological condition,” says Said Goueli, senior research fellow and cell signaling group leader at Promega Corporation. “Once validated, screening for modulators of kinase activity is typically the next step in the drug discovery time line.”

Researchers use purified kinase enzymes to screen for kinase modulators. “This is where ADP-Glo is principally used,” Goueli says. “The ADP-Glo Kinase Assay measures ADP, the universal product formed from all kinase reactions.” In this assay, ADP is converted into ATP, which is converted into light by Ultra-Glo Luciferase. “The luminescent signal positively correlates with kinase activity,” Goueli explains. “The ADP-Glo Kinase Assay has a high dynamic range and produces a strong signal at low ATP-to-ADP conversion, making it well suited for screening low-activity kinases, such as growth factor receptor tyrosine kinases.”

This approach also provides accuracy and versatility. “The assay produces minimal false hits and Z´ values of greater than 0.8,” Goueli says. “The ADP-Glo platform can be used in all stages, except clinical trials, of drug discovery programs to search for the next promising drug.” As examples, he points out that ADP-Glo can be used for target validation, monitoring kinase activity during compound library screening and in lead optimization, by using Promega’s ADP-Glo-based Kinase Selectivity Profiling System (KSPS). Profiling is usually carried out to minimize off-target effects of a drug during final stages of its approval by the U.S. Food and Drug Administration.

Advances from interference

The Fraunhofer Institute for Molecular Biology and Applied Ecology IME includes “a fully integrated industrial HTS system that is used in a variety of target and cell-based bio-assays against small-molecule libraries—in 384-well format against up to 500,000 compounds,” says Sheraz Gul, head of assay development and screening. “The HTS system is equipped with two Thermo Fisher Scientific Multidrop Combi dispensers that perform the bulk liquid handling of a variety of reagents.” This includes proteins, cells and detection components. Gul adds, “These liquid handlers are employed to dispense volumes between 5 and 50 microliters in assay plates, although the actual volume depends upon the assay being run.”

This facility also includes multiple stand-alone Thermo Fisher Scientific Multidrop dispensers. These can be used, Gul explains, “to dispense reagents into assay plates off-line when necessary.”

To get the most out of these platforms, the technology must be easy to learn. “We regularly have interns and students in our facility who are not familiar with our HTS system,” Gul explains. “Therefore, some training is necessary before any screening can be performed, and this can delay the initiation of projects, especially those that utilize expensive or precious reagents.” That has not been a problem with the Multidrop platforms, which are “very easy to become familiar with, and our new staff can learn to operate these devices and begin automation of and initiate screening campaigns with relative ease and generate data very soon after beginning any laboratory work,” Gul says.

In fact, Gul and his colleagues have been using this technology to develop filters that detect compounds that often get missed because on interference in an assay. In describing these compounds, Gul says, “Some of these have recently been reported in the literature as pan-assay interference compounds (PAINS), which have [a] tendency to appear as actives in many HTS campaigns, regardless of the target being investigated.” His team has started to find assays that work around the interference.

Acoustic improvements

The method of handling compounds also plays a crucial role in HTS. Labcyte uses high-frequency sound energy to eject 2.5-nl droplets into the wells of a microplate. Larger volume transfers are accommodated by multiple droplets, up to 500 droplets per second. Using this technology, Labcyte’s Echo Liquid Handlers work with various fluid types and reagents; contact-free transfers eliminate the potential for compounds adhering to pipette tips.

Labcyte has a collaboration with multinational pharmaceutical company AstraZeneca that will use acoustic tubes for sample storage. “Storing compounds in tubes has several advantages,” Grimley explains. “For targeted screens, assay-ready plates can be created more quickly since you pull only the compounds of interest from the compound store.” He adds, “Previously, an entire plate would be pulled from a store just to access a few, or even a single compound.”

Acoustic transfer also reduces the amount of compound used. “By going straight from storage tubes into assay-ready plates, the need for intermediate plates is eliminated, which saves time [and] compound and reduces costs,” Grimley says.

Integrating mass spec

A second collaboration between Labcyte and AstraZeneca combines acoustic-loading technology with mass spectrometry (MS). “We’ve been working with AstraZeneca for a couple of years on this project,” Grimley reports. “Screening using mass spectrometry is appealing because it’s label-free, and that greatly reduces the cost and provides more biologically relevant data.”

To date, throughput has been a limiting factor in the adoption of mass spectrometry within HTS. “The existing approaches have limited throughput, resulting in the need for multiple systems running in parallel in order to complete a screen in an acceptable amount of time,” Grimley explains. “The resulting high capital-equipment cost has limited the adoption of the technology.” Acoustic loading using the Labcyte system solves the throughput issue. “We’re running at close to three samples per second, which is an order of magnitude faster—a game changer,” Grimley says.

The RapidFire 365 High-Throughput MS system from Agilent is an alternative offering that integrates automated HTS workflows with MS, enabling label-free detection of a wide array of targets, including lipids, fatty acids, peptides, proteins and oligonucleotides. Agilent’s Benchbot robot handles the transfer of reagents and samples to stacks (up to 63) of multiwell plates. Samples are automatically processed on a solid-phase extraction (SPE) cartridge, and the material is then transferred to one of Agilent’s Triple-Quad, TOF (time-of-flight) or Q-TOF MS systems for analysis.

Expanding acoustic options

At AstraZeneca, scientists started using Labcyte’s acoustic technology in the early 2000s when they experienced an increase in compounds they wanted to screen and required assays that delivered more throughput, according to Grimley. The Echo also enabled the scientists at AstraZeneca to move assays from 384- to 1,536-well plates, while improving the quality of the data.

This technology also saves money. In some cases at AstraZeneca, an Echo platform replaced multiple traditional platforms, which also saves on reagents. “Using the Echo, AstraZeneca saves hundreds of thousands of dollars a year in DMSO, which it previously used in a wash step for tip-based dispensers,” Grimley says.

Now, AstraZeneca uses this technology in assays ranging from biochemical screens to cell-based assays, and the company’s scientists keep expanding the applications. As demonstrated in the collaboration with Labcyte, acoustic dispensing can be used with MS. The acoustic technology generates a droplet spray that goes directly into a mass spectrometer. This raises throughput from 384 to 4,000 samples per hour, and the level is expected to soon reach 6,000 to 7,000.

With continued innovations like these to enhance HTS for compound screening, patients around the world should benefit. 

For now, researchers are benefiting from improvements to reagents and instrumentation that are making HTS more efficient and potentially more sensitive in terms of detecting targets—and enabling faster time to results.