BETTER SCREENING TOOLS

CELLULAR ASSAYS GAINING FAVOUR IN DRUG-DISCOVERY RESEARCH

By Deborah Komlos

Drug-discovery efforts have a lot on their plates. As if finding effective molecules isn’t enough work, there are increasing demands to make the process faster and to better mimic in vivo drug responses.

Cellular, or cell-based assays are increasingly stepping in to address these needs.
Over the next few years, drug discovery is going to be the main thrust in the cellular assays market, says Alek Bituin, a San Mateo, Calif.-based senior analyst with Navigant Consulting Inc. (Chicago, IL). “It does have a few potential applications in diagnostics and some other minor applications. But really, the big push right now is going to be toward general drug discovery and the whole lead identification and validation process.”
Bituin co-authored a report released late December 2003 called Cellular Assays 2004: Industry Trends and End-User Analysis. Focused on the application of cellular assays in the field of drug discovery, the report gives an overview of the worldwide market for cellular assays from 2004 to 2009.
Although a sluggish economy is predicted, with recovery starting in 2007, the report estimates a worldwide market increase for the cellular assays field, rising from an estimated $470 million US in 2004 to $700 million in 2009. Fluorescence-based cellular assays are expected to remain the dominant detection method through 2009.
Bituin highlights amalgamation within the industry as a prevalent trend.
“The biggest thing that we’ve noticed is the consolidation, where the huge instrumentation firms were starting to integrate vertically and buy out the smaller assay firms, so they incorporate that technology into their own imaging systems,” Bituin says.
The acquisition of Molecular Probes Inc. (Eugene, OR) by Invitrogen Corp. (Carlsbad, CA) in 2003 is a deal that Bituin says is likely typical of what’s to come.
While the report cites Amersham Biosciences — now part of GE Healthcare (Chalfont St. Giles, U.K.) — PerkinElmer Inc. (Wellesley, MA) and BD Biosciences (San Jose, CA) as top players in the cellular assays field, Canadian efforts are also stepping notably into this sphere.

A New Skin
Biotech firm PharmaGap Inc. (Ottawa, ON) is one such example.
A National Research Council of Canada (Ottawa, ON) spinoff, PharmaGap has developed a unique 3-D human cell ADMET (absorption, distribution, metabolism, excretion and toxicity) model based on its proprietary wound-healing and skin-growth medium.
Using this medium — which does not contain animal products — the firm selects stem cell-like keratinocytes from the epidermis and makes stocks of these cells, explains PharmaGap CSO Jenny Phipps, PhD. Adult skin surgical discards are used as the source material.
“What is really original is the procedure itself because we also developed optimized media for cryopreservation and for thawing in such a way that we keep the cells undifferentiated, that is, with the optimum proliferation features,” Phipps says.
She explains that PharmaGap’s skin mimicry system uses inserts on which the cells are grown. Nutrients are initially provided through submergence of the inserts into the culture medium, followed by provision of the medium from below.
“After they have grown submerged for some time, we would change the medium and work at the air/liquid interface to redifferentiate cells,” Phipps says. The result of this setup is a multi-layer of cells with basal cells at the bottom and increasingly differentiated cells appearing outward.
Typically, skin constructs contract at some point, Phipps says, but PharmaGap has found a way to treat the insert to prevent this from happening. This prevention is needed for ADMET testing, Phipps explains, because contraction reveals space on the insert that would allow added compounds to avoid having to pass through the skin construct being tested.
In addition to the use of a proprietary growth medium, the growth of cell stocks originated from isolated primary adult cells, and the prevention of contraction, PharmaGap’s model is also unique for its applicability in high throughput screening.
The classical approach to growing epidermis constructs involves growth “on a raft model, and then people cut off pieces of this raft and put it into different devices depending on what they want to do,” Phipps says. “It is very tedious and time-consuming, while this construct we made could be used for high throughput.”
The construct has been optimized for high throughput in 12- or 24-well plates, she adds.
PharmaGap signed a deal last January with a major U.S. pharmaceutical company to develop a working model that meets the firm’s needs for screening a range of compounds to assess transdermal permeability and metabolism.
Finding ways to improve upon steps in the drug-discovery process is critical, says PharmaGap’s executive vice-president and COO Simon Goulet, as firms want to make the lengthy process more efficient.
“Clearly this is something different and unique because large U.S. companies are coming up North looking for technologies,” Goulet says of PharmaGap’s work.
Though the pharma industry tends to be cautious with novel technology, the big drug firms are “dipping their toe into this area,” Goulet says. “In fact, Novartis is sponsoring a 3-D research project in Switzerland. So they all see the benefit, it’s just a matter of getting conservative biologists and chemists and companies to try something different.”
Goulet says PharmaGap expects to be attracted to other drug firms as well as cosmetics companies that are aiming to get away from animal screening. “The EU has banned animal screening by 2009, so there’s a great market potential.”

Elucidating Relationships
Cellular assays are also finding application in the academic milieu, as with the case of James W. Dennis, PhD.
The Canada Research Chair in Glycobiology and senior investigator in molecular biology and cancer at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital (Toronto, ON), Dennis is conducting research on cell surface glycosylation of proteins that might be involved in cancer.
Using scan array immunofluorescence imaging, Dennis’s team is measuring cytonuclear translocation to study tumour cells and characterize the presence and function of cell surface receptors.
“A lot of signalling requires that something gets phosphorylated near the membrane and, of course, things like ERK and other proteins get phosphorylated and go to the nucleus where they regulate gene expression,” explains Dennis, also a professor in the University of Toronto’s (U of T) (Toronto, ON) department of medical genetics and microbiology.
With cellular imaging, Dennis says, molecules’ movement from the cytoplasm to the nucleus can be monitored and quantified.
“They are all developing wonderful algorithms for calculating the fluorescent localization of proteins in different compartments and also their movement around in the cell,” Dennis says of firms working on high throughput fluorescence cellular imaging.
“There are these assays where you can see (the proteins) at the cell surface and then move into endosomes,” he continues. “They can move from there to the nucleus or they can be destroyed, usually as part of the cycle. So, essentially, that can allow you to graph each one of those phases of the protein’s movement in the cell and get a quantification of that.”
Rapid data collection from use of cellular imaging assays has permitted Dennis’s team to refine a new computational model of cytokine-receptor regulation by endocytosis and protein N-glycosylation.
The team has applied its results in a systems-biology approach, he says, to develop a model where outcomes could be predicted if model parameters, such as rate of receptor production, were changed.
“That’s quite fun because it’s not only cancer that it applies to but also stem cell biology . . . the stem cells rely on these cytokine receptors for their survival and growth,” Dennis explains. “As we age, there’s a tendency for receptors to be lost from the cell surface and, in fact, that dominates the biology, and then the cells senesce and you lose stem cells . . . we basically lose potential for renewal that way.”
Dennis says another goal for his team is to measure 30 or 40 parameters on the same set of cells simultaneously. For instance, after finding the outcome of drug effect on a cell, “you can predict what pathways that might actually be affecting based on previous patterns of cellular phenotypes for other drugs and so on,” he says.
Biology patterns derived from the use of cellular imaging will be predictive of not only drug responses, Dennis says, but also differentiating between cell states, such as cancerous and benign. “I think the imaging may have diagnostic applications in a few years that are quite powerful.”

Making It Easy
Pharma firms seeking novel therapeutic targets are strongly focusing on G protein-coupled receptors (GPCRs), says Brian O’Dowd, PhD.
A professor in U of T’s department of pharmacology and head of molecular pharmacology at the Centre for Addiction and Mental Health (Toronto, ON), O’Dowd has been working on cloning GPCRs since the 1990s.
“In all of the big drug companies today, these receptors are being intensively looked at, particularly the new ones that have been deorphanized,” O’Dowd says.
For some receptors, the endogenous ligand — the naturally occurring molecule that binds the receptor — is known, O’Dowd explains. For others — called orphan receptors — that ligand is unknown. Reverse pharmacology can determine the ligand for an orphan receptor, he continues, by using the receptor to trap the endogenous molecule that activates it.
“Once you’ve essentially deorphanized the receptor, where you have both the receptor and you have this endogenous ligand,” O’Dowd explains, “you can now determine the pharmacology and eventually some of the functions that are related to this entirely novel receptor . . . Many of these GPCRs are expressed both in the central nervous system and in the peripheral tissue.”
Along with U of T colleague Dr. Susan George, O’Dowd has developed a GPCR-based assay that they’ve called MOCA (multipurpose original cellular assay). The assay provides a unique method to identify chemical compounds, which serve as synthetic novel ligands, that activate or block GPCRs.
There are an estimated 367 GPCRs, but only about 20 have so far been used to generate therapeutically and commercially successful drugs, O’Dowd says. Meanwhile, he adds, about 50 per cent of all prescribed drugs target a GPCR.
For MOCA, he explains, the GPCR is modified by insertion of a small motif called a nuclear translocation sequence. The receptor is thereby internalized and translocated to the nucleus, from where it cannot recycle to the cell surface. When a compatible ligand interacts with the modified GPCR, it is prevented from translocating to the nucleus and is measured as protein retained on the cell surface.
O’Dowd says MOCA has features that aren’t covered by existing technologies. For instance, he says, the assay can identify both agonists and antagonists. It can also identify receptor complexes called hetero-oligomers, which he says are currently one of the “hot topics” regarding GPCRs. It has been shown that closely related receptor subtypes and unrelated receptors can form these complexes, which have unique properties.
This understanding “provides the drug companies with additional targets, even on top of the 367 receptors,” O’Dowd says. The hope, he says, is to be able to target these new targets with unique drugs that would not bind to the individual receptors or to homo-oligomers — groupings of like receptor types.
O’Dowd and George are commercializing their patent-pending invention through patoBios Ltd. (Toronto, ON), which they co-founded last January. He says patoBios has already received a great deal of interest from the major pharma firms and is in various stages of negotiation with many of them.
“What we’re working on is sort of the tip of the iceberg of the potential of the assay,” O’Dowd says.
“The technology is always changing, so there’s a period of time when your assay is state of the art, but then something better is always around the corner,” he says. “The idea is to get it into the marketplace and have people use it and see the usefulness and success of it. Then it will join the ranks of the other good assays.”
There are an estimated 367 GPCRs that are potential drug-development targets, but only about 20 have so far been used to generate therapeutically and commercially successful drugs, O’Dowd says. Meanwhile, he adds, about 50 per cent of all prescribed drugs target a GPCR.
For MOCA, he explains, the GPCR is modified by insertion of a small motif called a nuclear translocation sequence. The receptor is thereby internalized and translocated to the nucleus, from where it cannot recycle to the cell surface. When a compatible ligand interacts with the modified GPCR, it is prevented from translocating to the nucleus and is measured as protein retained on the cell surface.
O’Dowd says MOCA has features that aren’t covered by existing technologies. For instance, he says, the assay can identify both agonists and antagonists. It can also identify receptor complexes called hetero-oligomers, which he says are currently one of the “hot topics” regarding GPCRs. It has been shown that closely related receptor subtypes and unrelated receptors can form these complexes, which have unique properties.
This understanding “provides the drug companies with additional targets, even on top of the 367 receptors,” O’Dowd says. The hope, he adds, is to be able to target these hetero-oligomers with unique drugs.
O’Dowd and George are commercializing their patent-pending invention through patoBios Ltd. (Toronto, ON), which they co-founded last January. He says patoBios has already received a great deal of interest for MOCA from the major pharma firms and is in various stages of negotiation with many of them.
“What we’re working on is sort of the tip of the iceberg of the potential of the assay,” O’Dowd says.
“The technology is always changing, so there’s a period of time when your assay is state of the art, but then something better is always around the corner,” he says. “The idea is to get it into the marketplace and have people use it and see the usefulness and success of it. Then it will join the ranks of the other good assays.”