The Skaggs Institute
for Chemical Biology

Scientific Report 2006

Catalysis, Cancer, and the Regulation of Genes: Inventing Molecules With Defined Functions

C.F. Barbas III, K. Albertshofer, L. Asawapornmongkol, T. Bui, S. Eberhardy, R.P. Fuller, B.A. Gonzalez, R. Gordley, J. Guo, M. Imai, D.H. Kim, J. Mandell, W. Nomura, M. Popkov, L.J. Schwimmer, S.V. Sripada, F. Tanaka, R. Thayumanavan, U. Tschulena, N. Utsumi, Y. Ye, K.S. Yi, Y. Yuan, 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 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. Recent studies revealed the potential of catalytic antibodies to catalyze unfavored Michael reactions and to serve as triggers for potent tethered prodrugs.

Organocatalysis: Enzymelike Chemistry with Small Organic Molecules

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 measure 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 advanced 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. A significant 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 enable us to synthesize particular diastereoisomers previously not accessible (Fig. 1).

Fig. 1. Organocatalysis and the design of novel catalysts. Our studies in organocatalysis with the natural amino acid proline have led to the efficient asymmetric syntheses of a variety of important synthons, including Mannich-type products (A). We have translated our understanding of proline catalysis to enable us to create novel catalysts (B) that allow the synthesis of anti-Mannich products not accessible through proline catalysis.

Small Molecules and Proteins: Targeting Cancer

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. The resulting chemically programmed antibody had significant advantages compared with small molecules or antibody alone in studies of metastatic melanoma (Fig. 2) and breast cancer. With catalytic antibodies, we are also developing strategies to activate drugs in a highly specific fashion at the site of cancer.

Fig. 2. Chemically programmed antibodies. By 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. The chemically programmed antibody 38C2/SCS completely protects mice from death by melanoma metastasis, beating the traditional antibody LM609, the small molecule SCS alone, and the unprogrammed antibody 38C2.

Designer Transcription Factors and Recombinases

From the simplest to the most complex, proteins that bind nucleic acids are involved in orchestrating gene expression. 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.

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 enzymes with sequence specificity of our own design through the creation of zinc finger recombinases (Fig. 3). We think these new enzymes will allow us to insert or delete genes with surgical precision within the genome.

Fig. 3.Programmable zinc finger recombinases. Through a combination of rational and evolutionary design, we have created zinc finger recombinases that function in human cells.

Publications

Cheong, P.H-Y., Zhang, H., Thayumanavan, R., Tanaka, F., Houk, K.N., Barbas, C.F. III. Pipecolic acid-catalyzed direct asymmetric Mannich reactions. Org. Lett. 8:811, 2006.

Chowdari, N.S., Ahmad, M., Albertshofer, K., Tanaka, F., Barbas, C.F. III. Expedient synthesis of chiral 1,2- and 1,4-diamines: protecting group dependent regioselectivity in direct organocatalytic asymmetric Mannich reactions. Org. Lett. 8:2839, 2006.

Eberhardy, S.R., Goncalves, J., Coelho, S., Segal, D.J., Berkhout, B., Barbas, C.F. III. Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site. J. Virol. 80:2873, 2006.

Gordley, R.M., Smith, J.D., Graslund, T., Barbas, C.F. III. Evolution of programmable zinc finger recombinases with activity in human cells. J. Mol. Biol., in press.

Guo, F., Das, S., Mueller, B.M., Barbas, C.F. III, Lerner, R.A., Sinha, S.C. Breaking the one antibody-one target axiom. Proc. Natl. Acad. Sci. U. S. A. 103:11009, 2006.

Mandell, J., Barbas, C.F. III. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 34(Web server issue):516, 2006.

Mase, N., Nakai, Y., Ohara, H., Yoda, H., Takabe, K., Tanaka, F., Barbas, C.F. III. Organocatalytic direct asymmetric aldol reactions in water. J. Am. Chem. Soc. 128:734, 2006.

Mase, N., Watanabe, K., Yoda, H., Takabe, K., Tanaka, F., Barbas, C.F. III. Organocatalytic direct Michael reaction of ketones and aldehydes with β-nitrostyrene in brine. J. Am. Chem. Soc. 128:4966, 2006.

Mitsumori, S., Zhang, H., Cheong, P.H-Y., Houk, K.N., Tanaka, F., Barbas, C.F. III. Direct asymmetric anti-Mannich-type reactions catalyzed by a designed amino acid. J. Am. Chem. Soc. 128:1040, 2006.

Nathan, S., Rader, C., Barbas, C.F. III. Neutralization of Burkholderia pseudomallei protease by Fabs generated through phage display. Biosci. Biotechnol. Biochem. 69:2302, 2005.

Popkov, M., Rader, C., Gonzalez, B., Sinha, S.C., Barbas, C.F. III. Small molecule drug activity in melanoma models may be dramatically enhanced with an antibody effector. Int. J. Cancer 119:1194. 2006.

Segal, D.J., Crotty, J.W., Barbas, C.F. III, Horton, N.C. Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA. J. Mol. Biol. 363:405, 2006.

Suri, J.T., Mitsumori, S., Albertshofer, K., Tanaka, F., Barbas, C.F. III. Dihydroxyacetone variants in the organocatalytic construction of carbohydrates: mimicking tagatose and fuculose aldolases. J. Org. Chem. 71:3822, 2006.

Swan, C.H., Buhler, B., Tschan, M.P., Barbas, C.F. III, Torbett, B.E. T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther. 13:1480, 2006.

Tan, W., Dong, Z., Wilkinson, T.A., Barbas, C.F. III, Chow, S.A. Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells. J. Virol. 80:1939, 2006.

Zhang, H., Mifsud, M., Tanaka, F., Barbas, C.F. III. 3-Pyrrolidinecarboxylic acid for direct catalytic asymmetric anti-Mannich-type reactions of unmodified ketones. J. Am. Chem. Soc. 128:9630, 2006.