NEWS

Almost Serendipity: Alcoholism Drug Reverses Drug Resistance In Vitro

Brian Vastag

Sometimes in science, luck and an open mind count as much as diligence and dedication. Just ask David Clarke, Ph.D., and Tip Loo, M.D., Ph.D., protein structure researchers at the University of Toronto.



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Dr. David Clarke

 
Their story starts with a spicy meal, includes a fortuitous encounter with a mislabeled drug, and ends with a discovery that could open an unexpected research agenda. In this issue of the Journal (p. 898), Clarke and Loo report that disulfiram (AntabuseTM), an inexpensive anti-alcoholism drug, blocks cancer drug resistance in cell cultures.

While still preclinical, the Toronto work presents an entirely new route for overcoming one of cancer therapy’s major stumbling blocks. Drug resistance stymies treatment in half or more of all cancer patients. Tumors often shrink during initial chemotherapy, only to regrow more resistant to therapy.

"Reversing drug resistance is one of the pseudo-Holy Grails of medical oncology. If we could just get that reversed, then our therapeutic index would go up dramatically," said Tom Anderson, M.D., who is helping to test an anti-resistance drug in leukemia patients at the Medical College of Wisconsin, Milwaukee.

But for a number of reasons, finding and testing anti-resistance agents has been a slow struggle (see News, Aug. 5 and 19, 1998). Evolution has equipped our cells with biologic shields against toxins. A key player in this defensive scheme is P-glycoprotein, a pump that straddles the cellular membrane and expels poisons. P-glycoprotein is remarkably catholic in its ability to reject toxic compounds; it spits out molecules of all shapes and sizes, including many cancer drugs. While recent research draws drug resistance as a tangled skein extending far beyond a "single pump" model, it is clear that P-glycoprotein plays a key role in the process.



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A hypothetical model of P-glycoprotein, an integral plasma membrane protein that causes multidrug resistance when overexpressed.

Image courtesy Michael Gottesman, M.D.

 
Seminal work in this area from the laboratory of Michael Gottesman, M.D., chief of the Laboratory of Cell Biology at the National Cancer Institute, shows that "at least 50%" of human tumors overexpress P-glycoprotein. Other research from Piet Borst, M.D., Ph.D., and colleagues at the Netherlands Cancer Institute, Amsterdam, confirms that transgenic mice without any P-glycoprotein have normal lifespans. These two factors make the molecule a keen target for extensive anti-resistance drug development efforts. Several drug companies, along with NCI, are vested in bringing anti-P-glycoprotein drugs to market.

Most of these efforts focus on finding drugs that bind P-glycoprotein at the cell surface to disarm its toxin pump. The only drug approved for overcoming chemotherapy resistance, cyclosporin A, works like this. Cyclosporin improves chemotherapy response rates in retinoblastoma, a type of eye tumor most common in children, and extends cancer-free survival times in some leukemias. Despite these successes, cyclosporin’s immunosuppressive and liver-toxic properties limit its use.

It’s in the Fold

Clarke and Loo discovered that unlike agents that were developed via high-priced modern drug design, disulfiram, which has been on the market for half a century and retails for pennies a pill, impedes P-glycoprotein in an entirely different way. Said Clarke: "Antabuse seems to block the movement of the protein from where it is synthesized in the cell to the cell membrane, where it’s needed to function. If it’s not at the cell membrane, then the chemotherapy drugs can get inside and do their damage."

Explaining this surprise takes a few steps back to the mid-1990s. Clarke and Loo were turning their attention to how P-glycoprotein and similar proteins form, or fold, during their construction. The field of protein folding was gaining followers as a host of diseases, including cystic fibrosis, sickle cell anemia, and mad cow disease, were found to be mediated by misshapen proteins.

One tiny crinkle, bend, or kink in a protein’s structure, and there is a good chance that molecular traffic cops will shunt it to the cellular junkyard for recycling. This is exactly what happens to the protein involved in cystic fibrosis. A close cousin of P-glycoprotein, the cystic fibrosis protein also sits on the cell membrane, serving as a gate or channel. But instead of pumping poisons out of the cell, cystic fibrosis protein regulates salt levels by shuttling chloride ions and other essentials.

Curried Brainstorm

During a trip to their local Indian restaurant, Clarke and Loo choked down bowlfuls of curry. Their noses ran and ran, and as the napkins piled up, they had a revelation of sorts. "We thought, these CF patients have a problem and their mucous is not flowing," said Clarke. "Then we thought, what is the active ingredient that makes your nose run, makes your mucous flow?"

That magical, spicy kick is capsaicin, well known to pepper lovers. Loo wanted to sprinkle it on cystic fibrosis cells.

"Loo kept after me for 6 months, and I kept saying no, no, you’re wasting your time," recalled Clarke. "He finally did the experiment himself."

But the lab did not have a supply of cells that expressed the defective cystic fibrosis protein. Fortunately, they did have plenty of cells engineered to make mutant, misfolded P-glycoprotein. Like the cystic fibrosis protein, P-glycoprotein, when misfolded, remains trapped inside cells.

"We thought we would try throwing some capsaicin on the P-glycoprotein mutants just to find the optimal concentration," said Clarke. "And lo and behold, all of our misfolded P-glycoprotein mutants popped to the cell surface. For a week we thought, ‘Wow, this must be a cure for cystic fibrosis!’ "

When the pair acquired cells that made mutant cystic fibrosis protein, they were immediately disappointed. Nothing happened despite the researcher’s visions of the proteins bobbing to the cellular surface.

But they had made an important discovery nonetheless—misfolded proteins could be rescued. Soon they tested cyclosporin and a raft of other cancer drugs, concluding that these compounds, too, revived misshapen P-glycoprotein, findings they disclosed in 1997 in the Journal of Biological Chemistry.

Although important for cystic fibrosis research, the work was not directly applicable to the problem of cancer drug resistance. However, it led to the next logical step: If mutant P-glycoprotein molecules could be fixed, could normal versions be crippled?

The answer, published in a 1999 FASEB Journal article, is a short yes. But there remained a big problem—the drugs Clarke and Loo used to block P-glycoprotein’s surface migration were extremely toxic. They needed something more tolerable.

A search through the pharmacopeia led them to a bottle of disulfiram. The drug was cheap and in widespread use, definite pluses. But the information label really caught their eye. It said that disulfiram shuts down an alcohol metabolic pathway by reacting with cysteine residues, important amino acid building blocks of an alcohol-reducing enzyme. P-glycoprotein also contains key cysteine residues, so disulfiram should disable it, they reasoned.

"We thought it would react at the cell surface and shut off P-glycoprotein there," said Clarke. "But disulfiram doesn’t work like that at all. It was because they labeled their compound wrong that we gave it a try."

When Clarke and Loo dosed kidney cell lines with disulfiram, they instead noticed that very few P-glycoprotein molecules surfaced. Further inspection revealed that disulfiram reacts with the molecules as they form inside the cell. Crumpled and broken, they never migrate to the cell membrane. The result: cell lines with increased sensitivity to standard chemotherapy drugs like vinblastine.

Gottesman calls the research "a clever new idea," adding, "The issue of course is will this be clinically useful? It depends on how much of the drug you need and whether it [inhibiting P-glycoprotein folding] is a specific phenomenon." Disulfiram may interfere with other key proteins, said Gottesman, something to be sorted out in further preclinical and clinical studies.

For Clarke, future plans include testing disulfiram in actual cancer cells that overexpress P-glycoprotein (he and Loo were using kidney cells transfected with a recombinant form of the protein) and performing animal studies to see how disulfiram really works in a living system.

Then there’s the obvious. "Of course, if someone got a whole bunch of alcoholics on Antabuse to volunteer, that would be worthwhile," said Clarke, tongue somewhat in cheek. "We need somebody to round them up and test their P-glycoprotein activity."



             
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