SCIENCE & TECHNOLOGY PERFECT FIT To specifically target a small-molecule inhibitor (carbon is green; nitrogen is blue) to the ATPbinding pocket of the kinase c-Src, researchers enlarge the binding pocket by replacing a threonine residue (yellow mesh) with glycine (red). The protein surface is shown in gray.
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UNTANGLING THE SIGNALING WEB Various chemical approaches are being used to tease apart signal transduction pathways AMANDA YARNELL, C&EN WASHINGTON
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talk goes on in a cell: Information is relayed by proteinprotein interactions, protease cascades, and highly choreographed stages of phosphorylation and dephosphorylation. Untangling these signaling pathways has mostly depended on the tools of genetics—deleting or altering a gene in yeast or mice and then observing the effects on signaling. But the relatively young field of chemical genetics is making its mark on signal transduction. "Chemists are developing a rich variety of chemical approaches that enable us to understand and manipulate signal transduction pathways," says KevanM. Shokat, professor of cellular and molecular pharmacology at the University of California, San Francisco, professor of chemistry at
U C Berkeley, and winner of this year's Eli Lilly Award for Fundamental Research in Biological Chemistry Shokat organized a symposium sponsored by the Division of Biological Chemistry at the American Chemical Society national meeting in Boston last month that showcased the work of a diverse group of young chemists who are using small molecules to probe and control various modes of signaling. The regulation of a variety of signaling proteins—including receptors, kinases, and proteases—depends on their interactions with other proteins. Timothy P. Clackson, senior vice president for science and technology at Ariad Pharmaceuticals in Cambridge, Mass., described his company's work to control protein-protein interactions in cells. "Our goal is to be able
to specifically induce a given protein-protein interaction at will, in real time," Clackson said. To do so, Ariad has turned to chemical dimerizers, small molecules that act to bridge two proteins. The idea isn't new to nature: The natural product rapamycin— used clinically to prevent rejection of transplanted organs—exerts its immunosuppressive effect by dimerizing two proteins, FKBP and FRAP, that don't interact in the absence of the drug. Although Clackson predicts that, within the next half century, "chemists will be able to make small molecules that bring together any two proteins," he admits that such designer dimerizers pose a formidable design challenge for synthetic chemists. SO FOR NOW, he and his coworkers are fusing a mutant form of FKBP—already the target of a designed dimerizer d r u g to a signaling protein of interest. W h e n cells that express the fusion FKBP-signaling protein are treated with the drug, it brings together the two copies of the signaling protein via their FKBP domains. Clackson's team members and their collaborators are using this dimerizer system to dissect the fibroblast growth factor (FGF) signaling pathway thought to be involved in prostate cancer. Dimerization of the receptor for FGF, signaled by FGF binding, is thought to induce the early stages of tumor growth. To study the details of this signaling pathway, the team is using mice that express chimeric receptorFKBP proteins in their prostate tissue. When the mice are treated with dimerizer, they undergo rapid prostate cell proliferation that precedes tumor growth. This inducible mouse model of prostate cancer is being used to study the early stages of tumorigenesis. Ariad is also engineering nature's rapamycin-FKBP-FRAP system to control red blood cell levels in primates—a first
"When we perturb a kinase with a small molecule instead of genetics, we can see how the cellular signaling system responds in seconds instead of months/' 26
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step toward clinical use to treat anemia in humans. Red blood cell levels are regulated by the hormone erythro poietin. To control the production of erythro poietin, the team fuses FKBP and FRAP to D N A binding and tran scription activation domains, respectively. W h e n the chimeric proteins are introduced into monkeys along with an erythropoietin gene altered to be con trolled by the transcrip tion factor domain, the Cravatt addition of rapamycin induces hormone production. In the presence of rapamycin, the mon keys quickly and reversibly increase ery thropoietin production—and red blood cell levels. The monkeys have remained sensitive to rapamycin for as long as four years. Since rapamycin can be administered orally, the method is an attractive alterna tive to hormone injections. MOST PROTEINS involved in signal trans duction contain both protein-protein in teraction domains and domains that cat alyze reactions such as proteolytic cleavage and phosphate transfer. But although a great deal of data on protein-protein in teractions have been compiled, less is known about the activity and regulation of signaling proteins' catalytic domains. Associate professor Benjamin F. Cravatt of Scripps Research Institute has developed a detection strategy that can be used to probe how enzymatic activities such as pro teolytic cleavage and phosphate transfer activate signaling pathways. His strategy depends on small-molecule probes direct ed to the active sites of members of enzyme classes involved in signaling, including pro teases, kinases, and phosphatases. Proteomics methods have allowed pro filing of the total abundance ofproteins in different cells and disease states. "But we think the real story is likely to be not which proteins are present but which proteins are active," Cravatt said. So instead of look ing at protein abundance, Cravatt's detec tion strategy profiles which proteins are active in a particular cell or disease state. Cravatt's first target was a class of pro teases, esterases, and lipases known as ser ine hydrolases that makes up 1 to 2% of eukaryotic proteins. By tethering a fluorescent tag to a fluorophosphonate inhibitor known to bind specifically and covalently in the HTTP://PUBS.ACS.ORG/CEN
active sites of serine hydrolases, Cravatt's team created a probe that can be used to tag only those serine hydrolases that are active. Cravatt and graduate stu d e n t N a d i m Jessani have shown that their fluorophos phonate probes can be used in melanoma and breast can cer cell lines to distinguish noninvasive from in vasive t u m o r cells [Proc. Nat. Acad. Set USA, 9 9 , 10335 (2002)]. "The inva sive cell lines share serine hydrolase ac tivity signatures that reflect their invasive nature more closely than their cellular origin," Cra vatt tells C&EN. Interestingly, activity signa tures for serine hydrolases that remain inside the cell do not Shokat p r e d i c t t u m o r invasiveness well, but activity signatures from se- I creted and membrane-bound serine hy drolases can accurately differentiate be- I
tween invasive and noninvasive cell lines. Cravatt hopes that probes can be found to profile the activity of other important signaling enzyme families such as kinas es—crucial players in signaling cascades that regulate cellular processes ranging from transcription to apoptosis. La Jolla, Calif.-based ActivX, a company Cravatt cofounded, has designed fluorescently la beled probes—based on natural products, endogenous ligands, and small-molecule inhibitors —that co valently bind in the active sites of a broad range of kinases, ac cording to David A. Campbell, vice presi dent for chemistry Campbell and his coworkers have also found a few probes that, instead of being broadly reactive across the kinase family, are highly specific for several key signaling kinases. Such smallmolecule inhibitors would allow chemists
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