FKBP12 rapamycin binds effector, inhibiting cell proliferation

Growth factor

^ 3 & Effector Α ί * 1 ψ^ protein

FKBP12-rapamycin Cell proliferation

Binding of growth factor such as interleukin-2 to receptor (red) on cell surf ace triggers multistep signaling process leading to cell proliferation. Ef­fector protein plays a key role in this signaling pathway. Binding of FKBP12-rapamycin complex to effector protein blocks signaling pathway, in­hibiting cell proliferation.

In work that could lead to the development of new drugs and a better understanding of

chemical signaling in cells, sever­al research groups have identified a previously unknown mammali­an protein as the long-sought mo­lecular target of the immunosup­pressant drug rapamycin.

Rapamycin, a bacteria-derived natural product, is undergoing Phase II clinical trials as an im­mune system suppressant for or­gan transplantation and is be­lieved to have other possible clinical applications. But the di­rect molecular target, or effector, of the drug in mammalian cells has been a mystery.

Now, in an extraordinary com­petition that reflects the intense interest swirling around rapamy­cin, four research groups have in­dependently isolated and identi­fied that target, which is struc­turally related to lipid kinase enzymes.

Rapamycin is being tested as a possible alternative to cyclospor­in A and FK506, the two immu­nosuppressant drugs currently used for organ transplantation. Cyclosporin A, made by Sandoz, has been an approved immuno­suppressant for more than a de­cade. However, it has serious side ef­fects including diabetes, hypertension, abnormal hair growth, and kidney, liv­er, and nerve damage.

FK506, produced by the Japanese drug maker Fujisawa, was approved early this year for treatment of liver transplant patients. It is more potent than cyclosporin A but shares many of that drug's deleterious side effects.

The side effects of cyclosporin A and FK506 are similar because the two drugs have the same mechanism of ac­tion: inhibition of an enzyme called cal-cineurin. Rapamycin will likely show different—and, it is hoped, less se­

vere—side effects than cyclosporin A and FK506 because it has a different mechanism of action. Rapamycin also is believed to have potential clinical applicability to autoimmune diseases and asthma, in addition to its use for immunosuppression.

Inside cells, the immunosuppres­sants bind to enzymes called immu-nophilins: Cyclosporin A binds to cy-clophilin, and FK506 and rapamycin bind to FKBP12 (FK506-binding pro­tein 12). Hence, many researchers ini­tially speculated that the drugs acted just by inhibiting the immunophilin enzymes.

But in 1991, chemistry profes­sor Stuart L. Schreiber of Har­vard University, Irving Weiss-man of the pathology depart­ment at Stanford University's School of Medicine, and cowork­ers discovered that cyclosporin A and FK506 don't just inhibit the immunophilins. They found that the cyclophilin-cyclosporin A and FKBP12-FK506 complexes have an additional function: inhi­bition of calcineurin, a phospha­tase enzyme in the signaling path­way that leads to activation of Τ cells.

Rapamycin interferes with a different signaling pathway— one in which the binding of growth factors such as interleu­kin-2 to growth factor receptors on cell membranes initiates an intracellular signaling process leading to cell proliferation.

The target of FKBP12-rapamy-cin—the molecule with which it binds, causing blockage of the signaling pathway—remained unknown. However, last year, biochemistry professor Michael N. Hall and coworkers at the University of Basel, Switzerland, identified and cloned the genes for two proteins that were shown genetically to be the like­

ly targets of FKBP12-rapamycin in yeast. The proteins, TORI and TOR2 (targets of rapamycin 1 and 2), are structurally similar to the phosphatidylinositol kin­ases, enzymes required for normal cell-cycle function.

Four independent groups have now found that a protein closely related to TORI and TOR2 is also the direct tar­get of FKBP12-rapamycin in mammali­an cells. First to publish are Eric J. Brown, Mark W. Albers, Tae Bum Shin, Kazuo Ichikawa, Curtis T. Keith, and Schreiber of the department of chemis­try and Howard Hughes Medical Insti­tute at Harvard University and Wil-

6 JULY 4,1994 C&EN

IMMUNOSUPPRESSANT DRUGS Rapamycin target protein found

NEWS OF THE WEEK

liam S. Lane of the Harvard Micro-chemistry Facility.

In a paper last week in Nature [369, 756 (1994)], Schreiber and coworkers report isolation and cloning of a hu­man protein called FRAP (FKBP12-rapamycin associated protein) and the complete 2,549-amino acid sequence of the protein. By correlating the ability of rapamycin variants to bind FRAP and inhibit cell proliferation, they provide evidence that FRAP is rapamycin's functional target.

A second group—David M. Sabatini and Solomon H. Snyder of the depart­ment of neuroscience at Johns Hopkins University School of Medicine and Hediye Erdjument-Bromage, Mary Lui, and Paul Tempst of the molecular biolo­gy program at Memorial Sloan-Kettering Cancer Center, New York City—isolated the protein from rat brain cells and cloned the rat version of the gene. In a paper to appear in the July 15 Cell they call the protein RAFT (rapamycin and FKBP12 target).

Meanwhile, Candace J. Sabers, Josie M. Williams, and Robert T. Abraham of the department of immunology at the Mayo Clinic, Rochester, Minn., with Gregory Wiederrecht, Mary M. Martin, and Francis J. Dumont of the depart­ment of immunology research at Merck Research Laboratories, Rahway, N.J., also isolated the protein from rat brain cells, naming it mTOR (mammalian tar­get of rapamycin). In a paper submitted to the Journal of Biological Chemistry, they report a partial sequence of the protein and binding data supporting its role as the functional target of rapamycin.

Moreover, Yanqiu Chen and Koji Nakanishi of the department of chemis­try at Columbia University, Katherine L. Molnar-Kimber of the inflammatory dis­eases division of Wyeth-Ayerst Research (Princeton, N.J.), and coworkers have submitted a paper to Biochemical & Bio­physical Research Communications report­ing isolation of an FKBP12-rapamycin effector protein (called p210) from T- and B-cell clones and primary human Τ cells. Wyeth-Ayerst senior research fellow Suren N. Sehgal, who discovered rapa­mycin in a soil sample in 1975, says the group is currently sequencing the protein.

The work of all these groups reveals that the endogenous (naturally occur­ring) protein EKBP12 binds with the drugs FK506 and rapamycin to form complexes that, in turn, bind two sig­naling molecules—calcineurin, which

helps control cell activation, and the FKBP12-rapamycin effector protein, which helps regulate cell proliferation. 'There has to be some reason that bind­ing of these natural products to a ubiqui­tous protein like FKBP12 has such spe­cific, critical effects on the cell cycle/'

says Schreiber. He predicts that future studies may well focus on finding endo­genous substances that are possibly be­ing mimicked by FK506 and rapamycin and on elucidating the normal function ofFKBP12incells.

Stu Borman

Fears eased about CFC substitutes' breakdown Research by U.S. Geological Survey (USGS) scientists has eased a nagging worry about the environmental accept­ability of some important substitutes for ozone-destroying chlorofluorocarbons (CFCs). The work shows that a potential­ly toxic degradation product of the substi­tutes is decomposed by soil microbes.

CFCs are being replaced by hy-drofluorocarbons (HFCs) and hydro-chlorofluorocarbons (HCFCs) deliber­ately designed to break down in the lower atmosphere. Although much saf­er for the ozone layer, the alternatives' reactivity has raised concern that their degradation products might them­selves harm the environment.

Unlike CFCs, the hydrogen atoms on HFCs and HCFCs make them suscepti­ble to attack by hydroxyl radicals in the troposphere. Trifluoroacetic acid (TFA) is the primary degradation product of HFC-134a (CH2FCF3) and HCFC-123 (CHC12CF3), two alternatives that play

major roles in the CFC phaseout re­quired by the Montreal Protocol on Substances That Deplete the Ozone Layer (see page 12).

What little is known about TFA's tox­icity indicates it is not very toxic to hu­mans, plants, or animals, says Forrest G. Chumley, DuPont's research manager for environmental biotechnology and head of a chemical industry consortium that is studying TFA's environmental fate. But initial studies uncovered no chemical or biological degradation mechanisms for the molecule, implying TFA could accumulate in the global bio­sphere with unexpected consequences.

Now, USGS researchers Pieter T. Vis-scher, Charles W. Culbertson, and Ro­nald S. Oremland report that TFA is de­graded by bacteria present in lake and salt marsh bottom sediments [Nature, 369, 729 (1994)]. Under anaerobic condi­tions, the microbes remove all the fluo­rine atoms to produce acetic acid, which

Nature warps meaning of trifluoroacetic add finding For methane expert Ronald S. Orem­land, finding that soil bacteria break trifluoroacetic acid (TFA) into carbon dioxide and methane or fluoroform (CHF3) is good news for the environ­ment. It implies that TFA, a major degradation product of certain chloro-fluorocarbon substitutes, is unlikely to accumulate in the biosphere.

But Nature changed the paper by Oremland and his colleagues at the U.S. Geological Survey so it mistak­enly conveys the opposite impres­sion. Nature's editorial staff added a sentence saying—wrongly—that fluo­roform is "a potential ozone-deplet­ing compound with a much longer atmospheric lifetime than the parent compounds/'

"I'm infuriated/' Oremland tells C&EN. "Nature inserted a sentence that I did not write. They did not flag the change so we didn't pick it up on the galleys. They changed the text,

then highlighted the change in a press release."

Fluoroform does have a long atmo­spheric lifetime and may prove to be of concern as a potential greenhouse gas. And the potential for molecules containing the unusually stable trifluo-romethyl group (CF3) to destroy ozone through reaction cycles involving alkoxy (CF30) and peroxy (CF302) radicals was a significant worry to the atmospheric science community a year ago. But definitive work published in Science in January [263, 71 (1994)] found fluoroform does not deplete ozone (C&EN, Jan. 10, page 5).

Gabrielle Walker of Nature's edito­rial staff says the sentence was added to the Oremland paper on the advice of one of the paper's referees. The journal has already issued a corrected press release and will publish a cor­rection to the paper in a few weeks, she adds.

JULY 4,1994 C&EN 7