Scientific Report 2006

Molecular Biology

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

C.F. Barbas III, M. Ahmad, K. Albertshofer, L. Asawapornmongkul, N.S. Chowdari, S. Eberhardy, R. Fuller, B. Gonzalez, R. Gordley, J. Guo, D.H. Kim, R.L. Lerner, C. Lund, J. Mandell, S. Mitsumori, W. Nomura, M. Popkov, D.B. Ramachary, S.S.V. Ramasastry, L.J. Schwimmer, D. Shabat,* F. Silva, J. Suri, F. Tanaka, U. Tschulena, N. Utsumi, Y. Ye, Y. Yuan, H. Zhang

* Tel Aviv University, Tel Aviv, Israel

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 approaches 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). 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.

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

Other advances in this area include the development of the first peptide aldolase enzymes. By using both design and selection, we have created small peptide catalysts that recapitulate many of the kinetic features of large enzyme catalysts. These smaller enzymes allow us to address the relationship between the size of natural proteins and the proteins’ catalytic efficiency.

Organocatalysis: A Bioorganic Approach to Catalytic Asymmetric Synthesis

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. Although aldolase antibodies are superior catalysts in terms of the kinetic parameters, these more simple catalysts are enabling us quantify the importance of pocket sequestration in catalysis.

Furthermore, many 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 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. 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 extensions of these concepts, we designed novel amino acid derivatives that direct the stereochemical outcome of reactions in ways not possible with proline (Fig. 2). In other studies, we created the first asymmetric small-molecule aldol catalysts that are highly effective with water and seawater as solvent. We think that our results are also relevant to the prebiotic synthesis of the molecules of life. For example, we have shown that our amino acid strategy can be used to synthesize carbohydrates directly, thereby providing a provocative prebiotic route to the sugars essential for life.

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 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.

Fig. 2. Design of the new catalyst (3R,5R)-5-methyl-3-pyrrolidinecarboxylic acid (right) allows efficient access to anti-Mannich products not accessible through proline catalysis (left).

We are also developing genetic methods to halt HIV by 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 obviate 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 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. Our studies 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. 3). This reaction cascade is not catalyzed by any known natural enzyme.

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/
antibody/antibody.mov.

Fig. 3. 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.

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 (Fig. 4). 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, colon cancer, and melanoma.

Fig. 4. Designed small-molecule targeting agents (SCS-873 as shown) program the specificity of the antibody 38C2 (A). The resulting chemobodies (cp38C2, B) have characteristics that are often superior to either those of either the small molecule or the antibody alone.

Zinc Finger Gene Switches

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. 5). 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.

Fig. 5. A designed polydactyl zinc finger binds 18 bp of DNA. A single zinc finger domain is highlighted. With this approach, we can now construct more than a billion gene switches and use them to specifically turn genes on or off in multiple organisms. Further elaboration of the approach allows every gene in the genome to be either turned on or upregulated or downregulated, providing a new approach to probe gene function across the genome.

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 promise a major impact on basic and applied biology.

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 aliments and provide the basis for a new strategy of 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 with this approach. Using a library of transcription factors, we developed a strategy that effectively allows us to turn on and 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

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.

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.

Cheong, P.H.-Y., Zhang, H., Thayumanavan, R., Tanaka, F., Houk, K.N., Barbas, C.F. III. Pipecolic acid-catalyzed direct asymmetric Mannich reactions. Org. Lett. 8:811, 2006.

Corte-Real, S., Collins, C., Aires da Silva, F., Simas, 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.

Dreier, B., Fuller, R.P., Segal, D.J., Lund, C.V., 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 human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site. J. Virol. 80:2873, 2006.

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.

Mandell, J., Barbas, C.F. III. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 34(Web server issue):W516, 2006.

Mase, N., Nakai, Y., Ohara, H., Yoda, H., Takabe, K., Tanaka, F., Barbas, C.F. III. Organocatalytic direct asymmetric aldol reactions in water. J. Am. Chem. Soc. 128:734, 2006.

Mitsumori, S., Zhang, H., Cheong, P.H.-Y., Houk, K.N., Tanaka, F., Barbas, C.F. III. Direct asymmetric anti-Mannich-type reactions catalyzed by a designed amino acid. J. Am. Chem. Soc. 128:1040, 2006.

Nathan, S., Rader, C., Barbas, C.F. III. Neutralization of Burkholderia pseudomallei protease by Fabs generated through phage display. Biosci. Biotechnol. Biochem. 69:2302, 2005.

Popkov, M., Rader, C., Gonzelez, B., Sinha, S.C., Barbas, C.F. III. Small molecule drug activity in melanoma models may be dramatically enhanced with an antibody effector. Int. J. Cancer 119:1194, 2006.

Suri, J.T., Mitsumori, S., Albertshofer, K., Tanaka, F., Barbas, C.F. III. Dihydroxyacetone variants in the organocatalytic construction of carbohydrates: mimicking tagatose and fuculose aldolases. J. Org. Chem. 71:3822, 2006.

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

Swan, C.H., Buhler, B., Tschan, M.P., Barbas, C.F. III, Torbett, B.E. T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther. 13:1408, 2006.

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 bias integration of viral DNA into a predetermined region in human cells. J. Virol. 80:1939, 2006.

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

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.