March 15, 2005 (Vol. 25, No. 6)

Evaluating new approaches for more efficient and effective target identification

Screening of compounds for drug activity and other properties is an important discipline involving chemistry, biochemistry, and physics. Fast, effective, and informative screening technology can enable companies to fail early and cheap when it comes to putting forward candidates for drug development. Many innovative approaches, covering safety and efficacy screening as well as target identification, were under discussion at Select Conferences’ “Screening Europe” meeting, which was held in Geneva.

Serono ( works on kinase, phosphatase, and cell-based receptor targets and takes a parallel approach to screening, using both molecular (enzyme and substrate) and cell-based approaches.

“Cell-based techniques are becoming the screening format of choice,” commented Didier Leroy, Ph.D., scientist in the screening technologies department at the Serono Pharmaceutical Research Institute.

Cell-based assays allow access to the major therapeutic targets and screening for function under more physiological conditions than do molecular assays. Toxicology and metabolism issues are addressed earlier in the drug discovery process with cell-based assays.

Serono is using the Array Scan II, a cell analyzer from Cellomics (, to look at morphological changes such as neurite outgrowth in cells in response to drug molecules and also at nuclear translocation of key proteins.

Ongoing development work is focused on multiplexing the assays, looking at the number of cells needed to get a result, and optimizing the time of the assay.

Kinase Targets

Many other companies are looking at kinase targets, a trend that can only have been strengthened by the emergence of recent successes like Novartis’ (www. kinase inhibitor Gleevec. However, there is an urgent need to screen for more selectivity among kinase inhibitors.

Doris Hafenbradl, Ph.D., director of drug discovery technologies at Axxima Pharmaceuticals ( described the ongoing development of an assay that allows quantitative selectivity testing of compounds against a panel of kinases. “True selectivity comes from optimized assays,” said Dr. Hafenbradl.

The company is looking at variations in assay parameters such as ATP concentration, kinase concentration, reaction time, substrate choice, and concentration which are proving to be the way forward in finding the true selectivity of potential kinase inhibitors.

DiscoveRx (www.discoverx. com) is another company developing a range of assays for kinase inhibitors. They are also looking at another popular target classG-protein coupled receptors (GPCR). Their approach to GPCR activation assays involves the measurement of second messengers such as cyclic AMP and inositol trisphophate.

Split and Mix Techniques

Assays are being developed in order to tackle other challenging problems. Michael Organ, Ph.D., of the department of chemistry, York University, Toronto, pointed out that screening mixtures of compounds has presented difficulties in the past because of an averaging effect of assay results which makes it hard to single out those compounds with true potential.

“This has produced a tendency to shy away from mixtures,” said Dr. Organ. Yet not rising to this challenge may mean that companies are missing out on valuable combinatorial chemistry collections synthesized by “split and mix” techniques and natural product extracts.

Dr. Organ’s team has been developing a technique called Frontal Affinity Chromatography (FAC), in which the target is trapped within a matrix, bypassing problems posed by chemical binding of the protein to the column. Then the mixture of compounds to be screened against the target is infused onto the column.

The most weakly bound compound comes off first, and the mixture is analyzed on a mass spectrometer (MS) which allows real-time analysis. FAC/MS has shown promise with a mixture of 100 tripeptides binding onto thrombin, and the team has also looked at Factor Xa, a clotting protein that is deactivated by conventional affinity chromatography.

Another new technology, with many potential screening applications, is Respirometric Screening Technology (RST) from Luxcel Biosciences (, which uses fluorescence quenching of a probe to give a measure of oxygen uptake by cells, organisms, oxygen-dependent enzymes, and metabolites.

“RST is amenable for the development of very sensitive cell-based assays,” said Richard Fernandes, Ph.D., founder and CEO of Luxcel.

Oxygen uptake is an important biomarker, giving a very direct assessment of mitochondrial function. It is used as an early indication of apoptosis, metabolic activity, and can also give a good indication of the mechanism of toxicology. Luxcel is working with many Big Pharma companies, applying RST in toxicological screening and ADME studies.

hERG Screening

In recent years, there has been increasing emphasis upon the potential cardiotoxicity of drugs, which often arises from blockage of hERG, an important ion channel protein in the electrical activity of the human heart. The problem seems to arise because the sequence and structure of hERG are such as to bind many small molecule drug candidates.

Drugs blocking hERG are the the major cause of prolongation of a feature called the QT interval in the electrocardiogram, an abnormality often linked to cardiac arrhythmia.

Elke Guenther, Ph.D., head of electrophysiology at the Natural and Medical Sciences Institute (NMI) at the University of Tbingen, Germany, described a new approach to assess the impact of compounds on the QT interval of the electrocardiogram.

According to Dr. Guenther, assays of this kind are now seen as an important part of safety pharmacology. The NMI QT-Screen system places a miniature electrode in the well of a 96-well plate containing beating myocytes (heart cells) and gives a readout like an ECG so that the impact of a compound on the QT interval can be assessed.

Martin Traebert, Ph.D., group head of in vitro safety pharmacology in preclinical development at Novartis Pharma described how hERG screening can be automated with the Flyscreen 8500 patch clamp robot which creates whole cell recordings. The system compares well with conventional approaches, as seen by experiments with E4031, a drug that is known to block hERG.

Novel Cell-Based Assays

There are other innovative approaches to cell-based screening. At the University of Southampton, Rosario Sanchez-Martin, Ph.D., and colleagues have created microspheres that can deliver probes into cells for use in assays and other applications.

Meanwhile, Promega’s (www. HaloTag technology is a new method of labeling target proteins within the cell that can be used in functional assays. It allows imaging of cells over a long period of time so that cellular events, such as those happening on exposure to a potential drug, can be analyzed.

Some of the new cell-based assays are targeting specific pathways important in cancer. BioImage ( uses redistribution, an approach to drug discovery based on targeting the function of intracellular signaling protein targets.

BioImage is quantifying the translocation behavior of such targets on exposure to potential drugs by labeling with Green Fluorescent Protein. Redistribution is being applied to the discovery of small molecules that inhibit the AKT/p13K pathway which regulates cell proliferation, growth, and survival.

Invitrogen (www.invitrogen) is working on Jak-STAT, another signalling pathway that is important in cancer. The company has developed three high throughput cell-based screens using a beta-lactamase reporter, which have been validated by work with known pathway agonists.

In cell-based assays, however, labels can interfere with the drug-target interactions they are meant to reveal. Kai Hoettges, Ph.D., and colleagues at the University of Surrey, U.K., have developed an assay based upon a phenomenon called dielectrophoresis which relies on changes of electrical properties within a cell when it is exposed to a drug.

Novel three-dimensional electrodes can be constructed as conventional multiwelled plates into which cells are dispensed and exposed to a light beam probe. The technology has already been used to investigate apoptosis in cancer cells, which is a useful screen for anticancer drugs. The researchers are now trying to put this technology onto a chip with liquid handling to create a high throughput system.

Yeast-Based System

In another approach, Li Zhu, Ph.D., president and CEO of Genetastix ( presented a novel yeast-based technology platform for screening and generation of fully human monoclonal antibodies. They put the “bait” (a protein target) and an antibody together in a yeast cell and linked to a reporter gene.

The system can look at any type of protein target including intracellular proteins, secreted proteins, membrane proteins, and viral proteins. Work so far has involved screening antibodies to CXCR4, which is overexpressed in many metastatic cancers, and CCR5, a co-receptor for HIV.

Many targets can be looked at simultaneously. The aim is to carry out contract screening work with U.S. and Japanese companies and also to discover their own therapeutic antibodies.

Screening for Drug Delivery

And it is not just drugs themselves which need to be screened. Martinus Capelle of the school of pharmacy, University of Geneva, described a screening platform for drug delivery systems in order to find the best system to release a protein drug into the body either systemically or locally.

Experiments have been carried on the model protein r-hirudin, using its fluorescence to track its release from an agarose gel delivery vehicle.

The protein is released into a solution whose pH can be altered to mimic the route the drug would take in the body. This process can be automated and run from 96-well plates and offers the potential for faster optimization of other drug delivery systems such as nanoparticles and microspheres.

Screening is also allowing access to new targets like ion channels. “Ion channels remain relatively unexploited,” said Adrian Kinkaid, Ph.D., head of hit discovery biology at BioFocus Discovery (www. “But new technologies make them more accessible.”

The approaches BioFocus is using to investigate ion channels include high throughput atomic spectroscopy and automated electrophysiology. “This technology is phenomenal compared with traditional patch clamp techniques,” said Dr. Kinkaid. The combination of the technologies is allowing exploration of targets that have previously been inaccessible. “Ion channels are here and ready to be exploited,” he added.

Cell-based assays can also be used to investigate targets emerging from genomic work, as described by Dorit Hello Arit, Ph.D., of the German Cancer Research Center in Heidelberg.

The center creates constructs from open reading frames and looks at the impact the corresponding over-expressed protein has in various cell lines. Applications so far include a proliferation, apoptosis, and MAP kinase assay, all of which are relevant to the discovery of cell cycle modulators as new targets for cancer research.

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