Scientific Report 2008
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
|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.
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).
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.
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.
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
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
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
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.
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
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.
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.
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.