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The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000


Cancer, AIDS, Catalysis, and the Regulation of Genes: Building Molecules With Defined Functions


C.F. Barbas III, R. Lerner, R. Beerli, J. Berry, P. Blancafort, T. Bui, J. Chung, B. Dreier, M. Elia, R. Fuller, J. Flippin, S. Gobuty, C. Lund, L. Magnenat, J. McDunn, J. Neves, W. Notz, M. Popkov, C. Rader, R. Reisfeld, K. Sakthivel, D. Segal, D. Shabat, S. Sinha, J. Stege, P. Steinberger, F. Tanaka, J. Turner, J. Velker, S. von Berg, G. Zhong

The ability to directly design proteins that efficiently perform predefined tasks would have a profound impact on science and on the everyday life of human beings. Designer proteins might enable us to dissect biological pathways and mechanisms, rapidly create and synthesize new drugs, use natural resources efficiently, and create new agricultural products. These proteins might help explain our past and define our future.

Catalytic Antibodies

In our laboratory, we are extending and refining approaches to catalytic antibodies by using novel recombinant strategies coupled with reactive immunization, chemical-event selections, and the design of unique multiturnover selection chemistries. We are developing in vitro selection and evolutionary strategies as routes for obtaining antibodies of defined biological and chemical activity. This strategy involves the directed evolution of human as well as rodent and synthetic antibodies. Essentially, we are evolving proteins to function as efficient catalysts, a task that was performed naturally over eons, and one that we aim to complete in weeks. The approach is a blend of chemistry, enzymology, and molecular biology.

A major focus of our research is the development of strategies to produce antibodies that efficiently form and break carbon-carbon bonds. Much of this work centers on the chemistry of imines and enamines and the development of antibodies that use covalent catalysis. We are examining the aldol, the Michael, and the Diels-Alder reactions and related reactions such as the Mannich reaction. Many of these catalysts may someday be important in the synthesis of enantiomerically pure drugs. Using novel catalytic antibodies, we showed the efficient asymmetric synthesis and resolution of a variety of molecules, including tertiary and fluorinated aldols, carbohydrates, insect pheromones, and derivatives of the epothilone anticancer drugs.

This past year we reported a new approach to catalytic antibodies that involves combining transition-state theory with reactive immunization (Fig. 1). Using this approach, we produced a large array of highly active catalytic antibodies, including antibodies with stereoselectivities antipodal to our original aldolase antibodies. The catalytic proficiency of the best of these antibodies is almost 1014, a value 1000 times that of the best catalytic antibodies reported to date and overall the best of any man-made protein catalyst to date. These new catalysts have enabled S. Sinha, the Skaggs Institute, to develop new highly efficient syntheses of the anticancer epothilone drugs.

Catalysts Large And Small

Recently, we reported the chemical synthesis of a new class of DNA bases that can be used to construct proteinlike DNAs. These new bases contain side chains common to amino acids used by proteins. The bases have enabled us to begin to explore the versatility of functionalized DNA in catalysts and to create the first proteinlike DNA enzyme, a general-purpose RNA-cleaving enzyme that is superior to "natural" DNA enzymes.

To further explore the principles of catalysis, we are studying amine catalysis as a function of catalytic scaffold. Using insights garnered from our studies of aldolase antibodies, we prepared simple chiral amines to catalyze aldol and related imine and enamine chemical actions such as Diels-Alder reactions. We also studied small amine-bearing peptides that are catalytic. Attempts to create aldolase DNAs are in progress. Although aldolase antibodies are superior catalysts, the simpler catalysts are enabling us to measure the importance of pocket sequestration in catalysis (Fig. 2).

Targeting Cancer

Catalytic antibodies can be used not only to synthesize anticancer drugs but also to deliver the drugs in a highly specific fashion to the cancer itself. Typically, the application of highly potent chemotherapeutic agents is limited by nonspecific toxic effects associated with the inability to direct these agents to the appropriate targets. We showed that a wide variety of clinically relevant anticancer drugs such as doxorubicin, camptothecin, and etoposide can be modified to less toxic prodrugs that can be specifically activated by catalytic antibody 38C2 to kill cancer cell lines. With R. Reisfeld, The Scripps Research Institute, we showed the efficacy of this approach in animal models of cancer (Fig. 3). Currently, we are developing more potent drugs as well as novel antibodies that will enable us to target a range of cancers and HIV. On the basis of our preliminary findings, we think that our approach can become a key tool in selective chemotherapeutic strategies.

Software for the Genome

In all organisms, from the simplest to the most complex, proteins that bind nucleic acids control the expression of genes. The nucleic acids DNA and RNA are the molecules that store the recipes of all life forms. The fertilized egg of a human contains the genetic recipe for the development and differentiation of a single cell into 2 cells, 4 cells, and so on, finally yielding a complete individual. The coordinated expression or reading of the recipes for life allows cells containing the same genetic information to perform different functions and to have distinctly different physical characteristics. Proteins that bind nucleic acids enable this coordinated control of the genetic code. Lack of coordination due to genetic defects or to viral seizure of control of the cell results in disease. Viruses are the most common cause of human ailments, from the common cold to AIDS. Viruses, the simplest of all organisms, cause the diseases that we are most poorly prepared to treat.

One project in our laboratory involves the development of methods to produce proteins that bind to specific nucleic acid sequences. The production of these new proteins will enable us to address fundamental questions about this binding. These proteins will be used as specific genetic switches to turn on or turn off genes on demand, creating an operating system for genomes.

To this end, we made significant progress in selecting and designing specific zinc finger domains that will constitute an alphabet of 64 domains that will allow any DNA sequence to be bound selectively (Fig. 4). The prospects for this "second genetic code" are fascinating and should have a major impact on basic and applied biology.

We showed that these domains are functionally modular and can be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. We now have at hand a family of zinc finger domains sufficient for the construction of 17 million novel proteins that bind the 5´-(GNN)6-3´ family of DNA sequences.

The goal of this work is to develop a new class of therapeutic proteins that inhibit or enhance the synthesis of proteins, providing a new strategy for fighting diseases of either somatic or viral origin. We are developing proteins that will inhibit the growth of tumors, halt replication of HIV, and even make healthier corn. We showed that we can use our alphabet of proteins to specifically turn on or turn off genes at will.

One of our targets in cancer is the oncogene erbB-2. ErbB-2, a member of the ErbB receptor family, is overexpressed as a result of gene amplification and/or transcriptional deregulation in a high percentage of human adenocarcinomas, including those of the breast, ovary, lung, stomach, and salivary gland. We showed that we can shut this gene off in a highly specific fashion and induce a growth arrest in cancer cells, providing a promising new strategy for cancer therapy.

Using combinatorial antibody strategies, we are attempting to discover new ways to fight disease with antibodies. To this end, we are continuing our development of novel means of antibody selection and evolution to create new classes of anti-HIV drugs that act by inhibiting viral entry into cells and anticancer drugs that target tumors for destruction. When combined with activation of prodrugs by catalytic antibodies, these disease-targeting antibodies will have powerful disease-fighting potential. We hope to take these molecules all the way to clinical trials.

Publications

Andris-Widhopf, J., Rader, C., Steinberger, P., Fuller, R., Barbas, C.F. III. Optimized methods for the preparation of chicken monoclonal antibody fragments by phage display. J. Immunol. Methods 242:159, 2000.

Barbas, C.F. III, Burton, D., Silverman, D., Scott, J. (Eds.). Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, in press.

Barbas, C.F. III, Rader, C., Segal, D.J., List, B., Turner, J.M. From catalytic asymmetric synthesis to the transcriptional regulation of genes: In vivo and in vitro evolution of proteins. Adv. Protein Chem. 55:317, 2000.

Beerli, R.R., Dreier, B., Barbas, C.F. III. Selective positive and negative regulation of endogenous genes by designed transcription factors. Proc. Natl. Acad. Sci. U. S. A. 97:1495, 2000.

Beerli, R.R., Schopfer, U., Dreier, B., Barbas, C.F. III. Chemically regulated zinc finger transcription factors. J. Biol. Chem. 275:32617, 2000.

Bui, T., Barbas, C.F. III. A proline-catalyzed asymmetric Robinson annulation reaction. Tetrahedron Lett. 41:6951, 2000.

Dreier, B., Segal, D.J., Barbas, C.F. III. Insights into the molecular recognition of the 5´-GNN-3´ family of DNA sequences by zinc-finger domains. J. Mol. Biol. 303:489, 2000.

Karlström, A.H., Zhong, G., Rader, C., Larsen, N., Heine, A., Fuller, R., List, B., Wilson, I.A., Barbas, C.F. III, Lerner, R.A. Using antibody catalysis to study the outcome of multiple evolutionary trials of a chemical task. Proc. Natl. Acad. Sci. U. S. A. 97:3878, 2000.

List, B., Lerner, R.A., Barbas, C.F. III. Enantioselective aldol-cyclodehydrations catalyzed by antibody 38C2. Org. Lett. 1:59, 1999.

List, B., Lerner, R.A., Barbas, C.F. III. Proline-catalyzed direct asymmetric aldol reactions. J. Am. Chem. Soc. 122:2395, 2000.

Notz, W., Sakthivel, S., Bui, T., Barbas, C.F. III. Amine-catalyzed direct asymmetric Mannich-type reactions. Tetrahedron Lett., in press.

Rader, C., Ritter, G., Nathan, S., Elia, M., Gout, I., Jungbluth, A.A., Cohen, L.S., Welt, S., Old, L.J., Barbas, C.F. III. The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies. J. Biol. Chem. 275:13668, 2000.

Santoro, S.W., Joyce, G.F., Sakthivel, K., Gramatikova, S., Barbas, C.F. III. RNA cleavage by a DNA enzyme with protein-like functionality. J. Am. Chem. Soc. 122:2433, 1999.

Segal, D., Barbas, C.F. III. Design of novel sequence-specific DNA-binding proteins. Curr. Opin. Chem. Biol. 4:34, 2000.

Sinha, S.C., Sun, J., Miller, G., Barbas, C.F. III, Lerner, R.A. Sets of aldolase-antibodies with antipodal reactivities: Synthesis of epothilone E by resolution of thiazole aldols. Org. Lett. 1:1623, 1999.

Steinberger, P., Andris-Widhopf, J., Bühler, B., Torbett, B.E., Barbas, C.F. III. Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion. Proc. Natl. Acad. Sci. U. S. A. 97:805, 1999.

Steinberger, P., Sutton, J.K., Rader, C., Elia, M., Barbas, C.F. III. Generation and characterization of a recombinant human CCR5-specific antibody: A phage display approach for rabbit antibody humanization. J. Biol. Chem. 275:36073, 2000.

Tanaka, F., Lerner, R.A., Barbas, C.F. III. Reconstructing aldolase antibodies to alter their substrate specificity and turnover. J. Am. Chem. Soc. 122:4835, 2000.

Tanaka, F., Lerner, R.A., Barbas, C.F. III. Thiazolium-dependent catalytic antibodies produced using a covalent modification strategy. Chem. Commun. 1383, 1999.

Turner, J.M., Bui, T., Lerner, R.A., Barbas, C.F. III, List, B. An efficient benchtop system for multigram-scale kinetic resolutions using aldolase antibodies. Chem. Eur. J. 6:2772, 2000.

Zhong, G., Lerner, R.A., Barbas, C.F. III. Broadening the aldolase catalytic antibody repertoire by combining reactive immunization and transition state theory: New enantio- and diastereo-selectivities. Angew. Chem. Int. Ed. 37:2481, 1999.

 

 







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