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The Skaggs Institute
for Chemical Biology

Scientific Report 2008




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 Nature’s 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.

Catalytic Antibodies

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.

Organocatalysis

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 Nature’s 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.
Fig. 1. Organocatalysis with natural and designed amino acids leads to a variety of efficient asymmetric syntheses previously approachable only via enzyme catalysis. A–C, Design considerations for a family of organoaldolases that allow large families of carbohydrates to be readily synthesized.

Chemically Programmed Antibodies

In 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.
Fig. 2. 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.

In 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.
Fig. 3.Through 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.

Publications

Alonso, 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. III. N-Tosylimidates in highly enantioselective organocatalytic Michael reactions. Tetrahedron Lett. 50:145, 2009.

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.

Zhang, 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.

 

Carlos F. Barbas III, Ph.D.
Professor
Janet and W. Keith
  Kellogg II Chair in
  Molecular Biology

Barbas Web Site