News and Publications

From Catalytic Asymmetric Synthesis to the Regulation of Genes: In Vivo and In Vitro Evolution of Proteins

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 eventually create new agricultural products. These proteins might help explain our past and define our future.

Two problems that thwart the realization of this goal are the protein-folding problem and the chemistry of catalysis. An alternative approach to the production of designer protein catalysts was developed in 1986 by the laboratories of Lerner and Schultz. This work gave rise to a new area of investigation: catalytic antibodies. A large part of this work is built on the Haldane-Pauling hypothesis of transition-state stabilization as a primary effector of catalysis.

In our laboratory, we are extending and refining approaches to catalytic antibodies by using novel recombinant strategies coupled with reactive immunization and chemical-event selections. 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 antibodies. Essentially, we are evolving proteins to function as efficient catalysts, a task that Nature has performed in 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 work 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 enamines and the development of antibodies that use covalent catalysis (Fig. 1).

The specific reactions we are examining are the aldol, the Michael, the Diels-Alder, and a variety of decarboxylation reactions. Many of these catalysts may someday be important in the synthesis of enantiomerically pure drugs. Using novel catalytic antibodies, we have shown the efficient asymmetric synthesis and resolution of a variety of molecules, including tertiary and fluorinated aldols and a variety of natural products. On the basis of the structure of one of our antibody catalysts, we created a plethora of enamine-forming antibodies and are evolving the antibodies toward ever greater efficiency. This past year, we also learned how to extend the complex covalent chemistry of proteins to nucleic acids and created the first functionalized DNA enzymes.

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 this 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. Recently, we created the first class of polydactyl proteins that recognize 18 contiguous base pairs of DNA. We have also made marked 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. 2). The prospects for this "second genetic code" are fascinating, and the code could have a major impact on basic and applied biology. 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 and others that will inhibit the expression of a protein known as CCR5, which is a key to infection of human cells by HIV type 1. This past year we showed that we can use our alphabet of proteins to specifically regulate genes important in cancer, such as erbB-2 (Fig. 3) and the integrin ß₃ genes.

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. In the past year, we produced human antibodies that should cause the selective starvation of a wide variety of cancers by inhibiting angiogenesis and antibodies that will be used to deliver radioisotopes to colon cancers to destroy the tumors. We hope to see some of these antibodies in clinical trials with our collaborators at the Memorial Sloan-Kettering Cancer Center in New York City in the next year.

Publications

Barbas, C.F. III, Heine, A., Zhong, G., Hoffmann, T., Gramatikova, S., Björnestedt, R., List, B., Anderson, J., Stura, E.A., Wilson, E.A., Lerner, R.A. Immune versus natural selection: Antibody aldolases with enzymic rates but broader scope. Science 278:2085, 1997.

Barbas, C.F. III, List, B. Alchemy, enzymes, and the blind-watchmaker. Nature Biotechnol. 16:423, 1998.

Beerli, R.R., Segal, D.J., Dreier, B., Barbas, C.F. III Towards controlling gene expression at will: Specific regulations of the erB-2/HER-2 promoter using polydactyl zinc finger proteins constructed from modular building blocks. Proc. Natl. Acad. Sci. U.S.A., in press.

Bera, T.P., Kennedy, P.E., Berger, E.A., Barbas, C.F. III, Pastan, I. Specific killing of HIV infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein. Mol. Med., in press.

Finn, M.G., Lerner, R.A., Barbas, C.F. III. Cofactor induced refinement of catalytic antibody activity: A metal-specific allosteric effect. J. Am. Chem. Soc. 120:2963, 1998.

Gauduin, M.-C., Parren, P.W.H.I., Weir, R., Allaway, G.P., Maddon, P.J., Barbas, C.F. III, Burton, D.R., Koup, R.A. Passive Immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1. Nature Med. 3:1389, 1997.

Hoffmann, T., Zhong, G., List, B., Shabat, D., Anderson, J., Gramatikova, S., Lerner, R.A., Barbas, C.F. III. Aldolase antibodies of remarkable scope. J. Am. Chem. Soc. 120:2768, 1998.

Lerner, R.A., Barbas, C.F. III, Jana, K.D. Making enzymes. Harvey Lect. Ser. 92:1, 1998.

Lin, C.-H., Hoffmann, T.Z., Wirsching, P., Barbas, C.F. III, Janda, K.D., Lerner, R.A. On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use the enamine mechanism. Proc. Natl. Acad. Sci. U.S.A. 94:11773, 1997.

Lin, E.C.K., Ratnikov, B.I., Tsai, P.M., Carron, C.P., Myers, D.M., Barbas, C.F. III, Smith, J.W. Identification of a region in the integrin ß₃ subunit that confers ligand binding specificity. J. Biol. Chem. 272:23912, 1997.

List, B., Shabat, D., Barbas, C.F. III, Lerner, R.A. Enantioselective total synthesis of some brevicomins using aldolase antibody 38C2. Chem. Eur. J. 4:881, 1998.

Rader, C., Cheresh, D., Barbas, C.F. III. Phage display approach for rapid antibody humanization: Designed combinatorial V gene libraries. Proc. Natl. Acad. Sci. U.S.A. 95:8910, 1998.

Sakthivel, K., Barbas, C.F. III Expanding the potential of DNA for binding and catalysis: Delineation of a class of highly functionalized dUTP derivatives that are substrates for thermostable DNA polymerases. Angew. Chem., in press.

Shulman, A., Shabat, D., Barbas, C.F. III, Keinan, F.J. Teaching catalytic antibodies to undergraduate students: An organic chemistry lab experiment. Chem. Educ., in press.

Sullivan, N., Sun, Y., Binley, J., Lee, J., Barbas, C.F. III, Parren, P.W.H.I., Burton, D.R., Sodroski, J. Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies. J. Virol. 72:6332, 1998.

Wong, N.C., Mueller, B.M., Barbas, C.F., Ruminski, P., Quaranta, V., Lin, E.C.K., Smith, J.W. α_V Integrins mediate adhesion and migration of breast carcinoma cell lines. Clin. Exp. Metastasis 16:50, 1998.

Zhong, G., Shabat, D., List, B., Anderson, J., Sinha, S.C., Lerner, R.A., Barbas, C.F. III Catalytic enantioselective retro-aldol reactions: Kinetic resolution of ß-hydroxyketones using aldolase antibodies. Angew. Chem., in press.