Lab Automation Supplement
After a sometimes painful period of trial and error, high-throughput screeners have settled on a few winning classes of targets, with a special emphasis on kinases.
About four billion years ago, Earth’s first high-throughput screening program got underway. It required no automation, because the assay was so brutally simple—any result that couldn’t reproduce itself just vanished. After a few billion years of testing, the campaign finally yielded a blockbuster: the metazoan body plan. Since then, most of the effort’s products have been follow-ons.
While biologists seldom think of the history of life in drug development terms, the analogy helps put the current state of high-throughput screening in perspective. After years of expensive, often unsuccessful campaigns, screeners are now starting to focus on a relatively small set of target classes that seem to be the most promising. Among these favored targets, kinases look set to become superstars, feeding a Cambrian explosion of new products.
“The pipeline in the pharmaceutical industry is drying up, and there are not many new drugs coming out except . . . the kinase inhibitors,” says Said Goueli, PhD, a lead research and development scientist at Promega, Madison, Wis. Goueli adds that the dozen or so kinase inhibitors currently on the drug market generate a combined total of nearly $15 billion in revenue for the industry. While basic scientists are adopting high-throughput techniques with somewhat different goals (see sidebar), the success of so many kinase-inhibiting drugs is prompting more and more industry screeners to train their sights on these enzymes.
Kinase targeting also addresses an urgent need, as these enzymes underlie many forms of cancer. Experts estimate that up to a third of all high-throughput screening programs now focus on finding kinase inhibitors, and more than 100 such drugs are currently in clinical trials. Many of the resulting products will end up hitting the same targets, but in oncology, these follow-ons could serve an important clinical purpose; tumors that respond well to one drug initially may become resistant to it during a later relapse.
Finding your assay
The centerpiece of any screening campaign is a reliable assay, and as kinase screening has mushroomed, researchers have gravitated toward assays that work for multiple targets. “A hallmark of kinase screening five to 10 years ago is they used five different techniques on five different kinases, because that’s the assay that was developed for that particular kinase,” says Mike Curtain, a senior product manager at Promega.
More recently, screening teams have been favoring general purpose assays that sense changes in ATP or ADP levels, rather than the phosphorylation of specific substrates. These newer assays are useful for screening inhibitors against a wide range of kinases, helping to make results comparable from one screen to the next.
Of course, reagent-makers quickly seized the opportunity to refine the general purpose assays. Promega, for example, decided to build on an established strategy of using the activity of a firefly luciferase enzyme as a biomarker for ATP concentration. Luciferase consumes ATP to generate light, so the assay’s bioluminescence declines predictably as kinases in a reaction compete for the ATP.
The company’s KinaseGlo system uses a genetically-engineered luciferase that is more chemical-resistant than the natural enzyme, making it less likely that a test compound will inhibit the luciferase and produce a false hit. “In addition, KinaseGlo is luminescent, so you don’t have to worry about fluorescent background,” says Curtain.
However, users still need to keep the system’s limitations in mind. The engineered luciferase has a linear response only at ATP concentrations around 10mM, which is appropriate for screening compounds that inhibit ATP binding to a kinase. To cater to the growing number of screeners testing compounds at higher ATP concentrations, the company recently introduced a “Plus” version of the enzyme that is linear up to 100mM, and Curtain says an even broader-range version, for reactions with up to 500mM ATP, should hit the market by the end of 2007.
Before worrying about ATP concentrations, though, researchers contemplating a kinase-screening campaign need to choose an overall strategy. “There are two different roads you can go down in looking into kinase activity and kinase profiling, and [they use] completely different sets of technologies,” says Anna Christensen, PhD, marketing manager for kinases at DiscoveRx, Fremont, Calif. This initial decision, Christensen says, is between a biochemical strategy that uses purified kinases and a cell-based strategy that measures biological activity directly.
Cell-based assays have much lower throughput than biochemical ones, but they can provide an early indication of a kinase inhibitor’s specificity. Indeed, many of the kinase inhibitors now on the market and in clinical trials were developed against specific kinases in biochemical assays, but later work has revealed that in vivo, they actually inhibit multiple kinases. While this broad-spectrum activity may explain some of the efficacy of the current crop of drugs, most drug developers would prefer to hit their targets more precisely.
To improve specificity, many screening campaigns are starting to cross-check their results with different assays. “Companies have been taking a whole set of techniques to look at their kinase inhibitors, rather than just one defined screening technique,” says Christensen.
That’s good news for product suppliers, as the success of one assay system won’t necessarily come at the expense of others. DiscoveRx markets a series of general-purpose kinase screening assays that nominally compete with Promega’s, but they use a different technique. Rather than measuring the depletion of ATP, assays such as DiscoveRx’s ADPHunter use a fluorescent readout to measure the accumulation of ADP, a related but distinct end point.
While cross-checking the assay is clearly a good idea, even using multiple assays in the early stages of screening doesn’t ensure that the resulting hits are selective. “For every kinase inhibitor you find, you then have to screen it again for 20 to 100 kinases to make sure you’ve got selectivity, and don’t just kill kinases in general,” says Can Ozbal, PhD, senior director of BioTrove, Woburn, Mass.
There are several ways to profile a compound’s activity against multiple kinases, but until recently most of them were too time-consuming even for medium-throughput work. Mass spectrometry, for example, is an excellent strategy for determining which kinase substrates stop being phosphorylated when a compound hits a complex mixture. Unfortunately, the technique is notoriously slow and finicky, requiring a laborious sample cleanup process before the analysis.
About seven years ago, though, Pfizer, New York, N.Y., funded a project at the Massachusetts Institute of Technology, Boston, Mass., to develop an automated system that would accelerate mass spectrometry sample preparation. “It was one of the first cases where we really built a solution for a problem, rather than build a cool machine and try to find applications for it,” says Ozbal. The project was a success, and the university created BioTrove to sell the resulting system. By automating a simple solid-phase extraction protocol, the company’s system shortens the procedure to about six seconds per sample. While still glacially slow by high-throughput standards, it brings mass spectrometry into the range of medium-throughput, suitable for following up on initial high-throughput hits.
Though not initially developed with kinases in mind, Ozbal hopes to market the machine for that purpose soon. One of the mass spectrometry’s big advantages is that it can distinguish phosphorylation events on different substrates in a complex mixture, without requiring separate reagents for each. “Mass spec is very binary, so I can actually multiplex a bunch of assays together into a single well,” says Ozbal, adding that “if I’ve got five different peptides, I can look at all of those phosphopeptide products, and I can also look at multiple phosphorylation events.”
The burgeoning crop of off-the-shelf assays and gear has been a boon for many screening programs, but groups with slightly unconventional needs may still prefer to build their own assays from scratch. “We were asked by [the National Institute for Allergy and Infectious Diseases] to develop assays for biochemical targets in Mycobacterium tuberculosis,” says Lucile White, manager of the High-Throughput Screening Center and Enzymology Laboratory at the Southern Research Institute, Birmingham, Ala.
That assignment was fairly typical for the nonprofit organization that works as a contractor for both government and private clients.
For their most recent tuberculosis project, the Institute’s scientists decided to target pantothenate synthase, a biosynthetic enzyme in the mycobacterium that consumes ATP to form pantothenate, an essential precursor for synthesizing coenzyme A and acyl carrier protein. On paper, the reaction looks like a good candidate for the kinds of assays industry screeners use for kinases: it consumes ATP and causes ADP to accumulate. In practice, however, Southern Research Institute’s team had better results with a less direct assay, measuring the change in NAD+/NADH ratios that accompanies the enzyme’s consumption of ATP. NADH absorbs light at 340nm, while NAD+ does not, so the researchers could measure the ratio with a quick, cheap spectrophotometric check. “Technically, all of the traditional types of kinases could be run through this type of assay, but they don’t generally have sufficiently high catalytic effiiency,” says White.
By industry standards, the tuberculosis screen was quite small: just over 4,000 compounds in 96-well plates. Nonetheless, the researchers found the first known inhibitor of the bacterium’s pantothenate synthase pathway, and they have now generated additional compounds that are even more potent against the enzyme. Southern Research Institute is now trying to turn these hits into leads for new tuberculosis drugs.
Meanwhile, industry screening campaigns seem likely to increase their reliance on kinase assays even further, for the simple reason that the strategy is working. As Promega’s Curtain puts it: “Kinases just seem to have a good track record right now, as far as being able to find successful drugs.”
About the Author
Originally trained as a microbiologist, Alan Dove has been writing about science and its interfaces with industry and government for more than a decade.
This article was published in the Lab Automation supplement to Drug Discovery & Development and Bioscience Technology magazines: November, 2007, pp. 4-8.
Filed Under: Drug Discovery