22 Apr 2012

Appreciating the Art of Assay Development

A good assay, they say, is the stuff of good science. Whether it’s a whole new type of test, a twist or a tweak of an old one, or a way of combining things that hasn’t been worked out before, assay development remains an integral—if sometimes behind-the-scenes and underappreciated—part of the discovery process.

Just ask participants of the Assay Development and Screening track of the recent “SLAS 2012” conference. Researchers there discussed the challenges of solubilizing fatty acids while avoiding toxicity, and ways to optimize conditions for high-throughput screens of libraries of molecular inhibitions to generate more reliable hit lists.

Some told of ways to circumvent or directly challenge the idea that some targets may be “undruggable”, while another spoke of uncovering previously unseen mechanisms of action by eliminating an inhibitor built into the standard assay.

Lawrence Wiater, Ph.D., is working on cell-based assays to systematically investigate and characterize metabolism of fatty acids in mammalian cells. Biolog, for which he is a senior scientist and group leader, currently offers a series of 96-well microplates (Phenotype MicroArrays™) that look at metabolic effects of carbon and nitrogen substrates, ions, hormones, metabolic effectors, and anticancer agents. The fatty acid metabolism assays, which are currently in beta testing, will be an extension of that platform.

The assays can help researchers understand what pathways cells use to metabolize substrates that are in the wells, to look at the effects substrates have on cell growth, or to see if they can increase the productivity of a metabolic product in bioprocessing.

“You can just add your cells to hundreds of wells and then you can kinetically monitor each well for increased or decreased productivity of your favorite molecule,” Dr. Wiater pointed out. “You typically don’t know what may affect your pathway of interest. These microplates allow you to screen hundreds of nutritional factors that could modulate that productivity.”

Disease research, too, can benefit from the company’s Phenotype MicroArrays. “Energy metabolism is linked to obesity, diabetes, nutrition, aging, mitochondrial diseases, drug toxicities—especially those that target mitochondria—and then cancer and cachexia,” he said. “Our fatty acid microplates offer additional pathways you can probe to look for relevance in any of these health problems.”

Biolog’s OmniLog™ instrument incubates the microplates at 37°C while it reads and records the linear reduction of a tetrazolium dye to a colored formazan, thereby measuring the rate of metabolism in each well. Currently techniques such as mass spectrometry and liquid chromatography can generate a snapshot of metabolic pathways pools, but Dr. Wiater “doesn’t know of any other technology platform that can measure metabolic rates of fatty acids and other cell energy sources.”

Inhibit the Inhibitor

Reactivation of telomerase allows cells to become immortal, and as such is seen as an attractive anticancer target. Yet the molecular architecture of the catalytic reverse transcriptase (hTERT) of telomerase makes it essentially undruggable. Despite 15 years of trying—and a lot of money, time, and effort—no small molecule-based inhibitors of hTERT have made it to the clinic.

Cancer Research Technology (CRT) medicinal chemist Jon Roffey, Ph.D., uses a more indirect approach to find inhibitors of telomerase in his cell-based assays. He and his collaborators set out to “look for a network of druggable pathways for therapeutic exploitation of a nondruggable target,” he explained.

Using the hTERT promoter cloned by principle investigator W. Nicol Keith, Ph.D., of the University of Glasgow as the basis of a standard luciferase reporter assay, they tested the effects of 79 well-characterized kinase inhibitors. “If you can inhibit anything in that pathway, and you can inhibit promoter activity, you can see a decrease in luciferase activity,” said Dr. Roffey.

Six compounds were found that did just that, three of which were known to inhibit the activity of the enzyme glycogen synthase kinase 3 (GSK3).

But the problem with cell-based assays and reporter-gene assays is that there are multiple pathways that can lead down to the promoter, and so specificity is key. They weeded out compounds that had nonspecific effects on luciferase such as general transcription factor inhibitors, and they utilized viability assays within the cascades because it should take multiple cell cycles before shutting down telomerase would kill the cells.

Ultimately they introduced their inhibitors to the endogenous system. Using both siRNA and small molecules in a panel of cancer cell lines, the researchers were able to show that inhibiting GSK3 led to a reduction in telomerase message, a reduction in telomerase catalytic activity, and ultimately (over the course of many days) a shortening of the telomeres themselves.

The GSK3 work, Dr. Roffey explained, was not the end in itself so much as a proof of principle. It was “basically saying that the promoter assays can be used to find compounds that can modulate the pathways that modulate telomerase expression.”


Some targets are considered “undruggable” because what’s known about their structure says that they lack traditional binding pockets for small molecules or the native ligand binds too tightly to be out-competed. X-ray crystallography, the preferred way of discerning the structure of a protein, freezes a protein into one particular conformation.

But proteins are plastic, points out Joshua Salafsky, Ph.D., CSO of Biodesy: “You’re not able to see all the conformations it’s adopting under physiological conditions, and therefore, just using crystallography, you’re unlikely to find drugs that perturb the conformation in specific ways.

“There are likely transient pockets that open up that aren’t visible in the crystal structure that you would like to develop a molecule to bind to and stabilize a particular conformation of the protein, to render it inactive, for example. Or you’d like to develop an allosteric drug, again that binds to a pocket that may or may not be visible in the crystal structure.”

While a post-doc at Columbia University, Dr. Salafsky developed a novel way to detect biomolecules, based on a technique used in physics and physical chemistry research called second harmonic generation (SHG), by labeling them with SHG-active dyes.

Subsequently, at Biodesy, the company he founded, he developed this advance into a tool to monitor a protein as its conformation changes in real time. Upon excitation, immobilized biomolecules labeled with SHG-active dyes will re-radiate two photons of red light as a single photon of blue light (the “second harmonic”).

The key, he said, is that the amount of blue light produced is very sensitive to the orientation of the dye, and so “we can detect a very small shift in the average orientation of the probe due to protein conformational change.”

By labeling a protein at a specific amine or cysteine, different parts of the protein can be monitored.

Biodesy recently used this technique to identify activators and inhibitors of Ras activity. “We not only were able to show that these compounds in fact did change the conformation of Ras directly, but that the label site also told you whether conformation changed at that particular site,” Dr. Salafsky said.

"It was really exciting that the two active compounds in our hands that changed conformation of Ras were also the two inhibitors that other people had found in cell-based assays, and that the third compound that had no effect in the cell-based assays had no effect on the conformation, in our case, either when Ras was labeled at the cysteine or at the amines.”

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