The Skaggs Institute
for Chemical Biology
Catalysis, Cancer, and the Regulation of Genes: Inventing Molecules With Defined Functions
C.F. Barbas III, K. Albertshofer, T. Bui, R.P. Fuller, T. Gaj, J. Gavrilyuk, C. Gersbach, B. Gonzalez, R.M. Gordley,
J. Guo, S. Juraja, D.H. Kim, A. Mercer, S. Mizuta, A. Onoda, S. Salahuddin, M. Santa Marta, L.J. Schwimmer, F. Tanaka, H. Uehara, N. Utsumi, U. Wuellner, K.S.
Yi, H. Zhang
We are concerned
with problems at the interface of molecular biology, chemistry, and medicine. Many
of our studies involve learning or improving on Natures strategies to prepare
novel molecules that perform specific functional tasks, such as regulating a gene,
destroying cancer, or catalyzing a reaction with small molecules in an enzymelike
manner. We hope to apply these novel insights, methods, and products to provide
solutions to human diseases, including cancer, HIV disease, and genetic diseases.
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.
These strategies involve the directed evolution of human, rodent, and synthetic
antibodies. Essentially, we are evolving proteins to function as efficient catalysts,
a task that is naturally performed 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. In addition to fashioning new enzymatic function to study the
chemistry of imines and enamines, we hope to apply these catalysts in novel therapies
against cancer and HIV type 1 infection that couple catalytic antibody activity
with activation of designed prodrugs.
In studying how proteins catalyze reactions,
we often examine how the constituent components react. These studies have led to
a new green approach to catalytic asymmetric synthesis that can be applied to the
synthesis of drugs and druglike molecules. Using insights garnered from our studies
of aldolase antibodies, we prepared simple chiral amino acids and amines to catalyze
aldol and related imine and enamine chemistries such as Michael and Mannich reactions.
We also studied small amine-bearing peptides that are catalytic. Although aldolase
antibodies are superior catalysts, simple chiral amino acids and amines are enabling
us to determine the importance of pocket sequestration in catalysis.
We showed that L-proline and other chiral
amines can be efficient asymmetric catalysts of a variety of important imine- and
enamine-based reactions. Studies from our laboratory and the contributions of others
have produced advances toward one of the ultimate goals in organic chemistry: the
catalytic asymmetric assembly of simple and readily available precursor molecules
into stereochemically complex products under operationally simple and, in some instances,
environmentally friendly experimental protocols. An important result of these studies
is the development of catalysts that allow aldehydes, for the first time, to be
used efficiently as nucleophiles in a wide variety of catalytic asymmetric reactions.
Previously, only naturally occurring enzymes were thought capable of this chemical
feat. With future efforts, small organic catalysts may match some of Natures
other heretofore unmatched synthetic prowess. These catalysts might help explain
the development of complex chemical systems in the prebiotic world and provide hints
toward yet-to-be discovered mechanisms in extant biological systems.
Using this method, we directly synthesized a wide variety of α and β
amino acids, carbohydrates, and lactams. Stereochemically complex molecules can
now be assembled by using small molecules in a manner analogous to that of natural
enzymes. Novel catalyst designs have enabled us to synthesize particular diastereoisomers
previously not accessible with proline, and we envision that this approach will
largely replace the use of aldolase enzymes in synthesis (Fig. 1). New and exciting
catalytic asymmetric reactions continue to emerge from these studies.
Organocatalysis with natural and designed amino acids leads to a variety of efficient
asymmetric syntheses previously approachable only via enzyme catalysis. AC,
Design considerations for a family of organoaldolases that allow large families
of carbohydrates to be readily synthesized.
Chemically Programmed Antibodies
targeting cancer, we take a multidisciplinary approach that involves gene regulation,
catalytic antibodies, drug design, and combinatorial antibody libraries. Using a
chemically programmed antibody strategy, we recently showed the power of combining
small-molecule chemistry with immunochemistry. We designed small-molecule integrin
antagonists to self-assemble into a covalent complex with antibody 38C2 (Fig. 2).
The resulting chemically programmed antibody had significant advantages compared
with small molecules or antibody alone in studies of metastatic melanoma, colon
cancer, and breast cancer. We recently developed a powerful new approach to a programmable
vaccine strategy based on a universal vaccination that elicits programmable antibodies.
Combining the power of small-molecule chemistry with the power of protein chemistry
and immunology, we have created a new and effective class of therapeutic molecules,
chemically programmed antibodies. A covalently bound diketone is shown in the active
site of an aldolase antibody. This covalent chemistry allows rapid antibody programming.
Designer Transcription Factors And Enzymes
From the simplest to the most complex,
proteins that bind nucleic acids are involved in orchestrating gene expression.
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. Lack of coordination due to genetic defects
or to viral seizure of control of the cell results in disease.
one project, we are developing methods to produce proteins that bind to specific
DNA sequences to control specified genes. As we showed earlier, these proteins can
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 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 should have a major impact on basic and applied biology.
Billions of transcription factors can
now be prepared by using our approach. Our goal 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.
Using a novel library of transcription
factors, we developed a strategy that effectively allows us to turn on and turn
off every gene in the genome. We recently extended this approach to enable us to endow
a variety of enzymes with sequence specificity of our own design (Fig. 3). In the
future, these new enzymes will enable us to insert, delete, or otherwise modify
genes with surgical precision within any genome.
a combination of rational and evolutionary design, we created a variety of zinc
finger enzymes that function in human cells. Novel enzymes such as recombinases,
methylases, nucleases, and integrases are under development. A designed zinc finger
recombinase enzyme is shown above the sequence on which it acts.
D., Kitagaki, S., Utsumi, N., Barbas, C.F. III. Towards
organocatalytic polyketide synthases with diverse electrophile scope: trifluoroethyl
thioesters as nucleophiles in organocatalytic Michael reactions and beyond. Angew.
Chem. Int. Ed. 47:4588, 2008.
Blancafort, P., Tschan, M.P., Bergquist,
S., Guthy, D., Brachat, A., Sheeter, D.A., Torbett, B.E., Edrmann, D., Barbas, C.F.
III. Modulation of drug resistance
by artificial transcription factors. Mol. Cancer Ther. 7:688, 2008.
Gordley, R.M., Gersbach, C.A., Barbas,
C.F. III. Synthesis of programmable
integrases. Proc. Natl. Acad. Sci. U. S. A., in press.
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 [published correction appears in
Gene Ther. 15:1246, 2008]. Gene Ther. 15:1223, 2008.
Massa, A., Utsumi, U., Barbas, C.F.
in highly enantioselective organocatalytic Michael reactions. Tetrahedron Lett.
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.
Tanaka, F., Hu, Y., Sutton, J., Asawapornmongkol,
L., Fuller, R., Olson, A.J., Barbas, C.F. III, Lerner, R.A.
Selection of phage-displayed peptides that bind to a particular ligand-bound antibody.
Bioorg. Med. Chem. 16:5926, 2008.
Utsumi, N., Kitagaki, S., Barbas,
C.F. III. Organocatalytic
Mannich-type reactions of trifluoroethyl thioesters. Org. Lett. 10:3405, 2008.
Zhang, 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. 350:791, 2008.
H.L., Mitsumori, S., Utsumi, N., Imai, M., Garcia-Delgado, N., 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.