News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998
From Catalytic Asymmetric Synthesis to the Regulation of Genes: In Vivo
and In Vitro Evolution of Proteins
C.F. Barbas III, H. Almer, J. Anderson, R. Beerli, B. Dreier, A. Karlström,
R. Lewis II, B. List, C. Rader, K. Sakthivel, D. Segal, D. Shabat, P. Steinberger,
F. Tanaka, S. Touami, J. Widhopf-Andris, G. Zhong
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.
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.
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. αV 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