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Scientific Report 2008

Molecular Biology

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

C.F. Barbas III, K. Albertshofer, T. Bui, R.P. Fuller, C. Gersbach, B. Gonzalez, R. Gordley, J. Guo, D.H. Kim, R.A. Lerner, W. Nomura, A. Onoda, S.S.V. Ramasastry, M. Santa Marta, L.J. Schwimmer, D. Shabat,* F. Tanaka, U. Tschulena, N. Utsumi, K.S. Yi, 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 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 their products to provide solutions to human diseases, including cancer, HIV disease, and genetic diseases.

Directing the Evolution of Catalytic Function

Using 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). These 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 to quantify the significance of pocket sequestration in catalysis.

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.

In extensions of these concepts, we designed novel amino acid derivatives that direct the stereochemical outcome of reactions in ways not possible with proline. 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.

We think that in time, research will show that organocatalysts or "aminozymes” (chiral amines or amino acids with biosynthetic roles) constitute components of an unseen biosynthetic apparatus at work in cells today. As we begin to appreciate the fascinating chemical transformations that are now possible through organocatalysis, and amino acid catalysis in particular, we need to look at cellular metabolism and biosynthesis in a new light. Classically, we are trained to search for a "protein” enzyme for each and every step in the synthesis of a natural product in vivo. We suggest that many of the more elusive metabolic enzymes are likely to be organocatalysts and, in many instances, simple amino acids. Because intracellular concentrations of amino acids can exceed 1 M, many wonderful and diverse exotic natural products may actually be synthesized in vivo with the aid of aminozymes and other forms of organocatalysts more complicated than amino acids (Fig. 2).
Fig. 2. Potential roles of aminozymes in the biosynthesis of the Daphniphyllum alkaloids (A) and the potential anticancer compound FR182877 (B).

Antibody Engineering: 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.

We are also developing genetic methods to halt HIV by gene therapy. We created unique human antibodies that can be expressed inside 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 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 radioisotopes 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, New York, New York.

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 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. 3). This reaction cascade is not catalyzed by any known natural enzyme.

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.

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 cancers 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.

Chemically Programmed Antibodies: 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 immunological 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 αvβ3 and αvβ5 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 the integrins αvβ3 and αvβ5, 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. Three novel chemically programmed antibodies, an entirely new class of drugs, are now in phase 1 clinical studies.
Fig. 4. Designed small-molecule targeting agents (SCS-873 is shown) program the specificity of the antibody 38C2 (A). The resulting chemobodies (cp38C2, B) have characteristics that are often superior to those of either the small molecule or the antibody alone.

Zinc Finger Gene Switches and Enzymes

The solutions to many diseases might be simply turning genes on or off in a selective way or adding or deleting genes. To accomplish all of these aims, we are studying molecular recognition of DNA by zinc finger proteins and methods of creating novel zinc finger DNA-binding proteins. We showed that proteins that contain zinc fingers can be selected or designed to 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 may have 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 (see www.zincfingertools.org). 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 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 and another transcription factor that upregulates fetal hemoglobin as a treatment for sickle cell anemia. More recently, we have focused on evolving zinc finger enzymes that modify the genome. These studies have led to the development of programmable zinc finger recombinases (Fig. 5) that promise to reshape the way scientists manipulate the genome for study and therapy of disease.

Fig. 5. Model of a zinc finger recombinase with programmable specificity created by using rational design and directed molecular evolution.


Barbas, C.F. III. Organocatalysis lost: modern chemistry, ancient chemistry, and an unseen biosynthetic apparatus. Angew. Chem. Int. Ed. 47:42, 2008.

Blancafort, P., Tschan, M.P., Bergquist, S., Guthy, D., Brachat, A., Sheeter, D.A., Torbett, B.E., Erdmann, D., Barbas, C.F. III. Modulation of drug resistance by artificial transcription factors. Mol. Cancer Ther. 7:688, 2008.

Jiang, L., Althoff, E.A., Clemente, F.R, Doyle, L., Röthlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F. III, Hilvert, D., Houk, K.N., Stoddard, B.L., Baker D. De novo computational design of retro-aldol enzymes. Science 319:1387, 2008.

Magnenat, L., Schwimmer, L.J., Barbas, C.F. III. Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches. Gene Ther. 15:1223, 2008.

Nomura, W., Barbas, C.F. III. In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase. J. Am. Chem. Soc. 129:8676, 2007.

Ramasastry, S.S.V., Albertshofer, K., Utsumi, N., Barbas, C.F. III. Water-compatible organocatalysts for direct asymmetric syn-aldol reactions of dihydroxyacetone and aldehydes. Org. Lett.10:1621, 2008.

Ramasastry, S.S.V., Albertshofer, K., Utsumi, N., Tanaka, F., Barbas, C.F. III. Mimicking fructose and rhamnulose aldolases: organocatalytic syn-aldol reactions with unprotected dihydroxyacetone. Angew. Chem. Int. Ed. 46:5572, 2007.

Tanaka, F., Fuller, R.P., Asawapornmongkol, L., Warsinke, A., Gobuty, S., Barbas, C.F. III. Development of a small peptide tag for covalent labeling of proteins. Bioconjug. Chem. 18:1318, 2007.

Utsumi, N., Imai, M., Tanaka, F., Ramasastry, S.S.V., Barbas, C.F. III. Mimicking aldolases through organocatalysis: syn-selective aldol reactions with protected dihydroxyacetone. Org. Lett. 9:3445, 2007.

Zhang, H., Mitsumori, S., Utsumi, N., Imai, M., Garcia-Delgado, M., Mifsud, M., Albertshofer, K., Tanaka, F., Barbas, C.F. III. Catalysis of 3-pyrrolidinecarboxylic acid and related pyrrolidine derivatives in enantioselective anti-Mannich-type reactions: importance of the 3-acid group on pyrrolidine for stereocontrol. J. Am. Chem. Soc. 130:875, 2008.

Zhang, H., Ramasastry, S.S.V., Tanaka, F., Barbas, C.F. III. Organocatalytic anti-Mannich reactions with dihydroxyacetone and acyclic dihydroxyacetone derivatives: a facile route to amino sugars. Adv. Synth. Catal. 350:791, 2008.


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

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