It's all good and well to sequence a gene and then identify the aminoacids in the protein it expresses. But good enough is no longer goodenough when it comes to designing drugs at the molecular level thatwill inhibit or promote that protein.

It's a truism that function follows form, and increasingly now thatmeans solving the 3-D structure of proteins with potential.

"If you just get an amino acid sequence," said structural biologist andmedicinal chemist Stephen Fesik, "that gives you very limitedinformation about the protein itself. And when you have the 3-Dstructure, you see the shape of the molecule, what amino acidresidues are on the outside of it, which ones you might mutate, spotthe hot spots where another protein might bind."

Fesik directs nuclear magnetic resonance (NMR) research at thePharmaceutical Discovery Division of Abbott Laboratories, in AbbottPark, Ill. He is senior author of an article in today's Nature, titled:"NMR structure and mutagenesis of the Fas (APO-1/CD95) deathdomain."

Just as life and death are two sides of the same coin, cell division andprogrammed cell death (apoptosis) mark every step of an organism'sexistence. And one of the main executioners in apoptosis is a trendyimmune-system cytokine called Fas.

Trendy because apoptosis is increasingly seen by drug discoverers asa key arbiter of wellness and illness, while Fas currently concentratesthe minds of patent attorneys the world over.

"Apoptosis gone awry contributes to many disease states, such ascancer, autoimmunity and neurodegeneration," Fesik told BioWorldToday. "And Abbott is interested in apoptosis, specifically in the areaof cancer."

He cited a recent finding that FasLigand increases in cancerous cells,"and through this increase, you can essentially kill surrounding cells.It has an effect," Fesik explained, "that could aid in treating tumormetastasis by killing the cells around it, by increasing FasLigand.This protein then interacts with the Fas receptor, which then goes ondownstream to kill the cell."

That stream is a cascade of post-Fas proteins that carry out itsapoptotic verdicts. Fesik describes how that relay system works:

"Different types of cells, particularly T cells, contain Fas, which isthe receptor for FasLigand. The portion of that receptor that lies inthe cell's cytoplasm includes an amino-acid sequence called theDeath Domain. This then interacts with a signaling partner fartherdownstream, called FADD (Fas-Associated Death Domain), whichhas its own Death Domain."

Fas and FADD then interact with each other and with FLICE(FADD-Like Interleukin 1b Converting Enzyme), a protein near theend of the run. It activates enzymes that executes the programmedcell death response.

Treatment of autoimmune diseases is another Fas-linked area: "Whenthe body has activated T cells to meet an immune challenge," Fesikobserved, "you'll have FasLigand being expressed. A normal thingthat happens is after the immune response is over, you must then stopit. If not, those T cells will go on and on, acting on all different partsof the body.

"The way that it's regulated," he continued, "the way to stop those Tcells, is getting this FasLigand that's expressed. They kill off theautoreactive T cells by binding to Fas as part of those T cellsthemselves, and then kill them.

"But if you have mutations that don't allow you to kill off the T cells,this defect displays itself as an autoimmune-like disorder."

Laying Bare Death Domain's 3-D Shape

The Fas protein is about 315 amino acids long. Fesik and his co-authors analyzed the 117-residue stretch, comprising its DeathDomain, near the tail of the molecule's C-terminal.

After portraying this region's 3-D structure, they went on to identify,by site-directed mutagenesis, the amino acids that are important forFas's interactions with itself and with FADD.

"Death Domains," Fesik pointed out, "are present on other proteins,such as the tumor necrosis factor's receptor (TNF-R)." Viamutagenesis, they noted that the TNF-R inactive mutants "weresprinkled across the entire protein, unlike the Fas mutants, whichseemed to be pretty well localized to one area."

It looked to Fesik as if mutants in the TNF-R molecule's DeathDomain "could be disrupting the protein fold, rather than directlyinhibiting protein-protein interaction."

Having now analyzed the 3D Death Domain structure of Fas, andearlier this year that of Bcl-XL an inhibitor of programmed cell deathin humans, Fesik is now primed to "identify targets that make themost sense" in applying apoptosis to drug discovery, "and to set upassays to find compounds that will attack cancer cells."

"As an example," he suggested, "one could think about designingmolecules that would cripple proteins, such as Bcl-XL, whichnormally inhibit apoptosis, and are upregulated in certain types oftumors. Those are the cancers that become resistant to chemotherapyagents, which act by enhancing apoptosis."

Treatment of autoimmune diseases are another Fas-linked area."When the body has activated T cells to meet an immune challenge,"Fesik observed, "you'll have FasLigand being expressed. A normalthing that happens," Fesik continued, "is after the immune response isover, you must then stop it. If not, those T cells will go on and on,acting on all different parts of the body.

"The way that it's regulated," he continued, "the way to stop those Tcells, is getting this FasLigand that's expressed. They kill off theautoreactive T cells by binding to Fas as part of those T cellsthemselves, and then kill them.

"But if you have mutations that don't allow you to kill off the T cells,this defect displays itself as an autoimmune-like disorder." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.