News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000
Cancer, AIDS, Catalysis, and the Regulation of Genes: Building Molecules
With Defined Functions
C.F. Barbas III, R. Lerner, R. Beerli, J. Berry, P. Blancafort, T. Bui, J.
Chung, B. Dreier, M. Elia, R. Fuller, J. Flippin, S. Gobuty, C. Lund, L. Magnenat,
J. McDunn, J. Neves, W. Notz, M. Popkov, C. Rader, R. Reisfeld, K. Sakthivel,
D. Segal, D. Shabat, S. Sinha, J. Stege, P. Steinberger, F. Tanaka, J. Turner,
J. Velker, S. von Berg, 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 create new agricultural products. These proteins might help explain our past
and define our future.
In our laboratory, we are extending and refining approaches to catalytic
antibodies by using novel recombinant strategies coupled with reactive immunization,
chemical-event selections, and the design of unique multiturnover selection chemistries.
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 and synthetic antibodies.
Essentially, we are evolving proteins to function as efficient catalysts, a task
that was performed naturally over 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 research 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 imines and enamines and the development of antibodies
that use covalent catalysis. We are examining the aldol, the Michael, and the
Diels-Alder reactions and related reactions such as the Mannich reaction. 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, carbohydrates, insect pheromones, and derivatives of the epothilone anticancer
This past year we reported a new approach to catalytic antibodies that involves
combining transition-state theory with reactive immunization (Fig. 1). Using
this approach, we produced a large array of highly active catalytic antibodies,
including antibodies with stereoselectivities antipodal to our original aldolase
antibodies. 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 man-made protein catalyst to date. These
new catalysts have enabled S. Sinha, the Skaggs Institute, to develop new highly
efficient syntheses of the anticancer epothilone drugs.
Catalysts Large And Small
Recently, we reported the chemical synthesis of a new class of DNA bases
that can be used to construct proteinlike DNAs. These new bases contain side
chains common to amino acids used by proteins. The bases have enabled us to begin
to explore the versatility of functionalized DNA in catalysts and to create the
first proteinlike DNA enzyme, a general-purpose RNA-cleaving enzyme that is superior
to "natural" DNA enzymes.
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 prepared simple chiral amines to catalyze aldol and
related imine and enamine chemical actions such as Diels-Alder reactions. We
also studied small amine-bearing peptides that are catalytic. Attempts to create
aldolase DNAs are in progress. Although aldolase antibodies are superior catalysts,
the simpler catalysts are enabling us to measure the importance of pocket sequestration
in catalysis (Fig. 2).
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. We showed that a wide variety of clinically relevant
anticancer drugs such as doxorubicin, camptothecin, and etoposide can be modified
to less toxic prodrugs that can be specifically activated by catalytic antibody
38C2 to kill cancer cell lines. With R. Reisfeld, The Scripps Research Institute,
we showed the efficacy of this approach in animal models of cancer (Fig. 3).
Currently, we are developing more potent drugs as well as novel antibodies that
will enable us to target a range of cancers and HIV. On the basis of our preliminary
findings, we think that our approach can become a key tool in selective chemotherapeutic
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 our 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 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. 4). The prospects for this "second
genetic code" are fascinating and should have a major impact on basic and applied
We showed that these domains are functionally modular and can be recombined
with one another to create polydactyl proteins capable of binding 18-bp sequences
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. We showed that we can use our alphabet of proteins to specifically turn
on or turn off genes at will.
One of our targets in cancer is the oncogene erbB-2. ErbB-2, a member
of the ErbB receptor family, is overexpressed as a result of gene amplification
and/or transcriptional deregulation in a high percentage of human adenocarcinomas,
including those of the breast, ovary, lung, stomach, and salivary gland. We showed
that we can shut this gene off in a highly specific fashion and induce a growth
arrest in cancer cells, providing a promising new strategy for cancer therapy.
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., Rader, C., Steinberger, P., Fuller, R., Barbas, C.F.
III. Optimized methods for the preparation of chicken monoclonal antibody
fragments by phage display. J. Immunol. Methods 242:159, 2000.
Barbas, C.F. III, Burton, D., Silverman, D., Scott, J. (Eds.). Phage
Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, in press.
Barbas, C.F. III, Rader, C., Segal, D.J., List, B., Turner, J.M. From
catalytic asymmetric synthesis to the transcriptional regulation of genes: In
vivo and in vitro evolution of proteins. Adv. Protein Chem. 55:317, 2000.
Beerli, R.R., Dreier, B., Barbas, C.F. III. Selective positive and
negative regulation of endogenous genes by designed transcription factors. Proc.
Natl. Acad. Sci. U. S. A. 97:1495, 2000.
Beerli, R.R., Schopfer, U., Dreier, B., Barbas, C.F. III. Chemically
regulated zinc finger transcription factors. J. Biol. Chem. 275:32617, 2000.
Bui, T., Barbas, C.F. III. A proline-catalyzed asymmetric Robinson
annulation reaction. Tetrahedron Lett. 41:6951, 2000.
Dreier, B., Segal, D.J., Barbas, C.F. III. Insights into the molecular
recognition of the 5´-GNN-3´ family of DNA sequences by zinc-finger
domains. J. Mol. Biol. 303:489, 2000.
Karlström, A.H., Zhong, G., Rader, C., Larsen, N., Heine, A., Fuller,
R., List, B., Wilson, I.A., Barbas, C.F. III, Lerner, R.A. Using antibody
catalysis to study the outcome of multiple evolutionary trials of a chemical
task. Proc. Natl. Acad. Sci. U. S. A. 97:3878, 2000.
List, B., Lerner, R.A., Barbas, C.F. III. Enantioselective aldol-cyclodehydrations
catalyzed by antibody 38C2. Org. Lett. 1:59, 1999.
List, B., Lerner, R.A., Barbas, C.F. III. Proline-catalyzed direct
asymmetric aldol reactions. J. Am. Chem. Soc. 122:2395, 2000.
Notz, W., Sakthivel, S., Bui, T., Barbas, C.F. III. Amine-catalyzed
direct asymmetric Mannich-type reactions. Tetrahedron Lett., in press.
Rader, C., Ritter, G., Nathan, S., Elia, M., Gout, I., Jungbluth, A.A.,
Cohen, L.S., Welt, S., Old, L.J., Barbas, C.F. III. The rabbit antibody repertoire
as a novel source for the generation of therapeutic human antibodies. J. Biol.
Chem. 275:13668, 2000.
Santoro, S.W., Joyce, G.F., Sakthivel, K., Gramatikova, S., Barbas, C.F.
III. RNA cleavage by a DNA enzyme with protein-like functionality. J. Am.
Chem. Soc. 122:2433, 1999.
Segal, D., Barbas, C.F. III. Design of novel sequence-specific DNA-binding
proteins. Curr. Opin. Chem. Biol. 4:34, 2000.
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. 1:1623, 1999.
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. 97:805, 1999.
Steinberger, P., Sutton, J.K., Rader, C., Elia, M., Barbas, C.F. III. Generation
and characterization of a recombinant human CCR5-specific antibody: A phage display
approach for rabbit antibody humanization. J. Biol. Chem. 275:36073, 2000.
Tanaka, F., Lerner, R.A., Barbas, C.F. III. Reconstructing aldolase
antibodies to alter their substrate specificity and turnover. J. Am. Chem. Soc.
Tanaka, F., Lerner, R.A., Barbas, C.F. III. Thiazolium-dependent catalytic
antibodies produced using a covalent modification strategy. Chem. Commun. 1383,
Turner, J.M., Bui, T., Lerner, R.A., Barbas, C.F. III, List, B. An
efficient benchtop system for multigram-scale kinetic resolutions using aldolase
antibodies. Chem. Eur. J. 6:2772, 2000.
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.