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Ordway s DiRusso Discusses a New Live-Cell Assay for Fatty Acid Uptake Inhibitors

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Concetta DiRusso
Professor
Center for Cardiovascular Sciences, Albany Medical College

At A Glance

Name: Concetta DiRusso

Position: Professor, Center for Cardiovascular Sciences, Albany Medical College, 2001-present; Senior Staff Scientist, Ordway Research Institute, 2003-present.

Background: Associate professor, biochemistry and molecular biology, Albany Medical College, 1996-2000; Assistant/Associate professor, biochemistry, University of Tennessee College of Medicine, 1986-1996; Postdoc, University of California, Irvine, 1982-1986; PhD, cell/molecular biology, University of Vermont, 1982.


It is safe to say that compounds to treat obesity are very high on pharma's list of potentially lucrative therapeutics; therefore, reliable assays for obesity-related biochemical pathways are also on pharma's wish list. Scientists from the Ordway Research Institute and Albany Medical College in Albany, NY, have recently developed a live-cell assay to screen for fatty acid uptake inhibitors, described earlier this year in Analytical Biochemistry [2005 Jan 1;336(1):11-19].

The assay, which uses yeast cells, is conducive to high-throughput screening campaigns, according to the researchers, who are now seeking to commercialize it. Last week, Concetta DiRusso, corresponding author on the paper, discussed the development of the assay with Cell-Based Assay News.

How did you develop your interest in cardiovascular and metabolic disease?

I've been working in fatty acid metabolism since I was a postdoc, investigating E. coli. My interest then was in fatty-acid dependent transcriptional control. My husband and collaborator, Paul Black, was looking at fatty acid transport at the time, and in bacteria, it's a very simple signal transduction system: There's an outer membrane transporter, and the activating enzyme, and the product is acyl-CoA, which then interacts with the transcription factor to actually inhibit DNA binding. This causes a cascade — an increase in degradative enzymes and a decrease in synthetic enzymes. It turns out that most eukaryotes have the same kind of response. Our interest in turning to yeast was to find a model organism for mammalian cells. Of course, in mammals, the major problem with fatty acid dysregulation, if you will, is obesity. That's our major disease interest, and is a contributing factor to so many other diseases that affect our society. Yeast are simple, yet have a lot in common with mammals. And one of those things is the fatty acid transporter, which in yeast is called FAT1. There's a gene that encodes the FAT1 protein, and this protein itself is required for fatty acid uptake. That actually winds up being essential in yeast when they're grown anaerobically, which they like to do when they're fermenting, for example.

The FAT1 gene has homologues in mammals, and that is the FATP protein. We deleted the FAT1 gene, and one of the first things we did was to show that the FATP gene of mice could complement that mutation. We've done a lot of work since then, about 10 years' worth, and we now know that FATP partners in a functional manner with an acyl-CoA synthetase, which is the activating enzyme. If you knock out the transporter and the major acyl-CoA synthetase activating enzyme — which is called FAA1 in yeast — then there is absolutely no uptake, and it's a very tight system, and you can express any of the mammalian enzymes that have these activities in yeast, and now look at their function in a very simple organism.

You said that obesity is the major human health implication …

Well there is something else that's very important to us. Our ultimate goal is not to inhibit all fatty acid uptake, but to force uptake to be selective. For example, we'd like to be able to increase the uptake of the omega-3 polyunsaturates and discourage the uptake of trans- and saturated fatty acids. Most evidence to date indicates this would be very beneficial to cardiac health and may prevent certain cancers.

How do people currently screen for compounds that might inhibit fatty acid uptake? Is this type of model a typical approach?

I think that most would use a mammalian line. The original FATPs were examined in Cos7 cells, for example. People also look at 3T3-L1 adipocytes, because they have a maximum uptake. One of the problems with those, of course, is the expense in culturing mammalian cells. You also don't have control over cellular genetics. Mammalian cells will typically express several FATP proteins, and several acyl-CoA synthetases. We can knock all of them out in yeast and just reconstitute pairs, or one or the other, and that's an advantage. Also, it's very inexpensive. Yeast are cheap, their media is cheap, you can put 50,000 cells in a 384-well format, it's very rapid, and it's extremely sensitive — our discriminating factors are very, very good — above 0.7.

In a drug-discovery setting, do you think you sacrifice relevancy?

Mammalian cells are essential in secondary screens. The advantage of the yeast is getting through 100,000 compounds or more, fast. Once you identify a set of potential target compounds then you can use a different variety of cells — even on the same plate, if you want.

It's still a live-cell approach, though. Is there a way to do this biochemically?

It's very difficult to screen transporters in vitro. You'd have to in some way put them into a native environment — a liposome or something. For fatty acid transport requiring two components, employing a live cell is certainly advantageous.

In the paper, you talked about adapting this to high-throughput screening. I'm assuming there's a pretty large market for this in pharma. Is this something you've looked at?

Definitely. Since the publication of that paper, we've refined it to 384 wells, we're expressing different FATPs, including some human FATPs and acyl-CoA synthetases, and we've been testing some of the small pilot libraries that are available. There's a 2,080-compound library from MicroSource called Spectrum Plus that we've tested, and we've been very happy with the results. Of course, our yeast-based system is also available for licensing, and we have submitted a patent.

Do you think it can be improved in terms of throughput, or if it were translated into more of a high-throughput pharma-type setting?

We're working on it every day. In screening these pilot libraries, we're learning how to eliminate false positives very rapidly. For example, some compounds that have intrinsic quenching, or have intrinsic fluorescence, can be eliminated very rapidly. And there are others that you can tell from the chemistry may have a strong detergent property, and make the membrane more permeable makes the membrane more permeable to the quenching agent and it looks like a false positive.

What else is next for your lab regarding this research?

In addition to the yeast-based system, we're working on several mammalian cell lines, such as Caco2 and HepG2, as well as some endothelial cells. We're interested in the cardiovascular implications here. Can we actually discriminate uptake, for example, across the blood-brain barrier, compared with an adipocyte? In one case, you don't want to necessarily be selective at all — fat cells take up any fatty acid — but in the brain, you are very much enriching for polyunsaturates, so our basic research is heading in that direction.

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