Lights, Camera, Action in the Membrane

Courtesy of AfCS-Nature Signaling Gateway (www.signaling-gateway.org) A WORK IN PROGRESS Complex as it is, this cell signaling map isn't finished. But since every interaction shown is a potential point for therapeutic intervention, understanding the wiring of these messaging systems could deliver new drugs to the clinic. Signal transduction wasn't exactly the first thing that came to mind when my mother told me that she had medullary thyroid cancer. Thoughts of not having my mother around when I finally got married or had my first baby were more immediate. Now that she's in remission, I've had the chance to marvel at how a mutation in a receptor known as c-Ret could cause the calcitonin-producing C cells of the thyroid to proliferate uncontrollably.1 Known as a receptor tyrosine kinase (RTK), membrane-bound c-Ret is the first player in one of the many signal transduction pathways of the C cells. In response to ligand binding, RTKs--such as the receptors for insulin, platelet-derived growth factor, and epidermal growth factor-- phosphorylate either themselves or nearby proteins to initiate a signaling cascade.2 Researchers now know that many diseases beyond cancer, including diabetes, heart disease, and asthma, ultimately stem from messaging systems gone awry. By coupling modern research tools with"the multidisciplinary attitude necessary to capitalize on that knowledge, investigators are accelerating the pace of discovery to augment the arsenal of disease-fighting drugs. CASE IN POINT Two-year-old Serenex, based in Durham, NC, blends proteomics with elements of combinatorial chemistry, genomics, and bioinformatics. This strategy has created a synergistic atmosphere that is boosting the company's research efforts, says Timothy Haystead, associate professor of pharmacology at Duke University and Serenex's scientific founder. "The sum is better than the parts." In sifting through cell lysates, Serenex researchers exploit the fact that every RTK contains an ATP-binding motif. By using a proprietary method to tether ATP to beads, Haystead can isolate those relatively few proteins--a so-called subproteome--that bind to adenosine or other purines. They then identify the resulting proteins, each a potential drug target, with mass spectrometry.3 "Our method helps us to find the needle without going into the haystack," says Paul Graves, a researcher in Haystead's Duke laboratory. "We can target the part of the proteome that we're interested in." The company is scrutinizing the adenosine-binding subproteome, looking for potential drug targets and therapeutics. Haystead explains that a candidate drug found to bind both the RTK and cytoplasmic kinases in the same signaling pathway may possibly serve as a more effective drug than one that binds only an RTK. "We're particularly interested in inhibitors that exhibit polypharmacology," he says, pointing to the success of Novartis' cancer drug Gleevec, which binds an RTK and other downstream kinases. Serenex hones its research using a multidisciplinary approach. After biologists perform the binding experiments, a team of nine in-house chemists use the information to fine-tune the ligands. Instead of aimlessly testing thousands of different compounds, Haystead explains, Serenex researchers may zone in on 100 and incrementally modify them to increase specificity and affinity, thus improving the pharmacokinetics and solubility. BEAD TOO Investigators need not have their own drug-development facility to study RTKs, as reagent manufacturers offer a vast array of products for signal transduction researchers. To get a high-level picture of phosphoprotein abundance and distribution, for instance, scientists can label cells with radioactive orthophosphate, but that's a dangerous and messy technique. Instead, they can use specific reagents, such as the fluorescent Pro-Q® Diamond phosphoprotein gel stains, developed by Molecular Probes of Eugene, Ore. Using Pro-Q Diamond technology, researchers can avoid the expense and risk of using radioactive phosphorus-32, says Joe Beechem, chief scientific officer of Molecular Probes, now a part of Invitrogen of Carlsbad, Calif. "For the first time, people can look at the global phosphoproteome and all the phosphorylation-coupled events," he says. They can then focus their work with more precise reagents, such as Beadlyte® assays from Upstate Cell Signaling Solutions of Charlottesville, Va. Beadlyte is based on xMAP® technology from Luminex in Austin, Texas. xMAP employs an array of 100 different polystyrene microspheres, each bearing a unique ratio of two dyes and, therefore, a distinct spectral signature, says Grant Gibson, product marketing manager at Luminex. Because each bead can be uniquely identified, researchers can test for as many as 100 different analytes per assay, instead of having to perform them one at a time. In Upstate's Beadlyte implementation, each Luminex bead is coupled to antibodies directed against a specific phosphorylated RTK or other phosphorylated kinase involved in signal transduction. "Besides the singular advantage of looking at multiple cellular responses at once, the Beadlyte pathway profiling allows scientists to look at the crosstalk between the pathways and to find new pathways that are activated from the cellular stimulus," says Gregory Moore, Upstate's director of business development. GOT GPCR? While RTKs can be prime targets for drugs, guanine protein-coupled receptors (better known as G protein-coupled receptors, or GPCRs) are the target of nearly half of all pharmaceutical drugs in clinical use today, says William Simonds, senior clinical investigator at the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. Approximately 1,000 GPCRs have been cloned, but the functions of many are still unknown. The challenge in elucidating GPCR-stimulated signaling pathways comes from the multiple binding events that occur after the ligand binds the receptor. The activated GPCR goes on to bind one of 20 alpha subunits of membrane-bound G proteins, which may have been associated with any of five beta subunits and 12 gamma subunits. All three, in part, determine which specific signaling cascade is touched off.4 "When you activate a G-protein, there are so many different possible expressions that cells might give you," says Simonds. "It's our ignorance that we can't predict which one just by looking at the DNA sequence of the GPCR." Researchers have developed several ways to study GPCRs and G proteins, which are involved in a broad spectrum of diseases as diverse as night blindness and cancer. The widely used beta-blocker drugs directly target beta-adrenergic receptors, GPCRs which were first cloned by Robert Lefkowitz, professor of medicine and biochemistry at Duke and a Howard Hughes Medical Investigator. He cofounded Norak Biosciences of Research Triangle Park, NC, to commercialize its Transfluor® technology, which was developed in the lab of his colleague and collaborator, Marc Caron, professor of medicine at Duke. The technology is based on the movement of ligand-bound GPCRs from the membrane into the cytosol, guided by the accessory protein beta-arrestin. By fusing beta-arrestin to green fluorescent protein, Transfluor makes it possible to track the movement of GPCRs,5 which indicates the cell's densensitization to the effects of a stimulant or drug and the downregulation or attenuation of a signal. At the University of New Mexico, regents professor Larry Sklar and research professor Bruce Edwards have developed a high-throughput method of studying GPCRs using flow cytometry. Called HyperCyt™, the team's invention connects an autosampler to the sample port of a flow cytometer, making flow cytometers compatible with the multiwell plate assays commonly found in high-throughput labs.6 Sklar and Edwards have developed a way to solubilize GPCRs. They attach the GPCRs to beads, providing access to both the extracellular and intracellular parts of the receptor. "This particle-based assay works both sides of the receptor so we can characterize the molecular action of drugs," says Sklar. With GPCRs isolated from the rest of the proteome, researchers can take advantage of timesaving multiplexed methods. Several particle-based assays, specific for different agonists and antagonists, can be evaluated simultaneously. Sklar and associate professor Eric Prossnitz are building a library of solubilized GPCRs. "It's hit and miss," Sklar says. "We screen a large number of de-tergents and figure out which one is best at solubilizing each GPCR." Currently, Sklar has solubilized just a handful of GPCRs and has set a goal of solubilizing about a dozen in the next two years.7 He is in the process of patenting HyperCyt and its application to solubilized receptors. ION CHANNEL SURFING Ligands sometimes deliver messages by binding to membrane-bound ion channels, which then allow ions to flood into the cell, causing a change in electric potential. Measuring the shift in membrane depolarization can offer evidence for the efficacy and binding specificity of a drug candidate, as many ion channels have been implicated in disease pathophysiology.8 Measuring electric potential has traditionally been accomplished using patch-clamp experiments, but these are far too slow for today's high-throughput needs. Gothenburg, Sweden-based Cellectricon developed its Dynaflow™ Chip, which relies on a microfluidic system to successively deliver different ligands to a single patch-clamped cell. The chip is designed with a series of 16 parallel channels, each filled with a different ligand. In between each channel lies another channel filled with a buffer, which washes the cell after its exposure to each ligand. Fluidic physics prevents the different ligands from mixing with one another.9 "You can expose the cell to 1,000 different drugs in one day, which is 10 to 100 times more than before," says Cellectricon cofounder Owe Orwar, who is also professor of physical chemistry at Chalmers University of Technology in Gothenburg. "We've really filled a need in the industry." PanVera of Madison, Wis., offers another way to measure changes in electric potential with its Voltage Sensor Probes, which utilize fluorescence resonance energy technology (FRET). The probes consist of a membrane-bound coumarin-phospholipid donor and an acceptor oxonol dye. Both are loaded into the cell membrane. When the electric potential changes, the negatively charged acceptor dye moves from the membrane's inner face toward the donor located at the membrane's outer face. This causes excitation of the oxonol dye and emission of a fluorescent signal. "With voltage-sensitive probes, you can truly run high-throughput screening for drugs that activate or inhibit ion channen activity," says John Printen, director of R&D at Invitrogen, which owns PanVera. Invitrogen is currently looking at ways to increase the speed of the probes' response, and it is developing dyes that display different voltage sensitivities and FRET wavelengths. OPTICAL SOLUTIONS Researchers can detect ligand-receptor and protein-protein binding via a variety of optical metrics, including fluorescence intensity, fluorescence polarization, homogeneous time-resolved fluorescence, luminescence, bioluminescence, bioluminescence resonance energy transfer, and absorbance. Researchers may gain an advantage, however, by measuring fluorescence lifetime (FLT), says David Yost, manager of strategic technical development at Tecan in Groedig, Austria. An inherent feature of every fluorophore, FLT is the average time a fluorescent molecule spends in an excited state before returning to the ground state. Rather than just assessing intensity, researchers who measure FLT can follow the rate of decay of an excited fluorophore, enabling them to ascertain the strength of ligand-receptor binding, Yost explains. Because FLT is a rate-constant measurement, it is not confounded by factors such as autofluorescence, variable volumes, and meniscus effects, which can affect intensity measurements. "It's a very powerful technique to measure the subtle changes in the environment of the binding," he says. Tecan recently introduced Ultra Evolution as one of a few instruments available that can measure FLT, as well as other forms of fluorescence and luminescence. The company is currently working on opportunities to allow FLT to become more commonplace in high-throughput screening laboratories, Yost says. THE BOTTOM LINE Screening of drug candidates has become more efficient than ever with the help of improved technologies and high-throughput, multiplexing methods. Still, developing a drug that will affect a signaling pathway is tedious work, says Jim Inglese, a senior research fellow at Merck Research Laboratories, North Wales, Pa. "There's no rulebook," he says, explaining that researchers must blaze new trails and find unique solutions with each new drug that they develop. Obstacles such as toxicity and bioavailability, which can't always be predicted early in the process, can pose real challenges. Inglese is not complaining, though. Having studied GPCRs for five years in Lefkowitz's Duke laboratory using traditional 20th-century approaches, he's relieved at having the convenience of today's latest technologies. "The advances over the last eight years [since I was a postdoc] have been quite fascinating and enabling," Inglese says. "They cut down dramatically on the amount of time needed in the screening process itself. We now talk in days and weeks as opposed to months and years." That's good news for my mom. Though regular imaging tests have revealed no additional tumors, her calcitonin levels remain sky-high, indicating abundant numbers of transformed thyroid C cells somewhere in her body. Finding a treatment to eradicate those cells would be a Godsend. Says her physician, "with the pace of science moving quickly, I'm optimistic that we'll have much-improved therapies." Laura Lane (laura_lane@yahoo.com) is a freelance writer in San Francisco. References 1. A. Machens et al., "Early malignant progression of hereditary medullary thyroid cancer," N Engl J Med, 349:1517-25, 2003. 2. T. Hunter, "Signaling--2000 and Beyond," Cell, 100:113-27, 2000. 3. P.R. Graves, T.A. Haystead, "A functional proteomics approach to signal transduction," Recent Prog Horm Res, 58:1-24, 2003. 4. W.F. Simonds, "G protein-regulated dysfunction in human disease," J Invest Med, 51:194-208, 2003. 5. L.M. Luttrell, R.J. Lefkowitz, "The role of beta-arrestins in the termination and transduction of G protein- coupled receptor signals," J Cell Sci, 115:455-65, 2002. 6. A. Waller et al., "High-throughput screening of G protein-coupled receptors via flow cytometry," Comb Chem High T Scr, 6:389-97, 2003. 7. L.A. Sklar et al., "Solubilization and display of G protein-coupled receptors on beads for real-time fluorescence and flow cytometric analysis," Biotechniques, 28:976-85, 2000. 8. S. Hatta et al., "Ion channels and diseases," Med Electron Microsc, 35:117-26, 2002. 9. A. Constans, "Probing ion channel function," The Scientist, 17[19]:41, Oct. 6, 2003">The Scientist, 17[19]:41, Oct. 6, 2003. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Lights, Camera, Action in the Membrane

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