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U Toronto s Brian O Dowd Discusses a New GPCR Cellular Assay


At A Glance

Name: Brian O’Dowd

Position: Professor, Dept. of Pharmacology, University of Toronto Department Head, Molecular Pharmacology, Centre for Addiction and Mental Health, Toronto

Background: Post doc, Howard Hughes Medical Institute, Duke University — 1987-1989; MS and PhD, Clinical Biochemistry, U. of Toronto — 1981-1986; BS, Biochemistry, London Polytechnic — 1975-1978

Brian O’Dowd has held several academic appointments with the University of Toronto’s pharmacology department over the last 15 years, and has simultaneously conducted pharmacology research in the areas of addiction and mental health for various Toronto-based institutions. Fresh off of a presentation at Cambridge Healthtech Institute’s Cell-Based Assays for HTS Screening conference in Philadelphia, O’Dowd will soon be speaking at various upcoming conferences about a new cell-based assay he developed for GPCRs. O’Dowd took some time to chat with Inside Bioassays about his work, efforts to commercialize the assay, and the evolution of cell-based assays for GPCRs.

How did you become interested in cell-based assays and G-protein coupled receptors?

My interest really started because I was a post doc with Bob Lefkowitz at Duke University. He’s a Howard Hughes investigator [at Duke], and he’s really one of the most cited scientists in pharmacology or biochemistry — a very well-known individual. And that was really my first exposure to GPCRs. And when I came to the University of Toronto, I was continuing with that interest, and we succeeded in the cloning of the D1 dopamine receptor and the D5 dopamine receptor. During the 1990’s, we really got involved in the discovery of many of the GPCRs — some of the subtypes — and the recognition that there were many receptors [called] orphan receptors for which the ligands are not known. We published a series of papers and reviews, and really tried to catalog the orphans. People were naming them things that meant something in an individual lab, but you couldn’t say whether receptor A and B from two different labs were the same or different receptor. So we devised a simple numerical scheme of naming them, like GPR 1,2,3,4, et cetera.

During that time, from an academic point of view, I think we discovered more of these receptors than any other lab. So at the end of the 1990s, we were left with a great deal of receptors for which the endogenous ligands were not known — probably 60 or 70 of them came from here. And in addition to that, we did actually find some receptors for which we found the endogenous ligand. But I think the fact that we had been so involved with the orphan receptors meant that we did consider the development of an assay that would allow us to find not necessarily the endogenous ligands, but also would enable us to find compounds from the chemical compound library. So the assay would let us find compounds that would interact with these orphan receptors, and also with the known receptors. The number of GPCRs today in the human genome is estimated to be around 367. [This is], I think, the number that people use now. This is independent of the olfactory receptors, which I’m not talking about, because they add considerably to the number. The receptors like the serotonin and dopamine — these types of receptors — there’s about 367 of them. And it’s not 50-50; there are probably more known receptors. But there are still a great deal — in excess of 100 of these are orphan receptors. So it was based on that interest that we developed the assay. When we look at the assay now, I think it will make the process of finding drugs, both at the known and orphan receptors, considerably easier.

The assay that you’re talking about is the MOCA (multipurpose original cellular assay) assay, correct? What is novel about this assay?

There are a number of things. One of the requirements of assays that are currently used, particularly for the GPCRs, is that it is helpful if you know the G-protein interacting with the receptor. You have the GPCR sitting at the cell surface, and they all interact with a G-protein. So the drug or agonist would be on the outside of the cell, the receptors on the cell surface, and it’s transferring the signal internally into the cell through the G-protein — so it helps if you know what this is.

But with our assay, it doesn’t matter. You don’t need to know anything about what type of G-protein is coupling to the receptor. So if you’re dealing with a brand new receptor, like the orphan receptors, it is a considerable advantage that you don’t have to know anything about the type of G-protein interacting. The second advantage is that in primary screening, you can screen directly with an antagonist. In some of the assays currently being used, you have to use an agonist to activate the receptor, and then you can find an antagonist by competing with that process. But with our assay, you can use an antagonist directly — you don’t have to pre-activate the receptor. So for an orphan receptor, essentially all you have is the receptor, and you don’t have to have an agonist against it. That’s not to say that the assay is useful only for the orphan receptors — it’s useful for all of the receptors, even the well-known ones.

Another advantage is that you can use transient cell lines — you don’t have to essentially waste time developing individual, permanently transfected cell lines. And the other aspect is that it’s universal for the GPCRs — particularly in the rhodopsin family, where there are hundreds of receptors. We do make a minor modification to the receptor, but it’s universal — it’s the same modification for each of the receptors. There is a little customization for each receptor, but the essential modification we make is the same, and it’s a minimal change, and it really doesn’t affect any of the pharmacology.

In your presentation, you mentioned that this is a very flexible assay. Can you expand on that?

The instrumentation that you can use — there’s great flexibility for it. It’s not tied into any expensive or exclusive instrument. You can use any instrumentation. We use an inexpensive fluorometer here. It does not require confocal microscopy. That too, depending on the lab, is somewhat of an advantage because there’s flexibility in the endpoint — the measuring of the receptor.

Also, we’ve tried a variety of different cell lines, and it does seem to be suitable to all of them. We’ve not found a cell line in which the assay doesn’t work. And also, it is not just for GPCRs, but it works very well for transporters, like the dopamine transporter or the serotonin transporter, which are pharmacologically very important transporters. And there’s also a family of transporters — a family of about 20 — in addition to those transporters, there’s also some orphan transporters for which the function is not known.

And lastly, one of the important things about GPCR pharmacology today is the fact that the receptors form hetero-oligomers. And that’s becoming important, even for drug discovery, because the oligomerization of two different receptors can form entirely new therapeutic targets. And the literature is showing the increasing importance of this. So the assay is very suitable for determining both receptors that form hetero-oligomers, and the ones that do not. So it will be quite practical to use the assay to understand the physiological rules governing which receptors are compatible in their ability to form these complexes.

And on top of all that, it’s a very easy assay to set up in a lab. The requirements necessary to utilize the assay are not complicated. Some of the assays are tied into visualization of green fluorescent protein, et cetera, but this does not require confocal visualization.

You’re involved with a company called PatoBios. Can you tell me what that is and how you are involved with it?

It’s a company that I founded with an academic partner here, Dr. Susan George, who’s also in the department of pharmacology, and both she and I founded the company to further the commercialization of this novel assay technology. Susan and I own the technology, and we’re the founding members of the company.

How long has the company been in existence?

Not so long — I would say a little less than six months. But the actual details of the assay were delayed until the patent submission was taken care of. The basic underlying discovery of the technology is patent-pending.

But in the several talks that we’ve given on the topic, I’ve had a lot of feedback. And I’m giving another talk at the upcoming GPCR meeting in Boston.

So you’re targeting partnerships with drug-discovery, pharmaceutical, and biotech companies?

We’ve had a great deal of interest from pharmaceutical companies, both big and small, and the biotech and assay companies. Just about everybody has shown some interest, and some companies have shown very strong interest in getting more information.

What do you see for the role of cell-based assays in drug discovery in the future?

I think that the future has to be cell-based assays, because of the complexity — the fact that the receptors have a very complex life in the cell. The number of jobs that each of the receptors is required to do, I think, can only be investigated in the context of the cell. Even for drug discovery, where it seems like a very simple process — “I’m looking for a drug, and do I, or do I not get an effect?” — Where we are now is that the golden age of drug discovery is really just beginning. If you contrast where we are now to where we were at the beginning of the 1990s, [there is] an unprecedented number of receptors — novel receptors that completely escaped classical pharmacology; that is, the pre-cloning era — receptors that no one had any idea existed. There are many examples of this — these are orphan receptors that have been ‘deorphanized.’ [For example], the apelin receptor — that was actually the first orphan receptor that we discovered. This receptor, which has really only been known to the world for four or five years, will really take its place in physiology and become as important as any other receptor, including the andrenenergic, dopamine, and serotonin receptors. So the 1990s gave us the subtypes of the receptors. I would say that if you had spoken to a serotonin scientist in 1985, he may have told you there are three or four serotonin receptors in the human genome, and it turns out there are 13 or 14 of them. [Looking at] the sheer number of receptors, the entirely novel receptors, and the subtypes of the receptors, pharmacology now has to deal with the question: “What are all these receptors doing?” The focus now is on the assays. There is a variety of assays now, but the assays that are really moving forward and that people are really paying attention to are the cell-based assays — and for good reason. This is really the first generation of cell-based assays, but as we move forward and understand the complexity of the receptors, such as the oligomerization — which until four or five years ago was still very controversial — where else are you going to find the physiological hetero-oligomers other than in a cell-based system? It’s only in the cell-based systems that these very complex ideas can be developed. It would be difficult for me to be convinced that there are better ways of looking at the receptors, be it for drug discovery or any aspect of receptor science.

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