functionalizing both ends of a twocarbon olefin with tin and then using the afunctional species to link the ends of a linear rapamycin precursor having io dines at both termini. The group is currently using stitching cyclization to create rapamycin analogs. "Now we have the ability to bring in dif ferent kinds of fragments ... to make a variety of analogs that differ from ra pamycin in this region," says Nicolaou. Rapamycin and another compound called FK506 are currently receiving considerable attention as possible cy closporin alternatives. Rapamycin, FK506, and cyclosporin all inhibit intra cellular signal-transduction pathways in Τ cells, key cells of the mammalian immune system. This inhibition inter feres with the cells' ability to proceed through the cell cycle of biosynthesis, DNA synthesis, and mitosis, thus caus ing immunosuppression. Cyclosporin and FK506 act by form ing complexes with immunophilins (immunosuppressant-binding proteins).
Binding of cyclosporin-immunophilin and FK506-immunophilin complexes to the enzyme calcineurin, a common tar get, is a key step leading to the immuno suppressive actions of these drugs. Rapamycin also forms an immunophilin complex. The target compound with which this complex binds is still unknown but is being very actively sought. Rapamycin analogs made available by total synthesis will be potentially useful as immunosuppressants. They could also find use for studies of the role played by rapamycin in signal transduction. Several other research groups are known to be pursuing or to have achieved (but not yet published) alterna tive methods for the total synthesis of rapamycin. Among them are those of chemistry professors Stuart L. Schreiber of Harvard University, Samuel J. Danishefsky of Yale University, Amir H. Hoveyda of Boston College, and Amos B. Smith III of the University of Pennsyl vania. Stu Borman
Polyclonal catalytic antibodies characterized In a clever twist on studies of catalytic antibodies, chemists at the University of Texas, Austin, have characterized the catalytic activity of the entire en semble of antibodies produced in re sponse to immunization with a mole cule designed to mimic the transition state of a desired reaction. Compared with monoclonal antibod ies, which have been the focus of most previous catalytic antibody research, these "polyclonal antibodies" offer a more rapid and cost-effective alternative for evaluating the catalytic activity of an tibodies, argues Brent L. Iverson, the University of Texas assistant chemistry professor who directed the research. 'This should allow us to assess in a quantitative way how to design better haptens, the molecules that elicit an im mune response," Iverson says. "By that, I mean haptens that are more 'catalogenic/ ones that will elicit antibodies with increased catalytic activity. That is not information that is generally available without studying a large number of monoclonal catalytic antibodies." Additionally, Iverson says, character izing polyclonal antibodies isolated di rectly from antisera is dramatically less expensive than producing and charac terizing monoclonal antibodies. And
Early research on catalytic antibodies focused on monoclonal antibodies for good reason, Iverson points out. Mono clonal antibodies are produced by tak ing antibody-producing cells from the spleen of an experimental animal and fusing them with a particular kind of tumor cell to produce what is known as a hybridoma. Individual hybridoma cells are selected and grown in culture to produce antibodies. Because the entire culture is derived from a single cell, or clone, the "mono clonal" antibodies produced by the cul ture are all identical. Thus, whereas the immunized animal responds to a single antigen by generating many different an tibody producing-cells—a polyclonal re sponse—hybridoma technology usually allows only a few of those antibodyproducing cells to be isolated and grown. In the studies that established that catalytic antibodies were feasible, Iver son points out, it was critical to use pure samples of monoclonal antibodies to eliminate the possibility of catalysis caused by trace enzymatic activity. Monoclonal antibodies also lend them selves to detailed mechanistic analysis. The downside of using monoclonal antibodies for catalytic antibody studies is that they provide "yes-no answers,"
understanding the catalytic activity of polyclonal antibodies is essential for evaluating strategies to harness catalytic antibodies as in-vivo therapeutic agents. Before arriving at Texas, Iverson was a postdoctoral fellow in the laboratory of Richard A. Lerner, director of Scripps Research Institute, La Jolla, Calif., and one of the discoverers of catalytic anti bodies. Iverson says he began investi gating polyclonal catalytic antibodies out of necessity. "About a year and a half ago, Richard and his collaborators published a won derful paper in J ACS [Journal of the Amer ican Chemical Society] on asymmetric pro tonation by a monoclonal catalytic anti body," Iverson recalls. "I looked at that paper, and I realized I couldn't afford to do the research. I calculated the cost, and I said there has to be a better way to get that answer, that an antibody can cata lyze an interesting reaction." Of Iverson's research, Lerner says, 'This is yet another step forward toward the use of active immunization to pre pare catalytic antibodies for disease in tervention." Lerner also notes that the approach "makes antibody catalysis available to researchers who do not want to become involved in hybridoma technology."
Phosphonium ion elicits catalytic antibodies · . . OCH
OCH* Tris(4-methoxyphenyl)(6-carboxyhexyl)phosphonium ion
. . . that catalyze hydrolysis of trityl ether OCHo
-OR
CH 3 0
OCH3 R = CH2CH2(OCH2CH2)50H
MAY 24,1993 C&EN
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SCIENCE/TECHNOLOGY Iverson observes. //What an experiment Iverson readily admits that for evendemonstrates is that this monoclonal an- tual applications, monoclonal catalytic tibody isolated on a given day gives a antibodies· may be preferred. "An overrate enhancement of x. But if we tweak riding assumption in all of this is that the hapten, can we quantitatively detect once we find out how to make a hapan improvement in the catalytic activity? ten more catalogenic, we can go back Monoclonal antibodies can't easily an- and isolate monoclonal antibodies that swer that question." are better catalysts," he says. "That is a To the extent that variability from scientific question that is interesting in animal to animal can be controlled, its own right." characterizing the catalytic activity of a Rudy Baum polyclonal response to a hapten can answer the question, Iverson says. In research supported by the Searle Foundation and a National Science Foundation Presidential Young Investigator award, Iverson and graduate student David B. Stephens immunized a rabbit A shareware laboratory information with a tris(4-methoxyphenyl)phospho- management system (LEVIS) developed nium hapten designed to mimic the pro- by an Irish chemist is breaking the price tonated species that occur during the barrier for small laboratories in need of acid-catalyzed hydrolysis of a trityl ether such a system. Shareware is software group [Biochem. Biophys. Res. Commun.,that is distributed free or at very low cost, with an additional fee requested or 192,1439 (1993)]. Periodically throughout the immuni- required for continued use. zation regimen, the chemists purified The LEVIS was developed by Cornelius polyclonal antibodies from the rabbit N. Hegarty, senior scientific officer of antiserum using a simple two-step pro- Dublin Corp., Dublin, Ireland. A fully cedure. They characterized the ability functional version of Hegarty's LIMS of the polyclonal antibodies to catalyze costs $350. Hegarty will provide copies the hydrolysis of a trityl substrate, and of the program for $25 (to cover duplifound that catalytic activity increased cating and mailing costs), and the disks throughout the immunization regimen. are available for the same price from a Late in the regimen, catalytic activity shareware library. In addition, anyone increased more steeply than simple an- with a copy of the program may duplitibody titer, Iverson says, suggesting cate it and distribute it without cost, or that the high-affinity antibodies as- at a reasonable price to cover only costs sumed to predominate during the lat- of preparing and mailing the disks. ter stages of the immune response are The program runs on 386SX (or highthe best catalysts. er) IBM-compatible personal computers Iverson notes that Gerard Gallagher with 4 megabytes of random access and coworkers in the department of memory and a recommended 40 megabiochemistry at the University of Lon- bytes of free hard disk space. It includes don reported last year on evidence of password access control, encrypted data catalytic activity in polyclonal antibod- tables (to prevent unauthorized alteries isolated from sheep [Biochem. ]., 284, ation of data), an audit trail with date 675 (1992)]. However, a number of fac- and time stamps for all changes made to tors made definitive interpretation of recorded data, and four levels of user access. An owner's manual can be printed that result difficult. "Polyclonal antibodies by definition out after installation of the program. The represent a cross-section of the entire program is based on Paradox, a relationimmune response, so results obtained al database program by Borland Internashould be more statistically accurate tional Inc. According to chemistry professor than analyzing a few monoclonal antibodies that may not faithfully represent and LIMS specialist Raymond E. Dessy the overall immune response elicited of Virginia Polytechnic Institute & State by a hapten/7 Iverson says. As such, University, Blacksburg, "There areTrtot catalytic polyclonal antibody experi- many products out there for the small ments could "form the basis of the first lab, and they're pretty hefty in price. systematic, accurate comparisons be- When people call me and say they're a tween the catalytic activity produced two-man lab, I don't really know what to tell them to do." Dessy says the lowby related haptens."
Lab management system offered as shareware
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MAY 24,1993 C&EN
est priced comparable system he knows of costs about $15,000—although that price may include training and service that would not be available with a shareware LIMS. More complex LIMS systems can cost $100,000 and more, but those would not be comparable to the shareware LIMS. The shareware method of distribution allows "each person who obtains a copy the opportunity to examine the application in detail, thereby gaining an understanding of the functionality and use of [the] LIMS," says Hegarty. The $350 registration fee should only be paid, he says, if the application is found to be useful. When the program is loaded onto a user's computer, it is initially fully functional. But after 30 days the manager module, a key section of the program, is deactivated. When access to the module is lost, the system's configuration becomes frozen and vital functions such as audit trail and archiving become unavailable. However, even in its frozen state, the software is useful as a demonstration program for students, and the program may continue to be used as an operational system in the laboratory with no loss of data. Access to the manager module can be restored by registering the software (for $350) to obtain a parallel port "key," a hardware device that provides access to the manager module. Hegarty says, "The text and shareware were written as a hobby in my spare time, albeit very closely related to my career." He has developed the file
Hegarty: shareware wntten as a hobby
