HTS Carving Out Hit-to-Lead Position

Pressure to discover more hits and develop more blockbuster drugs with less time and expense spent on target screening continues to spur companies to invest in high-throughput technologies earlier in the drug discovery process. In addition, there is a trend toward combining cell biology, imaging, and in vivo experimentation skills. Firms presented their newest screening methods at the recent “Screening Europe 2008” conference in Stockholm.

Drug screening against GPCR targets continues to be a major focus for many companies, accounting for more than 60% of prescription drugs currently on the market.

Researchers at Acadia Pharmaceuticals use its high-throughput screening platform, R-SAT® (receptor selection and amplification), to screen GPCRs, RTKs, and nuclear hormone receptors for targets. This platform is a cell-based assay where genes are transferred to cultured cells. Functional activities of the potential targets are evaluated via signal-transduction pathways that trigger cellular growth. Marker gene technologies are used to report the growth signals.

“This is a homogenous assay that allows you to better understand the pharmacology around GPCR targets,” noted Fabrice Piu, Ph.D., director, clinical genomics. “It also integrates all the signaling pathways that emanate from the receptor based on the action of the ligand. It detects any chemistries that have direct interaction between the receptor and the ligand.”

The technology also enables the identification of new mechanisms of action. Dr. Piu presented two examples of this. The first was that inverse agonism at the 5-HT2A receptor predicted antipsychotic activity. One agonist, pimavanserin, is currently in Phase III trials for treating psychosis in Parkinson’s disease. The second example was that a metabolite of clozapine, ACP-104, is a M1 muscarinic agonist. This suggested that it may have cognitive benefits. It is currently in a Phase II trial for schizophrenia.

Cryopreserved Cells

“Frozen cells are very much the industry trend at the moment,” according to Bob Kendall, Ph.D., development scientist at GE Healthcare Life Sciences. “Researchers are adopting cryopreserved cells for a number of assays, and it seems to be the goal to convert all assays to a frozen-cell format.” The main advantage, he added, is that it decouples scheduling of large batches of cells from actual screening and improves assay performance by eliminating batch-to-batch variation.

“It’s something we picked up on as being a potential niche market. We’re providing proprietary cryopreserved cells or we take customers’ cells and grow them to bulk in single batches.”

The company is using microcarriers in combination with bioreactors to culture cells, explained Dr. Kendall. The microcarriers have several advantages over roller bottles and stirred tanks, including increased surface area, improved exposure to nutrients, increased purity, protection from shearing forces, as well as a reduction in culture medium costs and labor, he reports.

A complete screen requires about 2–3x10¹⁰ cells and up to 15 m² of plastic, Dr. Kendall stated. “That’s why we’re interested in the microcarrier culture; it’s a small footprint with a controlled environment for growing cells.” Fully validated 10¹⁰ cryopreserved cells are available in six to eight weeks from a seed vial.

The company’s custom cell services called Cell Factory range from assay design and optimization, transient cell engineering, cell provision, membrane production, and high-content secondary screening to custom image analysis.

Compound Selectivity

One major roadblock in the drug discovery pipeline is whether a compound will be effective in a patient. “Our screening service can determine in preclinical stages whether a drug being developed interacts with other receptors that may cause side effects,” explained Blaine Armbruster, Ph.D., product manager at Millipore.

The GPCRProfiler service is the first complete, cell-based functional platform using a common, validated readout for more than 130 GPCRs, which represent all three GPCR classes and more than 55 ligand families, Dr. Armbruster pointed out. It is based on the company’s ChemiScreen™ Stable Calcium Optimized GPCR cell lines, which were developed for cell-based functional assays.

In the past, Dr. Armbruster noted, radio-ligand binding assays were used for drug specificity. “These are limited in that they can give you a yes or no answer in terms of whether a drug interacts with a receptor but not the functional consequence of that interaction.” An example is the 5H2b receptor. If a drug is developed that interacts with this receptor, it could lead to heart disease. An agonist will cause disease, but an antagonist won’t. The GPCRProfiler has the capability to distinguish this characteristic.

The company also offers a Flex Lab service, which is not part of the standard GPCR service but provides custom assays and screens. Millipore is also developing a screening platform to identify allosteric modulators, which work at different sites on GPCRs than most native ligands and can modulate the receptor in a noncompetitive manner. Dr. Armbruster said that these molecules are gaining more interest in cases where a disease state is due to low receptor activity.

Neglected Diseases

Merck Serono has been involved in a research effort with the WHO since 2004 to develop leads for neglected diseases like malaria and sleeping sickness. Academic groups suggest potential drug targets that are then forwarded to the company for screening.

“The plan was for us to take in some postdoc fellows and train them to perform the screens,” said Dominique Perrin, Ph.D., group leader, molecular and cellular lead discovery. “We chose a relatively low-tech approach, so if the fellows returned to developing countries, they could set up screening activities with low-tech technologies.”

For the program, three main potential therapeutic targets were presented. Two were for malaria: the kinase PfCDPK1 and a protease PfSUB1. The third target, a phosphatase called TbPTP1, was for sleeping sickness. The screening platform for the phosphatase and the protease was a fluorescence intensity-based assay, and a simple luminescence reader was used for the kinase. Dr. Perrin’s group performed the primary screen for the kinase, and positive dose response and analogues were synthesized.

Dr. Perrin said that his group will continue to characterize these compounds and then send them out for testing on parasite assays. One of the difficulties with the program, he noted, is that few of the labs proposing targets were willing or able to provide enough active protein for testing. “It’s a unique bottleneck that I think reflects the difference between the perspective of academia versus industry.”

Transcriptional Screening

Instead of focusing on the traditional, druggable targets, Avalon Pharmaceuticals developed a screening platform, HITS® (high-throughput integrated transcriptional screen), that allows for blind, initially hypothesis-free screening to discover targets and access novel mechanisms of action in key disease pathways, according to Reinhard Ebner, Ph.D., principal scientist.

“We believe there are instances where our platform can produce lead compounds faster than traditional approaches and discover compounds that affect targets that could not be discovered by traditional target-based screens.”

The process starts by generating signatures that report on desirable physiological changes that the compounds elicite. “One way we select the signatures is by comparing the gene expression of a cancer cell to a normal cell,” explained Dr. Ebner. “Then, we screen for compounds that make a cancer cell look more like a normal cell.”

Another way to do this involves pathways that are generally recognized as playing an important role in disease but have been overlooked because the targets are often adaptor molecules, which interact with many other proteins. “We mimic the knockout of key nodes in that pathway to see what a gene-expression profile looks like if the pathway is suppressed. Then we screen for compounds that elicit a similar profile change.” An example of this process is the company’s beta-catenin lead series program. Beta-catenin plays an important role in cancer and is key in colorectal cancer. It is not an enzyme with readily measurable activity. Using transcriptional screening, however, Dr. Ebner’s group discovered several lead families that interfere with this pathway and may be promoted for preclinical development.

Integrating HTS and Profiling

Researchers at the Novartis Institutes for BioMedical Research developed a protease platform that combines biology, medicinal chemistry, and structural biology. It enables target validation, protein production, assay development, protein crystallization, hit finding, hit validation, hit-to-lead chemistry, and lead optimization.

“It’s buoyed by institutional memory around a particular single-target family,” explained Lorenz Mayr, Ph.D., executive director, Novartis Pharma. The platform is organized to facilitate close interaction with other platforms as well as all disease areas.

Proteases are considered a difficult target family, because proteins can make significant confirmational changes and not all targets have easily accessible active sites. To address this challenge, the group uses tools that contain three main elements for lead discovery: HTS, fragment-based screening, and in silico structure-guided drug discovery.

Another challenge is that many compounds, when removed from an organic solvent and placed into an aqueous buffer, form aggregates and create artifacts. Many compounds also have fluorescent properties that may interfere with HTS and the readout of an enzyme. Dr. Mayr’s group developed several methods to reduce artifacts. These include using microfluidics instead of HPLC and enzymatic assays with a series of red-shifted dyes such as Rhodamine-110 that reduce interference.

The team also uses fluorescence lifetime (FLT) in HTS for effective compound profiling. For this technique, a high-energy laser is pulsed thousands of times per second, and the time a molecule remains in an excited state is the FLT. It can be used to follow the enzymatic cleavage of an enzyme substrate labeled with the appropriate dyes.

“We believe we are the first ones to use FLT on a routine basis in drug discovery in 384-well and 1,536-well microtiter plates,” Dr. Mayr stated. “It was previously used mostly in academic labs in single cuvettes for basic research.”

HTS has become a standard technology for finding hits. The step from hit to lead optimization, however, remains difficult, requiring a broad range of approaches. As companies continue to develop innovative HTS platforms, this will help ensure the discovery and development of novel drug candidates.