Scientific Report 2005

Molecular Biology

Studies at the Interface of Molecular Biology, Chemistry, and Medicine

C.F. Barbas III, B.A. Gonzalez, L. Asawapornmongkul, D.B. Ramachary, S. Eberhardy, R. Fuller, R. Gordley, J. Guo, B. Henriksen, C. Lund, J. Mandell, S. Mitsumori, R. Mobini, N.S. Chowdari, M. Popkov, D. Steiner, J. Suri, F. Tanaka, U. Tschulena, Y. Ye, Y. Yuan, G. Zhong

We are concerned with problems in 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 enzymelike efficiency. We hope to apply these novel insights, technologies, methods, and products to provide solutions to human diseases, including cancer, HIV disease, and genetic diseases.

Directing the Evolution of Catalytic Function

Using our concept of reactive immunization, we have developed antibodies that catalyze aldol as well as retro-aldol reactions of a wide variety of molecules. 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 synthetic protein catalyst. We have shown the efficient asymmetric synthesis and resolution of a variety of molecules, including tertiary and fluorinated aldols, and have used these chiral synthons to synthesize natural products (Fig. 1).

Fig. 1. A variety of compounds synthesized with the world’s first commercially available catalytic antibody, 38C2, produced at Scripps Research.

The results highlight the potential synthetic usefulness of catalytic antibodies as artificial enzymes in addressing problems in organic chemistry that are not solved by using natural enzymes or more traditional synthetic methods.

To further evolve these catalytic antibodies, we are developing genetic selection methods. Other advances in this area include the development of the first peptide aldolase enzymes. Using both design and selection, we created small peptide catalysts that recapitulate many of the kinetic features of large protein catalysts. With these smaller enzymes, we can address how the size of natural proteins is related to catalytic efficiency.

Organocatalysis: A Bioorganic Approach to Catalytic Asymmetric Carbon-Carbon Bond–Forming Reactions

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 determined the efficacy of simple chiral amines and amino acids for catalysis of aldol and related imine and enamine chemistries such as Michael, Mannich, Knoevenagel, and Diels-Alder reactions (Fig. 2). Although aldolase antibodies are superior in terms of the kinetic parameters, these more simple catalysts are enabling us to quantify the importance of pocket sequestration in catalysis.

Fig. 2. L-Proline and other organocatalysts developed for a variety of catalytic asymmetric syntheses via aldol, Michael, Mannich, Diels-Alder, and Knoevenagel reactions provide access to important classes of compounds. These catalysts make reactions that were once complex multistep reactions, simple 1-step reactions. A wide variety of medicinally important products can be assembled by using the Mannich reaction manifold alone.

Furthermore, many of these catalysts are cheap, environmentally friendly, and practical for large-scale synthesis. With this approach, we showed the scope and usefulness of the first efficient amine catalysts of direct asymmetric aldol, Mannich, Diels-Alder, and Michael reactions. The organocatalyst approach is a direct outcome of our studies of catalytic antibodies and provides an effective alternative to organometallic reactions that use severe reaction conditions and often-toxic catalysts.

We think that our discovery that the simple naturally occurring amino acids such as L-proline and other amines can effectively catalyze a variety of enantioselective intermolecular reactions will change the way many reactions will be performed. Furthermore, these catalysts are functional in related ketone addition reactions such as Mannich- and Michael-type reactions. As a testament to the mild nature of this approach, we developed the first catalytic asymmetric aldol, Mannich, Michael, and fluorination reactions involving aldehydes as nucleophiles. Previously, such reactions were considered out of the reach of traditional synthetic methods.

In an extension of these concepts, we invented a variety of novel multicomponent or asymmetric assembly reactions (Fig. 3).

Fig. 3. A few recently developed catalytic asymmetric assembly reactions. In these reactions, designed small organic molecules are used to synthesize complex molecules.

Our finding that a variety of optically active amino acids can be synthesized with proline catalysis in which an L-amino acid begets other L-amino acids suggests that this route may have been used in prebiotic syntheses of optically active amino acids. In addition, we showed that our strategy can be used to synthesize carbohydrates directly, thereby providing a provocative prebiotic route to the sugars essential for life.

Unlike most catalysts obtained via traditional approaches, our catalysts are environmentally safe and are available in both enantiomeric forms. The reactions do not require inert conditions or heavy metals and can be performed at room temperature without preactivation of the donor substrates. Because amines can act as catalysts via both nucleophilic (enamine based) and electrophilic (iminium based) activation, they have great potential in catalytic asymmetric synthesis.

Therapeutic Antibodies, In And Out of Cells

We developed the first human antibody phage display libraries and the first synthetic antibodies and methods for the in vitro evolution of antibody affinity. The ability to manipulate large libraries of human antibodies and to evolve such antibodies in the laboratory provides tremendous opportunities to develop new medicines. Laboratories and pharmaceutical companies around the world now apply the phage display technology that we developed for antibody Fab fragments. In our laboratory, we are targeting cancer and HIV disease. One of our antibodies, IgG1-b12, protects animals against primary challenge with HIV type 1 (HIV-1) and has been further studied by many other researchers. We improved this antibody by developing in vitro evolution strategies that enhanced its neutralization activity. By coupling laboratory-evolved antibodies with potent toxins, we showed that immunotoxins can effectively kill infected cells.

We are also developing genetic methods to halt HIV by using gene therapy. We created unique human antibodies that can be expressed inside human cells to make the cells resistant to HIV infection. In the future, these antibodies might be delivered to the stem cells of patients infected with HIV-1, allowing the development of a disease-free immune system that would preclude the intense regimen of antiviral drugs now required to treat HIV disease.

Using our increased understanding of antibody-antigen interactions, we extended our efforts in cancer therapy and developed rapid methods for creating human antibodies from antibodies derived from other species. We produced human antibodies that should enable us to selectively starve a variety of cancers by inhibiting angiogenesis and antibodies that will be used to deliver radionuclides to colon cancers to destroy the tumors. We hope that some of these antibodies will be used in clinical trials done by our collaborators at the Sloan-Kettering Cancer Center in New York City.

On the basis of our studies on HIV-1, we used intracellular expression of antibodies directed against angiogenic receptors to create a new gene-based approach to cancer. We are determining if this new approach can be applied in vivo to halt tumor growth. Our preliminary results indicate that this type of gene therapy can be successfully applied to the treatment of cancer.

Therapeutic Applications of Catalytic Antibodies

The development of highly efficient catalytic antibodies opens the door to many practical applications. One of the most fascinating is the use of such antibodies in human therapy. We think that use of this strategy can improve chemotherapeutic approaches to diseases such as cancer and AIDS. Chemotherapeutic regimens are typically limited by nonspecific toxic effects. To address this problem, we developed a novel and broadly applicable drug-masking chemistry that operates in conjunction with our unique broad-scope catalytic antibodies. This masking chemistry is applicable to a wide range of drugs because it is compatible with virtually any heteroatom. We showed that generic drug-masking groups can be selectively removed by sequential retro-aldol–retro-Michael reactions catalyzed by antibody 38C2 (Fig. 4). This reaction cascade is not catalyzed by any known natural enzyme.

Fig. 4. Targeting cancer and HIV with prodrugs activated by catalytic antibodies. A bifunctional antibody is shown targeting a cancer cell for destruction. A nontoxic analog of doxorubicin, prodoxorubicin, is being activated by an aldolase antibody to the toxic form of the drug.

Application of this masking chemistry to the anticancer drugs doxorubicin, camptothecin, and etoposide produced prodrugs with substantially reduced toxicity. These prodrugs are selectively unmasked by the catalytic antibody when the antibody is applied at therapeutically relevant concentrations. The efficacy of this approach has been shown in in vivo models of cancer. Currently, we are developing more potent drugs and novel antibodies that will allow us to target breast, colon, and prostate cancer as well as cells infected with HIV-1. On the basis of our preliminary findings, we think that our approach can become a key tool in selective chemotherapeutic strategies. To see a movie illustrating this approach, visit http://www.scripps.edu/mb/barbas/index.html.

Adaptor Immunotherapy: The Advent of Chemobodies

We think that combining the chemical diversity of small synthetic molecules with the immunologic characteristics of antibody molecules will lead to therapeutic agents with superior properties. Therefore, we developed a conceptually new device that equips small synthetic molecules with both the immunologic effector functions and the long serum half-life of a generic antibody molecule. For a prototype, we developed a targeting device based on the formation of a covalent bond of defined stoichiometry between (1) a 1,3-diketone derivative of an arginine–glycine–aspartic acid peptidomimetic that targets the integrins α_vβ₃ and α_vβ₅ and (2) the reactive lysine of aldolase antibody 38C2. The resulting complex spontaneously assembled in vitro and in vivo, selectively retargeted antibody 38C2 to the surface of cells expressing integrins α_vβ₃ and α_vβ₅, dramatically increased the circulatory half-life of the peptidomimetic, and effectively reduced tumor growth in animal models of human Kaposi sarcoma and colon cancer (Fig. 5). These studies have been extended to melanoma.

Fig. 5. Adaptor Immunotherapy dramatically slows tumor growth. A variety of cancer xenografts have been effectively treated with chemobodies, a combination of small-molecule drugs and antibodies. Chemobodies have characteristics that can be superior to those of either the small molecule or the antibody alone.

The solution to many diseases might be simply turning genes on or off in a selective way. In order to produce switches that can turn genes on or off, we are studying molecular recognition of DNA by zinc finger proteins and methods of creating novel zinc finger DNA-binding proteins (Fig. 6).

Fig. 6. A designed polydactyl zinc finger binds 18 bp of DNA. A single zinc finger domain is highlighted. With this direct approach, we can construct more than a billion gene switches and use the switches to specifically turn genes on or off in multiple organisms. With further elaboration of the approach, every gene in the genome can be either upregulated or downregulated, providing a new approach to probe gene function across the genome.

Because of their modularity and well-defined structural features, zinc finger proteins are particularly well suited for use as DNA-binding proteins. Each finger forms an independently folded domain that typically recognizes 3 nucleotides of DNA. We showed that proteins can be selected or designed that contain zinc fingers that recognize novel DNA sequences. These studies are aiding the elucidation of rules for sequence-specific recognition within this family of proteins. 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.

Zinc Finger Gene Switches

We showed the potential of this approach in multiple mammalian and plant cell lines and in whole organisms. With the use of characterized modular zinc finger domains, polydactyl proteins capable of recognizing an 18-nucleotide site can be rapidly constructed. Our results suggest that zinc finger proteins might be useful as genetic regulators for a variety of human ailments and provide the basis for a new strategy in gene therapy. Our goal is to develop this class of therapeutic proteins to inhibit or enhance the synthesis of proteins, providing a direct strategy for fighting diseases of either somatic or viral origin.

We are also developing proteins that will inhibit the growth of tumors and others that will inhibit the expression of a protein known as CCR5, which is a key to infection of human cells by HIV-1. We developed an HIV-1–targeting transcription factor that strongly suppresses HIV-1 replication. Genetic diseases such as sickle cell anemia are also being targeted. Using a library of transcription factors, we developed a strategy that effectively allows us to turn on and turn off every gene in the genome. With this powerful new strategy, we can quickly regulate a target gene or discover other genes that have a key role in disease. In the future, we hope to use novel DNA-modifying enzymes directed by zinc fingers to manipulate chromosomes themselves.

Publications

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.

Betancort, J.M., Sakthivel, K., Thayumanavan, R., Tanaka, F., Barbas, C.F. III. Catalytic direct asymmetric Michael reactions: addition of unmodified ketone and aldehyde donors to alkylidene malonates and nitro olefins. Synthesis 1509, 2004, Issue 9.

Blancafort, P., Segal, D.J., Barbas, C.F. III. Designing transcription factor architectures for drug discovery. Mol. Pharmacol. Rev. 66:1361, 2004.

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.

Chowdari, N.S., Suri, J.T., Barbas, C.F. III. Asymmetric synthesis of quaternary α- and β-amino acids and β-lactams via proline catalyzed Mannich reactions with branched aldehyde donors. Org. Lett. 6:2507, 2004.

Crotty, J.W., 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.

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.

Magnenat, L., Blancafort, P., Barbas, C.F. III. In vivo selection of combinatorial libraries and designed affinity maturation of polydactyl zinc finger transcription factors for ICAM-1 provides new insights into gene regulation. J. Mol. Biol. 341:635, 2004.

Mase, N., Thayumanavan, R., Tanaka, F., Barbas, C.F. III. Direct asymmetric organocatalytic Michael reactions of α,α-disubstituted aldehydes with β-nitrostyrenes for the synthesis of quaternary carbon-containing products. Org. Lett. 6:2527, 2004.

Notz, W., Tanaka, F., Barbas, C.F. III. Enamine-based organocatalysis with proline and diamines: the development of direct catalytic asymmetric aldol, Mannich, Michael, and Diels-Alder reactions. Acc. Chem. Res. 37:580, 2004.

Notz, W., Watanabe, S., Chowdari, N.S., Zhong, G., Betancort, J.M., Tanaka, F., Barbas, C.F. III. The scope of the direct proline-catalyzed asymmetric addition of ketones to imines. Adv. Synth. Catal. 346:1131, 2004.

Popkov, M., Jendreyko, N., McGavern, D.B., 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 cell 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.

Ramachary, D.B., Barbas, C.F. III. Towards organo-click chemistry: development of organocatalytic multicomponent reactions through combinations of aldol, Wittig, Knoevenagel, Michael, Diels-Alder and Huisgen cycloaddition reactions. Chemistry 10:5323, 2004.

Sinha, S.C., Li, l.-S., Watanabe, S., Kaltgrad, E., Tanaka, F., Rader, C., Lerner, R.A., Barbas, C.F. III. Aldolase antibody activation of prodrugs of potent aldehyde-containing cytotoxics for selective chemotherapy. Chemistry 10:5467, 2004.

Steiner, D.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.

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., in press.

Tanaka, F., Barbas, C.F. III. Organocatalytic approaches to enantioenriched β-amino acids. In: Enantioselective Synthesis of β-Amino Acids, 2nd ed. Juaristi, E., Soloshonok, V. (Eds.). Wiley-VCH, New York, 2004, 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, 2004, p. 304.

Tanaka, F., Flores, F., Kubitz, D., Lerner, R.A., Barbas, C.F. III. Antibody-catalyzed aminolysis of a chloropyrimidine derivative. Chem. Commun. (Camb.) 1242, 2004, Issue 10.<

Tanaka, F., Mase, N., Barbas, C.F. III. Determination of cysteine concentration by fluorescence increase: reaction of cysteine with a fluorogenic aldehyde. Chem. Commun. (Camb.) 1762, 2004, Issue 15.

Thayumanavan, R., Tanaka, F., Barbas, C.F. III. Direct organocatalytic asymmetric aldol reactions of α-amino aldehydes: expedient synthesis of highly enantiomerically enriched anti-β-hydroxy-α-amino acids. Org. Lett. 6:3541, 2004.

Zhong, G., Fan, J., Barbas, C.F. III. Amino alcohol catalyzed direct asymmetric aldol reactions: enantioselective synthesis of anti-α-fluoro-β-hydroxy ketones. Tetrahedron Lett. 45:5681, 2004.

Zhu, X., Tanaka, F., Hu, Y., Heine, A., Fuller, R., Zhong, G., Olson, A.J., Lerner, R.A., Barbas, C.F. III, Wilson, I.A. The origin of enantioselectivity in aldolase antibodies: crystal structure, site-directed mutagenesis, and computational analysis. J. Mol. Biol. 343:1269, 2004.