News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
From Catalytic Asymmetric Synthesis to the Regulation of Genes: In Vivo and
In Vitro Evolution of Proteins
C.F. Barbas III, R. Lerner, R. Beerli, J. Berry, P. Blancafort, T. Bui, B.
Dreier, M. Elia, R. Fuller, A. Karlström, R. Lewis, A. Li, B. List, C. Lund,
L. Magnenat, J. Neves, C. Rader, B.P. Rheiner, K. Sakthivel, U. Schopfer,* D.
Segal, D. Shabat, J. Stege, P. Steinberger, F. Tanaka, S. Touami, J. Turner,
J. Andris-Widhopf, J. Velker, S. von Berg, G. Zhong
* Novartis Pharmaceuticals, Basel, Switzerland
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 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 a route 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. 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 showed the efficient asymmetric synthesis and
resolution of a variety of molecules, including tertiary and fluorinated aldols.
Using an antibody-based synthon approach, we synthesized carbohydrates, insect
pheromones, and derivatives of the epothilone anticancer drugs.
Catalytic antibodies can be used not only to synthesize anticancer drugs but
also to deliver the drugs in a highly specific fashion to the cancer itself.
Typically, the application of highly potent chemotherapeutic agents is limited
by nonspecific toxic effects associated with the inability to direct these agents
to the appropriate targets. This year we showed that the clinically relevant
anticancer drugs doxorubicin and camptothecin can be modified to be less toxic
prodrugs that can be specifically activated by catalytic antibody 38C2 to kill
prostate and colon cancer cell lines (Fig. 1). We hope to advance to animal models
of cancer in the next year.
What if DNA could be endowed with the functionality of proteins? Might it
be a more versatile catalyst? Through chemical synthesis, we developed a new
class of DNA bases that can be used to construct proteinlike DNAs. These new
bases contain side chains common to amino acids. In a collaborative effort with
G. Joyce, the Skaggs Institute, we created the first proteinlike DNA enzyme (Fig.
2), a general-purpose RNA-cleaving enzyme that is superior to "natural" DNA enzymes.
This catalyst may be directly applicable as an antiviral agent. We hope to create "proteinlike" DNA
enzymes that perform the small-molecule chemistry of interest to synthetic chemists.
Software For the Genome
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. To this end, we have
made significant 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. 3). The prospects for this "second genetic code" are
fascinating, and the code could have a major impact on basic and applied biology.
We showed that these domains are functionally modular and can be recombined with
one another to create polydactyl proteins capable of binding 18-bpsequences with
subnanomolar affinity. We now have at hand a family of zinc finger domains sufficient
for the construction of 17 million novel proteins that bind the 5´-(GNN)6-3´ family
of DNA sequences.
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, halt replication of HIV, and even make healthier
corn. This year we showed that we can use our alphabet of proteins to specifically
turn on or turn off genes at will.
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. When combined with activation of prodrugs by catalytic
antibodies, these disease-targeting antibodies will have powerful disease-fighting
potential. We hope to take these molecules all the way to clinical trials.
Andris-Widhopf, J., Steinberger, P., Barbas, C.F. III. Bacteriophage
display of combinatorial antibody libraries. In: Encyclopedia of Life
Sciences. Macmillan Press, London, England, in press.
Beerli, R.R., Dreier, B., Barbas, C.F. III. Exquisitely selective positive
and negative regulation of endogenous genes by designed transcription factors.
Proc. Natl. Acad. Sci. U. S. A., in press.
Beerli, R.R., Segal, D.J., Dreier, B., Barbas, C.F. III. Towards controlling
gene expression at will: Specific regulations of the erbB-2/HER-2 promoter using
polydactyl zinc finger proteins constructed from modular building blocks. Proc.
Natl. Acad. Sci. U. S. A. 95:14628, 1998.
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. 4:384, 1998.
Crowe, J.E., Jr., Firestone, C.-Y., Crim, R., Beeler, J.A., Coelingh, K.L.,
Barbas, C.F. III, Burton, D.R., Chanock, R.M., Murphy, B.R. Monoclonal antibody-resistant
mutants selected with a respiratory syncytial virus-neutralizing human antibody
Fab fragment (Fab 19) define a unique epitope on the fusion (F) glycoprotein.
Virology 252:373, 1998.
List, B., Barbas, C.F. III, Lerner, R.A. Aldol sensors for the rapid
generation of fluorescence by antibody catalysis. Proc. Natl. Acad. Sci. U. S.
A. 95:15351, 1998.
List, B., Lerner, R.A., Barbas, C.F. III. Enantioselective aldol-cyclodehydrations
catalyzed by antibody 38C2. Org. Lett., in press.
List, B., Shabat, D., Zhong, G., Turner, J.M., Li, A., Bui, T., Anderson,
J., Lerner, R.A., Barbas, C.F. III. A catalytic enantioselective route to
hydroxy-substituted quaternary carbon centers: Resolution of tertiary aldols
with a catalytic antibody. J. Am. Chem. Soc. 121:7283, 1999.
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. 37:2872, 1998.
Segal, D., Barbas, C.F. III. Design of novel sequence-specific DNA-binding
proteins. Curr. Opin. Chem. Biol., in press.
Segal, D.J., Dreier, B., Beerli, R.R., Ghiara, J.B., Barbas, C.F. III. Towards
controlling gene expression at will: Selection and design of zinc finger domains
recognizing each of the 5´-GNN-3´ DNA target sequences. Proc. Natl.
Acad. Sci. U. S. A. 96:2758, 1999.
Shabat, D., List, B., Lerner, R.A., Barbas, C.F. III. A short enantioselective
synthesis of 1-deoxy-l-xylulose by antibody catalysis. Tetrahedron Lett. 40:1437,
Shabat, D., Rader, C., List, B., Lerner, R.A., Barbas, C.F. III. Multiple
event activation of a generic prodrug trigger by antibody catalysis. Proc. Natl.
Acad. Sci. U. S. A. 96:6925, 1999.
Shulman, A., Shabat, D., Barbas, C.F. III, Keinan, F. Teaching catalytic
antibodies to undergraduate students: An organic chemistry lab experiment. J.
Chem. Educ. 76:977, 1999.
Sinha, S.C., Barbas, C.F. III, Lerner, R.A. The antibody catalysis
route to the total synthesis of epothilones. Proc. Natl. Acad. Sci. U. S. A.
Sinha S.C., Sun, J., Miller, G., Barbas, C.F. III, Lerner, R.A. Sets
of aldolase antibodies with antipodal reactivities: Synthesis of epothilone E
by resolution of thiazole aldols. Org. Lett., in press.
Steinberger, P., Andris-Widhopf, J., Bühler, B., Torbett, B.E., Barbas,
C.F. III. Functional deletion of the CCR5 receptor by intracellular immunization
produces cells that are refractory to CCR5-dependent HIV-1 infection and cell
fusion. Proc. Natl. Acad. Sci. U. S. A., in press.
Tanaka, F., Lerner, R.A., Barbas, C.F. III. Catalytic single-chain
antibodies possessing ß-lactamase activity selected from a phage displayed
combinatorial library using a mechanism-based inhibitor. Tetrahedron Lett. 40:8063,
Tanaka, F., Lerner, R.A., Barbas, C.F. III. Thiazolium-dependent catalytic
antibodies produced using a covalent modification strategy. Chem. Commun., in
Zhong, G., Lerner, R.A., Barbas, C.F. III. Broadening the aldolase
catalytic antibody repertoire by combining reactive immunization and transition
state theory: New enantio- and diastereo-selectivities. Angew. Chem. Int. Ed., in
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.
Zwick, M.B., Bonnycastle, L.L.C., Noren, K.A., Venturini, S., Leong, E.,
Barbas, C.F. III, Noren, C.J., Scott, J.K. The maltose-binding protein as
a scaffold for monovalent display of peptides derived from phage libraries. Anal.
Biochem. 264:87, 1998.