August 1, 2009 (Vol. 29, No. 14)
K. John John Morrow Jr. Ph.D. President Newport Biotech
New Assays Streamline Scrutiny of Cellular and Molecular Binding Events
As the pharma and biotech industries search out new chemical entities, advances in drug screening technology have been sought as a way to control spiraling R&D costs. Newly perfected label-free technologies for drug screening are at the forefront, providing novel ways of interrogating cellular and molecular binding events. Academics and researchers from the industry discussed the current state of the art at “Assay and Screening Technologies” held recently in San Francisco.
“Invitrogen’s goal is to provide a rapid way to examine drug effects with functional cell-based assays that monitor specific ligand-receptor interactions,” said Richard Somberg, Ph.D., the company’s Wisconsin regional business manager. He discussed several products from Invitrogen, a division of Life Technologies, including a new cell-based assay known as LanthaScreen.
LanthaScreen is a time-resolved fluorescence resonance energy transfer (FRET) assay. It uses the rare earth cryptate element Terbium, which serves as donor, transferring energy to a fluorescent molecule when the two molecules are brought into close proximity.
In one of the LanthaScreen’s typical assays, a cell line is engineered with a green fluorescent protein gene fused to one of several kinase targets. The cells are subjected to a treatment that will stimulate phosphorylation, the cells are lysed, and a Terbium-labelled antibody specific to the phosphorylated molecule is added. This triggers the FRET reaction, and a powerful signal is produced. The beauty of this technology is that it has the properties of a single-step ELISA with high specificity and sensitivity, according to Dr. Somberg.
“We promote two different technologies—LanthaScreen for direct target modification measurements (e.g., phosphorylation, ubiquitination, acetylation) and CellSensors for total pathway activity measurements. Together, there are over 250 fully validated cell-based assays available off-the-shelf.”
Stem Cell Hemotoxicity
According to Ivan N. Rich, Ph.D., founder, chairman, and CEO at HemoGenix, conventional hemotoxicity evaluation is not an accurate predictive tool during preclinical animal testing.
The colony-forming assay was developed over 40 years ago as an in vitro means of testing cellular response of blood-forming cells to various agents. Cells are plated in petri dishes in a special medium containing methyl cellulose to immobilize them, and the number of colonies per cell plated can be measured as a function of the treatment. But the procedure suffers from a number of shortcomings, including subjectivity, long incubation periods, and a low-throughput performance.
For this reason, Dr. Rich and his colleagues have investigated the possibilities of using hemopoietic stem cells for a more meaningful and functional assay. These cells have the capacity to produce a number of lineages of mature functional cells, and offer the promise of a well-delineated model for testing of toxic compounds.
The company offers a variety of kits for measuring proliferation and cytotoxicity using its HALO®, ATP bioluminescence, and differentiation assays. This procedure has been validated against the Registry of Cytotoxicity Prediction Model, an information resource containing 347 compounds for which the LD50 and IC50 values are known.
HALO was designed for both 96- and 384-well screening to predict human in vitro hemotoxicity. The IC50 values obtained through the HALO assay correlate extremely well across a 5 log interval with cytotoxicity values generated for the registry, Dr. Rich said. The global response of drugs and other agents can be compared on seven different lympho-hematopoietic cell populations from various species.
In order to test drug-drug interactions with the HALO platform, the company has developed a 384-well plate high-throughput screening assay.
With this new assay, Dr. Rich and his colleagues have evaluated Verapamil, a p450 inhibitor, in the presence of cyclosporine. Verapamil may prevent cyclosporine from being broken down in the digestive system and the liver, leading to an increase in cyclosporine levels. These interactive patterns between the two compounds can be replicated in vitro using the HALO assay.
“The HALO assay is one of the most predictive toxicity screening tools available,” added Dr. Rich, “and combined with our other ATP-based assays allows ranking according to compound IC values, tissue, and species.”
Illuminating Nonspecific Activation
Natasha Thorne, Ph.D., a post-doctoral fellow at the NIH National Human Genome Research Institute, has carried out investigations of drugs targeting many conditions, especially human genetic disorders. Working in the NIH Chemical Genomics Center under the leadership of James Inglese, Ph.D., and Douglas Auld, Ph.D., Dr. Thorne and her colleagues have employed a high-throughput screening assay that uses firefly luciferase and Renilla luciferase to validate the purported nonsense codon suppression activity of compounds.
Part of the NIH Roadmap for Medical Research is directed toward the screening of chemical libraries of low molecular weight compounds in a search for innovative pharmaceuticals. The collection is vast, including more than 300,000 different substances aimed at the entire range of human illnesses. The results of these screening studies will be shared through peer-reviewed publications and the PubChem database.
Since traditional high-throughput screening presents a single concentration, with a subsequent detailed follow-up of promising candidates, many hopeful molecules may be lost due to inauspicious choices of the initial set-up conditions.
In the platform that Dr. Thorne and her coworkers have adopted, however, a quantitative high-throughput approach is put into play, which includes a seven-point titration in 1,536-well plates. Because of the volume and complexity of the data, a bioinformatics program is brought to bear on the analysis.
Firefly luciferase is used as an indicator of target activity, however the compounds being evaluated can interact directly with the luciferase, causing unanticipated and confusing artifacts. In fact, when the team investigated a 70,000 compound library they identified 3% of the molecules as being directly active against the luciferase.
An example of how these concerns were realized comes from studies on PTC124 (from PTC Therapeutics), which is believed to suppress nonsense codons. Duchenne muscular dystrophy and cystic fibrosis are sometimes caused by nonsense mutations that prematurely terminate the proteins specified by these genes. “Our data suggests that the initial discovery of PTC124 may have been biased by its direct effect on the luciferase reporter,” Dr. Thorne said. Nonsense codon suppression is under clinical investigation by other laboratories, but it is too early to rule on its clinical promise.
Target Proteins in Plasma
“We are investigating the use of mass spec-based biomarker screening as an alternative to liver biopsy in the diagnosis of liver diseases with Naga Chalasani’s group at the Indiana School of Medicine,” said Jinsam You, Ph.D., chief scientist at Monarch LifeSciences.
The company focuses its efforts on protein analysis, using mass spectrometry in order to develop new protein biomarkers for use as diagnostic tools.
An area of current interest for the firm is a panel of proteins, the Apolipoprotein family, which is essential for cholesterol transport. Through the development of mass spec-based assays the company intends to build rapid noninvasive assays that will allow investigators and clinicians to monitor liver and cardiovascular disease progression.
As a tool for developing high-throughput assays, mass spec has much to recommend itself. It can distinguish proteins that differ in as few as one amino acid, as well as different isoforms, and when the technology is installed and running it can be surprisingly economical.
“We are particularly interested in hepatic steatosis, a fibrotic disease of the liver brought on by high-fat diets,” Dr. You said. “At present this condition can only be diagnosed through the use of highly invasive and painful hepatic biopsies. At the end stage of the disease, when cirrhosis occurs, the condition is fatal, so it is extremely important to obtain early diagnosis.”
Dr. You and his colleagues found that there are significant gender-based quantitative differences in some of the apoproteins, so it is necessary to take this variable into account in developing a reliable assay. Sample preparation is also critical for good reproducibility, and the team has developed some novel approaches to limit variability.
The study was fostered by a growing clinical need. Non-alcoholic steatohepatitis (NASH) has mortality as high as one in four over the long term. In seeking an alternative to the liver biopsy, the Monarch group is studying individuals in the three stages of NASH, from early fatty liver (which frequently goes undiagnosed) through inflammation and finally fibrosis, with the overall goal of distinguishing these groups without resorting to biopsy.
When mass spec investigations were carried out, it was observed that Apo A1 was significantly higher in steatosis and NASH, Apo B was higher in NASH, and one of the lipoproteins (which Dr. You refrained from designating) was high in all three conditions.
“By building a multiplex panel of proteins we were able to diagnose with 100% accuracy the different stages of NASH in a group of 85 samples,” Dr. You said. “This was used to build a training set of discriminatory markers. Now we will continue into expanded studies with thousands of patient samples in order to provide definitive clinical support for this technology and the specific markers.”
Analyzing Altered Ion Channels
NMI Ion Channel Services investigates the function and dysfunction of ion channels for pharma and biotech companies, according to Timm Danker, Ph.D., a scientist with the firm. “Today although more than 300 ion-channel genes are known through the human genome project, only about 100 have been isolated and tested.”
Clearly, the automation and adaptation for high-throughput platforms would profoundly affect the capacity of investigators to screen, using electrophysiological approaches, promising ion channel drug candidates. The Roboocyte is an automated, flexible platform for voltage-clamp recordings on Xenopus laevis (the African tree frog) oocytes adaptable to a wide range of applications.
In the oocyte voltage clamp technique, two microelectrodes are pierced into the cells membrane. The cell can then be stimulated and current changes can be measured under different conditions, including treatment with candidate drugs. The Roboocyte is configured to allow the individual testing of large numbers of oocytes in conical wells.
Accurate, fast, and versatile, it can be left unattended overnight to yield up to 2000 data points in a month’s time, when three machines are employed simultaneously. The data is highly reproducible, with clean kinetics, he added.
Dr. Danker and his colleagues evaluated the selectivity of blockers of the closely related channels Kv1.3 (expressed in T lymphocytes) and Kv1.5, expressed in cardiomyocytes. Using standard molecular biology protocols, the genes for these channels and other proteins of interest can be cloned and expressed in the Xenopus oocytes. They showed highly reproducible dose response curves, which present a basis for selectivity-screening of molecules of interest.
On the drawing board are plans to extend these studies to point-mutated ion channels using a high throughput approach.
K. John Morrow Jr., Ph.D. ([email protected]), is president of Newport Biotech and a contributing editor for GEN. Web: www.newportbiotech.com.