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The Skaggs Institute
for Chemical Biology


Scientific Report 2005




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


C.F. Barbas III, L. Asawapornmongkol, P. Blancafort, N.S. Chowdari, R.P. Fuller, S. Eberhardy, B.A. Gonzalez, R. Gordley, J. Guo, C. Lund, L. Magnenat, R. Mobini, M. Popkov, D.B. Ramachary, D. Steiner, J. Suri, F. Tanaka, R. Thayumanavan, U. Tschulena, Y. Ye, Y. Yuan

We are concerned with problems at the interface of molecular biology, chemistry, and medicine. Many of our studies involve learning or improving on Nature’s strategies to prepare novel molecules that perform specific functional tasks, such as regulating a gene, destroying cancer, or catalyzing a reaction with small molecules in an enzymelike manner. We hope to apply these novel insights, methods, and products to provide solutions to human diseases, including cancer, HIV disease, and genetic diseases.

Catalytic Antibodies

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 activities. These strategies involve the directed evolution of human, rodent, and synthetic antibodies. Essentially, we are evolving proteins to function as efficient catalysts, a task 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. Recent studies revealed the potential of catalytic antibodies to catalyze unfavored Michael reactions and to serve as triggers for potent tethered prodrugs.

Organocatalysis: Enzymelike Chemistry with Small Organic Molecules

In studying how proteins catalyze reactions, we often examine how the constituent components react. These studies have led to a green approach to catalytic asymmetric synthesis that can be applied to the synthesis of drugs and druglike molecules. Using insights garnered from our studies of aldolase antibodies, we prepared simple chiral amino acids and amines to catalyze aldol and related imine and enamine chemistries such as Michael and Mannich reactions. We also studied small amine-bearing peptides that are catalytic. Although aldolase antibodies are superior catalysts, simple chiral amino acids and amines are enabling us to measure the importance of pocket sequestration in catalysis.

We showed that L-proline and other chiral amines can be efficient asymmetric catalysts of a variety of significant imine- and enamine-based reactions. Studies from our laboratory and the contributions of others have advanced one of the ultimate goals in organic chemistry: the catalytic asymmetric assembly of simple and readily available precursor molecules into stereochemically complex products under operationally simple and, in some instances, environmentally friendly experimental protocols. A significant result of these studies is the development of catalysts that allow aldehydes, for the first time, to be used efficiently as nucleophiles in a wide variety of catalytic asymmetric reactions. Previously, only naturally occurring enzymes were thought capable of this chemical feat. With future efforts, small organic catalysts may match some of Nature’s other heretofore unmatched synthetic prowess. These catalysts might help explain the development of complex chemical systems in the prebiotic world and provide hints toward yet-to-be-discovered mechanisms in extant biological systems.

Using this method, we directly synthesized a wide variety of α and β amino acids, carbohydrates, and lactams. Stereochemically complex molecules can now be assembled by using small molecules in a manner analogous to that of natural enzymes (Fig. 1).

Fig. 1. Our studies in organocatalysis have provided mimetics of threonine aldolases, providing routes to β-hydroxy-amino acids that are key constituents of many biologically active molecules and mimetics of the dihydroxyacetone family of aldolases key for the synthesis of carbohydrates.

Targeting Cancer

In targeting cancer, we take a multidisciplinary approach that involves gene regulation, catalytic antibodies, drug design, and combinatorial antibody libraries. Using a combinatorial antibody strategy, this year we prepared high-affinity antibodies that target key angiogenic receptors, and we engineered these antibodies to simultaneously block 2 angiogenic pathways in mice to produce a strong therapeutic effect against melanoma (Fig. 2).

Fig. 2. To explore the therapeutic potential of blocking both the Tie-2 receptor–interaction pathway and the vascular endothelial cell growth factor receptor 2 (VEGF-R2)–interaction pathway, we used an adenoviral vector to deliver the recombinant intradiabody shown here. This novel protein allowed us to demonstrate the therapeutic advantage of simultaneous blockade of Tie-2 and VEGF-R2 in cancer therapy.

Using catalytic antibodies, we are developing a strategy to activate drugs in a highly specific fashion at the site of cancer. We also advanced our chemotherapeutic approaches based on catalytic antibodies and prodrugs. Studies with chemically programmed antibodies continue to progress, providing routes to novel treatments of melanoma and breast and ovarian cancers.

Designer Transcription Factors

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.

In one project, we are developing methods to produce proteins that bind to specific DNA sequences to control specified genes. As we showed earlier, these proteins can 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 selected and designed specific zinc finger domains that will constitute an alphabet of 64 domains that will allow any DNA sequence to be bound selectively. The prospects for this “second genetic code” are fascinating and should have a major impact on basic and applied biology.

This year we developed the CNN family of zinc finger domains. Together with the GNN and ANN domains we have already developed, billions of transcription factors can now be prepared. Our goal 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.

Using a novel library of transcription factors, we have developed a strategy that effectively allows us to turn on and turn off every gene in the genome (Fig. 3).

Fig. 3. Phenotypic screens involving delivery of zinc finger libraries into cancer cells allows the selection of cells with distinct disease-related phenotypes such as the transformation of the parental cell on the left into the slender highly metastatic cell on the right.

With this powerful new strategy, we can quickly regulate a target gene or discover other genes key in disease. We are targeting a variety of other genes involved not only in cancer and HIV disease but also in genetic diseases such as sickle cell anemia. We hope to take this genetic strategy and our other molecular approaches all the way to clinical trials.

Publications

Alwin, S., Gere, M.B., Guhl, E., Effertz, K., Barbas, C.F. III, Segal, D.J., Weitzman, M.D., Cathomen, T. Custom zinc-finger nucleases for use in human cells. Mol. Ther. 12:610, 2005.

Amir, R.J., Popkov, M., Lerner, R.A., Barbas, C.F. III, Shabat, D. Prodrug activation gated by a molecular “OR” logic trigger. Angew. Chem. Int. Ed. 44:4378, 2005.

Blancafort, P., Chen, E.I., Gonzalez, B., Bergquist, S., Zijlstra, A., Guthy, D., Brachat, A., Brakenhoff, R.H., Quigley, J.P., Erdmann, D., Barbas, C.F. III. Genetic reprogramming of tumor cells by zinc finger transcription factors. Proc. Natl. Acad. Sci. U. S. A. 102:11716, 2005.

Blau C.A., Barbas, C.F. III, Bomhoff, A.L., Neades, R., Yan, J., Navas, P.A., Peterson. K.R. γ-Globin gene expression in chemical inducer of dimerization (CID)-dependent multipotential cells established from human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice. J. Biol Chem. 280:36642, 2005.

Chen, E.I., Florens, L., Axelrod, F.T., Monosov, E., Barbas, C.F. III, Yates, J.R. III, Felding-Habermann, B., Smith, J.W. Maspin alters the carcinoma proteome. FASEB J. 19:1123, 2005.

Chowdari, N.S., Barbas. C.F. III. Total synthesis of LFA-1 antagonist BIRT-377 via organocatalytic asymmetric construction of a quaternary stereocenter. Org. Lett. 7:867, 2005.

Corte-Real, S., Collins, C., Aires da Silva, F., Simas, J.P., Barbas, C.F. III, Chang, Y., Moore, P., Goncalves, J. Intrabodies targeting the Kaposi sarcoma-associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells. Blood 106:3797, 2005.

Crotty, J., Etzkorn, C., Barbas, C.F. III, Segal, D.J., Horton, N.C. Crystallographic analysis of Aart, a designed six-finger zinc finger peptide, bound to DNA. Acta Crystallogr. F61:573, 2005.

Dreier, B., Fuller, R.P., Segal, D.J., Lund, C., Blancafort, P., Huber, A., Koksch, B., Barbas, C.F. III. Development of zinc finger domains for recognition of the 5′ -CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem. 280:35588, 2005.

Eberhardy, S.R., Goncalves, J., Coelho, S., Segal, D.J., Berkhout, B., Barbas, C.F. III. Inhibition of HIV-1 replication with artificial transcription factors targeting the highly conserved primer binding site. J. Virol., in press.

Gräslund, T., Li, X., Popkov, M., Barbas C.F. III. Exploring strategies for the design of artificial transcription factors: targeting sites proximal to known regulatory regions for the induction of γ-globin expression and the treatment of sickle cell disease. J. Biol. Chem. 280:3707, 2005.

Haba, K., Popkov, M., Shamis, M., Lerner, R.A., Barbas, C.F. III, Shabat, D. Single-triggered trimeric prodrugs. Angew. Chem. Int. Ed. 44:716, 2005.

Jendreyko, N., Popkov, M., Rader, C., Barbas, C.F. III. Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesis in vivo. Proc. Natl. Acad. Sci. U. S. A. 102:8293, 2005.

Li, L.-S., Rader, C., Matsushita, M., Das, S., Barbas, C.F. III, Lerner, R.A., Sinha, S.C. Chemical adaptor immunotherapy: design, synthesis, and evaluation of novel integrin-targeting devices. J. Med. Chem. 47:5630, 2004.

Lund, C.V., Popkov, M., Magnenat, L., Barbas, C.F. III. Zinc finger transcription factors designed for bispecific coregulation of ErbB2 and ErbB3 receptors: insights into ErbB receptor biology. Mol. Cell. Biol. 25:9082, 2005.

Popkov, M., Jendreyko, N., McGavern, D., Rader, C., Barbas C.F. III. Targeting tumor angiogenesis with adenovirus-delivered anti-Tie-2 intrabody. Cancer Res. 65:972, 2005.

Popkov, M., Rader, C., Barbas C.F. III. Isolation of human prostate cancer reactive antibodies using phage display technology. J. Immunol. Methods 291:137, 2004.

Ramachary, D.B., Barbas, C.F. III. Direct amino acid-catalyzed asymmetric desymmetrization of meso-compounds: tandem aminoxylation/O-N bond heterolysis reactions. Org. Lett. 7:1577, 2005.

Steiner, D., Mase, N., Barbas. C.F. III. Direct asymmetric α-fluorination of aldehydes. Angew. Chem. Int. Ed. 44:3706, 2005.

Suri, J.T., Ramachary, D.B., Barbas. C.F. III. Mimicking dihydroxy acetone phosphate-utilizing aldolases through organocatalysis: a facile route to carbohydrates and aminosugars. Org. Lett. 7:1383, 2005.

Suri, J.T., Steiner, D.D., Barbas, C.F. III. Organocatalytic enantioselective synthesis of metabotropic glutamate receptor ligands. Org. Lett. 7:3885, 2005.

Tan, W., Dong, Z., Wilkinson, T.A., Barbas, C.F. III, Chow, S.A. Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc-finger protein E2C can catalyze site-specific integration in human cells. J. Virol., in press.

Tanaka, F., Barbas, C.F. III. Enamine-based reactions using organocatalysts: from aldolase antibodies to small amino acid and amine catalysts. J. Synth. Org. Chem. Jpn. 63:27, 2005.

Tanaka, F., Barbas, C.F. III. Organocatalytic approaches to enantioenriched β-amino acids. In: Enantioselective Synthesis of β-Amino Acids, 2nd ed. Juaristi, E. (Ed.). Wiley-VCH, New York, 2005, p. 195.

Tanaka, F., Barbas, C.F. III. Reactive immunization: a unique approach to aldolase antibodies. In: Catalytic Antibodies. Keinan, E. (Ed.). Wiley-VCH, New York, 2005, p. 305.

Tanaka, F., Fuller, R., Barbas, C.F. III. Development of small designer aldolase enzymes: catalytic activity, folding, and substrate specificity. Biochemistry 44:7583, 2005.

Tanaka, F., Fuller, R., Shim, H., Lerner, R.A., Barbas, C.F. III. Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection. J. Mol. Biol. 335:1007, 2004.

Weinstain, R., Lerner, R.A., Barbas, C.F. III, Shabat, D. Antibody-catalyzed asymmetric intramolecular Michael addition of aldehydes and ketones to yield the disfavored cis-product. J. Am. Chem. Soc. 127:13104, 2005.

 

Carlos F. Barbas III, Ph.D.
Professor
Janet and W. Keith
  Kellogg II Chair in
  Molecular Biology

Barbas Web Site