Christoph Boss, Ph.D.
Thomas Heinz, Ph.D.
Göran Hilmersson, Ph.D.
Carina Horn, Ph.D.
Byeang Hyean Kim, Ph.D.
Arne Luetzen, Ph.D.
Shihong Ma, Ph.D.
Tomas Martin, Ph.D.
Sandro Mecozzi, Ph.D.
Daniel Mink, Ph.D.
Derek Nelson, Ph.D.
Ulrike Obst, Ph.D.
Doris Pupowicz, Ph.D.
Christian Rojas, Ph.D.
Dmitry Rudkevich, Ph.D.
Javier Santamaria, Ph.D.
Tomas Szabo, Ph.D.
Yuji Tokunaga, Ph.D.
Boris Vauzeilles, Ph.D.
Siegfried Waldvogel, Ph.D.
Sabine Wallbaum, Ph.D.
* Joint appointment in Department of Chemistry
Richard A. Lerner, M.D.
The efforts of our nation's scientific community in the 20th century have been responsible for remarkable discoveries resulting in innovative medical treatments and important diagnostic tools, improving the quality of life and increasing life expectancy for all Americans. Further, basic research has boosted this nation's technology base, fostered the vibrant biotechnology industry and substantially reduced health care costs for certain diseases through the development of improved health-related strategies and procedures.
Although the fruits of scientific labors are plentiful and have led to a quality of life unimagined not many years ago, a great deal more needs to be done. The achievements in molecular biology have been numerous, and the output of scientists has been prodigious, but many questions are as yet unanswered.
The United States must maintain its preeminence in biomedical research. To do so, we must encourage talented, young people to pursue the exhilarating and sometimes difficult field of scientific inquiry. We must provide them with adequate resources to appropriately use the plethora of new technologies available to advance knowledge. While the National Institutes of Health continues to receive modest annual funding increases--in the midst of the drive toward a balanced federal budget and fierce competition for discretionary government funding--much more needs to be done. Scientists must communicate with their constituents such that private industry, foundations, and philanthropists understand the importance of their roles in the research effort in this country.
This year is the first in the innovative research alliance with Novartis, and by all accounts, it has already been a remarkably productive collaboration. This alliance is a great match of institutions, with numerous examples of complementary scientific interests and a strong start to what we hope will be a long-term relationship. We are confident that our combined efforts will spark important discoveries and insights that will be transferred into useful medical therapeutics, with consequences for improved health and quality of life.
The monumental commitment of $100 million from the Skaggs family could not have come at a more momentous time in the history of the scientific community and The Scripps Research Institute. Scientists are in the midst of an explosion of scientific knowledge, with a concomitant pace of research discovery that is unparalleled. The synergy that has been created by TSRI scientists who hold dual appointments at the Skaggs Institute, as well as the exceptional investigators who have been recruited this year to join the Institute's ranks, promises to yield results that will have profound consequences for generations to come.
Indeed, this organization is extremely fortunate to have attracted researchers of the caliber of those who have recently joined us. Their expertise will add significantly to the depth and scope of the scientific activities here.
They are Paul Schimmel, Ph.D., former John D. and Catherine T. MacArthur Professor of Biochemistry and Biophysics, Massachusetts Institute of Technology; Jeffery W. Kelly, Ph.D., former Professor, Department of Chemistry, Texas A&M; James R. Williamson, Ph.D., former Associate Professor, Department of Chemistry, Massachusetts Institute of Technology; Martha J. Fedor, Ph.D., former Assistant Professor, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center; and Erik J. Sorensen, Ph.D., former National Science Foundation Postdoctoral Fellow, Memorial Sloan-Kettering Cancer Center.
A member of the National Academy of Sciences, Dr. Schimmel's major research activities have concentrated on the decoding of genetic information, with emphasis on the rules of the universal genetic code that are established through aminoacylation reactions catalyzed by aminoacyl tRNA synthetases. These synthetases are thought by many to be among the first enzymes to arise on this planet in the early stages of the evolution of life forms. Dr. Schimmel is the recipient of numerous prestigious awards, the author of many scientific papers and of a widely used three-volume textbook on biophysical chemistry. Having a longstanding interest in the applications of basic biomedical research to human health, he holds several patents and is a cofounder of four biotechnology companies.
Dr. Kelly, Lita Annenberg Hazen Professor of Chemistry, is an expert in the structure of amyloid peptides and the dynamics of their aggregation as they relate to amyloid diseases, including Alzheimer's. By engineering compounds that could bind selectively to the amyloid proteins and block other compounds from binding, Kelly and others hope to develop a compound that could stop the dense plaque formation found in the brains of patients with Alzheimer's disease.
The research interests of Dr. Williamson focus on determining RNA structure by using nuclear magnetic resonance spectroscopy. Additional research activities include nucleic acid chemistry, kinetics of RNA folding, RNA-protein interactions, and interaction of ribosomal proteins with rRNA.
Dr. Fedor works primarily in the area of structure and function of catalytic RNA. Her numerous honors include the Leukemia Society of America Postdoctoral Fellowship and the Bank of America Giannini Foundation Postdoctoral Fellowship.
Dr. Sorensen recently completed a postdoctoral fellowship at Memorial Sloan-Kettering Cancer Center, but he is not a stranger to TSRI. He completed his graduate studies at TSRI and the University of California, San Diego, in the laboratory of K.C. Nicolaou. His research interests include the development of strategies and methods for the synthesis of biologically active natural products and the design and chemical synthesis of mechanism-based inhibitors of enzymes.
The extraordinary generosity and vision of the Skaggs family have enabled this organization to welcome these outstanding researchers to the scientific community at TSRI. In an environment in which excellence is the norm, these scientists and others provide this organization with an opportunity to unlock a deeper understanding of life than has ever been possible.
Vision of the Skaggs Institute
Julius Rebek, Jr., Ph.D., Director
The Skaggs Institute for Chemical Biology has now completed its second full year and, thanks to the extraordinary munificence of Aline and Sam Skaggs, is fully funded. The Institute consists of more than 20 principal investigators in six departments, including Chemistry, Molecular Biology, Cell Biology, Neurobiology, and Molecular and Experimental Medicine. The Institute also has a physical presence in the building formerly known as Molecular Biology. The researchers have broad expertise in determining the structure of biological macromolecules, devising chemical and antibody catalysts, synthesizing natural products and combinatorial libraries, effecting molecular recognition, and designing methods for molecular modeling. These programs give the Institute its research identity at the interface of chemistry and biology in the United States and worldwide.
The reports in this publication highlight the individual achievements of the principal investigators. These accomplishments include the determination of the crystal structure of the T-cell receptor, the synthesis of antitumor agents, the discovery of multipurpose antibodies, the characterization of lipidlike hormones, the regulation of cell adhesion molecules, and the invention of self-replicating peptides. A more subtle accomplishment is the synergy that the Skaggs Institute has made possible between research groups.
For example, an initiative in RNA chemistry and biology is emerging around the recent recruitment of Paul Schimmel, Martha Fedor, and James Williamson. Their intent is to develop an understanding of the structure and function of these key molecules of life that will ultimately lead to new therapeutic agents. As another example, four groups now work in molecular evolution, research related to the origins of life. The depth of this effort has made the Skaggs Institute the leading edge for research in this field. A third cohesive effort is in drug design, which brings the Institute's superb structural and computational facilities for proteins and nucleic acids together with the expertise in organic synthesis and combinatorial chemistry.
The capability of the Skaggs Institute to assume broad, long-term projects makes it unique, and as we enter our third year, we are eyeing strategic opportunities in newly emerging fields that blend chemistry with biology. Nowhere does this seem more likely than in the opportunities expected to emerge from the sequencing of the genomes of living organisms and from the conversion of biological information pouring out of such projects to a science at the molecular level. This conversion will involve determining the genes that encode the specific proteins, receptors, or nucleic acids associated with a particular state; unraveling the interactions of those genes; and ultimately controlling the interactions by means of appropriate synthetic agents. Because an enormous array of small molecules is already available (and more agents will become available) through combinatorial chemical synthesis, it seems inevitable that the disciplines of genomics and combinatorial chemistry will meet at the biological macromolecules--the therapeutic targets--of the disease.
We intend to maintain the Skaggs Institute as the model for research in chemical biology and to provide a nurturing environment for the next generation of scientists. Accordingly, we will continue to recruit and maintain the very best researchers, even if their scientific interests do not fall neatly into one of the existing research efforts. The ultimate research identity of the Skaggs Institute will be the scientists it produces.
From Catalytic Asymmetric Synthesis to the Regulation of Genes: In Vivo and In Vitro Evolution of Proteins
C.F. Barbas III, H. Almer, J. Anderson, R. Beerli, J. Ghiara, S. Gramatikova, K. Kustedjo, R. Lewis II, B. List, C. Rader, K. Sakthivel, D. Segal, D. Shabat, P. Steinberger, F. Tanaka, S. Venturini, J. Widhopf-Andais, 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 eventually create new agricultural products and organisms. These proteins might help explain our past and define our future.
Two problems that thwart the realization of this goal are the protein-folding problem and the chemistry of catalysis. An alternative approach to the production of designer protein catalysts was developed in 1986 by the laboratories of Lerner and Schultz. This work gave rise to a new area of investigation: catalytic antibodies. A large part of this work is built on the Haldane-Pauling hypothesis of transition-state stabilization as a primary effector of catalysis.
In our laboratory, we are extending and refining approaches to catalytic antibodies by using novel recombinant strategies coupled with reactive immunization and chemical-event selections. We are developing in vitro selection or evolutionary strategies as a route for obtaining antibodies of defined activity. This strategy involves the directed evolution of human as well as rodent antibodies. Essentially, we are evolving proteins to function as efficient catalysts, a task that nature has performed over eons, and one that we intend to perform in weeks. The approach is a blend of chemistry, enzymology, and molecular biology.
A major focus of our work 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 enamines and the development of antibodies that use covalent catalysis (Fig. 1).
The specific reactions we are examining are the aldol, the Michael, the Diels-Alder, and a variety of decarboxylation reactions. Many of these catalysts may someday be important in the synthesis of enantiomerically pure drugs. In the past year, we have obtained the structure of one of our antibody catalysts and expressed it in Escherichia coli, making it possible to generate an abundance of efficient aldolases.
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 two cells, four cells, and so on, finally yielding 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 this 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. Recently, we created a class of polydactyl proteins that recognize 18 contiguous base pairs of DNA. This new class of proteins will enable us to target unique sites within all known genomes. To this end, we have made significant progress in selecting 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 the code could have a major impact on basic and applied biology.
The goal of this work is to develop a new class of therapeutic proteins that inhibit the synthesis of proteins involved in diseases of either somatic or viral origin. We are 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 type 1. We are also continuing our development of novel means of antibody selection and evolution to create a new class of anti-HIV drugs that act by inhibiting viral entry into cells. Our starting point in this development is antibodies; our long-term goal is to develop small molecules that have the activity of the larger antibodies.
Barbas, C.F. III, Burton, D.R. Selection and evolution of high-affinity human anti-viral antibodies. Trends Biotechnol. 14:230, 1996.
Barbas, C.F. III, Heine, A., Zhong, G., Hoffmann, T., Gramatikova, S., Björnestedt, R., List, B., Anderson, J., Stura, E.A., Wilson, E.A., Lerner, R.A. Immune versus natural selection: Antibody aldolases with enzymic rates but broader scope. Science, in press.
Bjornestedt, R., Zhong, G., Lerner, R.A., Barbas C.F. III. Copying nature's mechanism for the decarboxylation of ß-ketoacids into catalytic antibodies by reactive immunization. J. Am. Chem. Soc. 118:11720, 1996.
Crowe, J.E., Jr., Firestone, C.-Y., Crim, R., Beeler, J.A., Coelingh, K.L., Barbas C.F. III, Burton, D.R., Chanock, R.M., Murphy, B.R. Monoclonal antibody resistant mutants selected with an respiratory syncytial virus (RSV) neutralizing human antibody Fab fragment (Fab19) define a unique epitope on the fusion (F) glycoprotein. J. Virol., in press.
Ditzel, H.J., Parren, P.W.H.I., Binley, J.M., Sodroski, J., Moore, J.P., Barbas, C.F. III, Burton, D.R. Mapping the protein surface of HIV-1 gp120 using human monoclonal antibodies from phage display libraries. J. Mol. Biol. 267:684, 1997.
Gauduin, M.-C., Parren, P.W.H.I., Weir, R., Allaway, G.P., Maddon, P.J., Barbas C.F. III, Burton, D.R., Koup, R.A. Passive immunization with recombinant immunoglobulin molecules protects hu-PBL-SCID mice against challenge by primary isolates of human immunodeficiency virus type 1. Nat. Med., in press.
Hu, D.D., Barbas, C.F. III, Smith, J.W. Evidence for an allosteric Ca2+ binding site on the ß3-integrins that regulate the dissociation rate for RGD ligands. J. Biol. Chem. 36:21745, 1996.
Kessler, J.A. II, McKenna, P.M., Emini, E.A., Chan, C.P., Patel, M.D., Gupta, S.K., Mark, G.E. III, Barbas, C.F. III, Burton, D.R., Conley, A.J. The recombinant human monoclonal antibody IgG1b12 neutralizes diverse human immunodeficiency virus type 1 primary isolates. AIDS Res. Hum. Retroviruses 13:575, 1997.
Lerner, R.A., Barbas, C.F. III. Using the process of reactive immunization to induce catalytic antibodies with complex mechanisms: Aldolases. Acta Chem. Scand. 50:672, 1996.
Li, A., Baba, T.W., Sodroski, J., Zolla-Pazner, S., Gorny, M.K., Robinson, J., Posner, M.R., Katinger, H., Barbas, C.F. III, Burton, D.R., Chou, T.-C., Ruprecht, R.M. Synergistic neutralization of a chimeric SIV/HIV-1 virus with combinations of human anti-HIV-1 envelope monoclonal antibodies or hyperimmune globulins. AIDS Res. Hum. Retroviruses 13:647, 1997.
Lin, C.-H., Hoffmann, T.Z., Wirsching, P., Barbas, C.F. III, Janda, K.D., Lerner, R.A. On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use the enamine mechanism. Proc. Natl. Acad. Sci. U.S.A., in press.
Liu, Q., Segal, D.J., Ghiara, J.B., Barbas, C.F. III. Design of polydactyl zinc finger proteins for unique addressing within complex genomes. Proc. Natl. Acad. Sci. U.S.A. 94:5525, 1997.
Mo, H., Stamatatos, L., Ip, J.E., Barbas, C.F. III, Parren, P.W.H.I., Burton, D.R., Moore, J.P., Ho, D.D. Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. J. Virol. 71:6869, 1997.
Parren, P.W.H.I., Fisicaro, P., Labrijn, A.F., Binley, J.M., Yang, W.-P., Ditzel, H.J., Barbas, C.F. III, Burton, D.R. In vitro antigen "challenge" of human antibody libraries for vaccine evaluation: The human immunodeficiency virus type 1 envelope. J. Virol. 70:9046, 1996.
Rader, C., Barbas, C.F. III. Phage display of combinatorial antibody libraries. Curr. Opin. Biotechnol. 8:503, 1997.
Zhong, G., Hoffmann, T., Lerner, R.A., Danishefsky, S., Barbas C.F. III. Antibody catalyzed enantioselective Robinson annulation. J. Am. Chem. Soc. 119:8131, 1997.
Single Gene Mutations
E. Beutler, T. Gelbart, A. Demina
The research program of the Department of Molecular and Experimental Medicine of The Scripps Research Institute is eclectic, and the support provided by the Skaggs Institute cuts across so many important areas that here we summarize only the work dealing with human genetics and the use of modern genetic techniques to solve health problems.
In the past few years, we have developed an extraordinary capacity to detect mutations that cause a variety of hereditary diseases. We have determined mutations that cause Gaucher disease (the most common disorder of lipid storage), glucose-6-phosphate dehydrogenase deficiency (the most common hereditary cause of anemia in humans), pyruvate kinase and glucosephosphate isomerase deficiencies (less common causes of hereditary anemia), hemochromatosis (a disease characterized by the accumulation of excess amounts of iron), and neurofibromatoses (disorders characterized by inappropriate growth of the sheaths of nerve cells). For some of these diseases, we have discovered more new mutations than all the other laboratories in the world combined.
The ability to detect mutations has several important benefits. On the clinical side, detection of mutations allows diagnosis in cases in which diagnosis was not formerly possible. From an investigational point of view, the ability to detect mutations increases our understanding of the epidemiologic features of these diseases and provides the potential for cure by gene transfer. A case in point is a study of hemochromatosis just getting under way. This disease is common in the Northern European population; 1015% of persons carry one copy of the mutant gene, and about 5 persons per 1000 have two copies and therefore have the hemochromatosis genotype. But in what percentage of these patients will the disease actually develop and necessitate the relatively simple treatment that controls hemochromatosis? We plan to answer this question by carrying out genetic analysis on 60,000 persons undergoing health screening. When we complete this study 4 years from now, medical science will know, for the first time, the role of mutation analysis in this disease. To do such a large study at a reasonable cost, we have had to develop innovative methods for handling large numbers of blood samples, collecting the data, and analyzing the results.
Postdoctoral fellows in the Department of Molecular and Experimental Medicine at TSRI continue to benefit from training obtained through Skaggs training grants. Anna Demina is learning to perform state-of-the-art mutation analysis in human diseases, and she has discovered a number of mutations that cause disorders such as Gaucher disease, pyruvate kinase deficiency, and glucose-6-phosphate dehydrogenase deficiency. Makoto Suzuki, a urologist from Japan, is learning to use phage-display technology in an effort to target drugs to prostate cancers. Jennifer Johnson is exploring the mechanism by which phagocytic cells generate oxidative bursts, an important defense mechanism of the body against invading microorganisms.
Makoto Nishizawa studies oncogenic transcription factors that belong to the BZIP family of proteins in an effort to enhance our understanding of the mechanisms by which cancers are formed. Randolph Pietrowitz is studying the mechanism by which blood vessels nourish breast cancers. The angiogenic factors produced by tumors are important in maintaining tumor growth; interdiction of these factors may be an important treatment for cancer. Valerie Pasquetto is studying the mechanism by which inflammatory cytokines such as the interferons can suppress the hepatitis virus. Understanding the body's defense against the hepatitis virus is important in attempting to control hepatitis, a disease that can irreparably damage the liver and can cause liver cancer, which kills millions of people every year. We look forward to further achievements by the Skaggs fellows and to offering advanced training to others in the biomedical sciences.
Synthetic and Bioorganic Chemistry
D.L. Boger, C. Andersson, B. Bollinger, R. Borzilleri, C. Boyce, H. Cai, S. Castle, B. Cravatt, R. Garbaccio, J. Goldberg, N. Han, N. Haynes, O. Hüter, M. Ishida, T. Jenkins, D. Johnson, M. Kapiamba, B. Lewis, M. Loncar, J. McKie, S. Nukui, R. Ozer, J. Patterson, T. Ramsey, K. Saionz, G. Schüle, K. Takahashi, C. Tarby, S. Teramoto, P. Turnbull, A. Vaupel, J. Zhou
The research interests of our group include the total synthesis of biologically active natural products, the development of new synthetic methods, heterocyclic chemistry, bioorganic and medicinal chemistry, combinatorial chemistry, the study of DNA-agent interactions, and the chemistry of antitumor antibiotics. We place a special emphasis on investigations to define the structure-function relationships of natural and designed agents in efforts to understand the origin of their biological properties.
As exploration of the properties of complex natural products becomes increasingly sophisticated with technological advances in screening and evaluation and as structural details of the products' interactions with biological targets become more accessible, the opportunities for providing unique solutions to complex biological problems have grown. A powerful complement to the examination of the naturally derived agents themselves is the preparation and subsequent examination of key partial structures, agents containing deep-seated structural modifications, and the corresponding unnatural enantiomers of the natural products. Well-conceived deep-seated structural modifications can be used to address the structural basis of the interactions of the natural products with biological targets and to define fundamental relationships between structure, functional reactivity, and properties. In these studies, we address the challenging problem of understanding the beautiful solutions and subtle design elements that nature has provided in the form of a natural product and work to provide more selective, more efficacious, or more potent agents designed specifically for the problem or target under investigation.
Central to such studies are the development of dependable synthetic strategies and the advent of new synthetic methods for the preparation of the natural products, key partial structures, and analogs incorporating deep-seated structural changes. The resulting efforts have reduced many difficult or intractable synthetic challenges to manageable problems and have provided an approach not only to the natural product but also to a series of structural analogs. Our research has enabled us to fully explore the origin of the properties of the natural products and to devise agents with improved selectivity and efficacy.
Since the discovery of oleamide (Fig. 1), a fatty acid primary amide with sleep-inducing properties, continued study of this prototypical member of a new class of endogenous chemical messengers has led to the detection of an enzyme (fatty acid amide hydrolase) responsible for its degradation and regulation. Further, two potential sites of action at which inhibition of cell-cell communication at the gap junction or potentiation of the activation of serotonin receptors may occur have been discovered. Effective inhibitors of fatty acid amide hydrolase have been designed, prepared, and characterized and should continue to aid in the study of the effects of oleamide.
Receptor activation by homodimerization, heterodimerization, and higher order oligimerization has emerged as a general mechanism of initiating intracellular signal transduction. Studies have been initiated to investigate the fundamental principles and structural features that are embodied in activation of the receptor for erythropoietin.
Complementary to the emerging techniques of solid-phase combinatorial chemistry for advancing drug discovery, we have been engaged in efforts to develop solution-phase approaches to the multistep preparation of combinatorial libraries that, for the proper applications, offer substantial advantages. For example, the direct dimerization linkage of combinatorial libraries of iminodiacetic acid diamides, which is precluded by solid-phase techniques, provides a unique approach to the discovery of agonists for the receptor dimerization and activation events detailed in the preceding paragraph (Fig. 2).
Boger, D.L. Applications of free radicals in organic synthesis. Isr. J. Chem. 37:119, 1997.
Boger, D.L., Bollinger, B., Hertzog, D.L., Johnson, D.S., Cai, H., Mésini, P., Garbaccio, R.M., Jin, Q., Kitos, P.A. Reversed and sandwiched analogs of duocarmycin SA: Establishment of the origin of the sequence selective alkylation of DNA and new insights into the source of catalysis. J. Am. Chem. Soc. 119:4987, 1997.
Boger, D.L., Borzilleri, R.M., Nukui, S., Beresis, R.T. Synthesis of the vancomycin CD and DE ring systems. J. Org. Chem. 62:4721, 1997.
Boger, D.L., Boyce, C.W., Garbaccio, R.M., Goldberg, J.A. CC-1065 and the duocarmycins: Synthetic studies. Chem. Rev. 97:787, 1997.
Boger, D.L., Boyce, C.W., Johnson, D.S. pH Dependence of the rate of DNA alkylation for (+)-duocarmycin SA and (+)-CCBI-TMI. Bioorg. Med. Chem. Lett. 7:233, 1997.
Boger, D.L., Chai, W., Ozer, R.S., Andersson, C.-M. Solution-phase combinatorial synthesis via the olefin metathesis reaction. Bioorg. Med. Chem. Lett. 7:463, 1997.
Boger D.L., Chen, J.-H. An exceptionally potent analog of sandramycin. Bioorg. Med. Chem. Lett. 7:919, 1997.
Boger, D.L., Garbaccio, R.M. Catalysis of the CC-1065 and duocarmycin DNA alkylation reaction: DNA binding induced conformational change in the agent results in activation. Bioorg. Med. Chem. 5:263, 1997.
Boger, D.L., Han, N. CC-1065/duocarmycin and bleomycin A2 hybrid agents: Lack of enhancement of DNA alkylation by attachment to noncomplementary DNA binding subunits. Bioorg. Med. Chem. 5:233, 1997.
Boger, D.L., Haynes, N.-E., Kitos, P.A., Warren, M.S., Ramcharan, J., Marolewski, A.E., Benkovic, S.J. 10-Formyl-5,8,10-trideazafolic acid (10-formyl-TDAF): A potent inhibitor of glycinamide ribonucleotide transformylase. Bioorg. Med. Chem. 5:1817, 1997.
Boger, D.L., Haynes, N.-E., Warren, M.S., Gooljarsingh, L.T., Ramcharan, J., Kitos, P.A., Benkovic, S.J. Functionalized analogs of 5,8,10-trideazafolate as potential inhibitors of GAR Tfase or AICAR Tfase. Bioorg. Med. Chem. 5:1831, 1997.
Boger, D.L., Haynes, N.-E., Warren, M.S., Ramcharan, J., Kitos, P.A., Benkovic, S.J. A multisubstrate analog based on 5,8,10-trideazafolate. Bioorg. Med. Chem. 5:1853, 1997.
Boger, D.L., Haynes, N.-E., Warren, M.S., Ramcharan, J., Kitos, P.A., Benkovic, S.J. Functionalized analogs of 5,8,10-trideazafolate: Development of an enzyme-assembled tight binding inhibitor of GAR Tfase and an irreversible inhibitor of AICAR Tfase. Bioorg. Med. Chem. 5:1839, 1997.
Boger, D.L., Haynes, N.-E., Warren, M.S., Ramcharan, J., Marolewski, A.E., Kitos, P.A., Benkovic, S.J. Abenzyl 10-formyl-trideazafolic acid (abenzyl 10-formyl-TDAF): An effective inhibitor of glycinamide ribonucleotide transformylase. Bioorg. Med. Chem. 5:1847, 1997.
Boger, D.L., Hertzog, D.L., Bollinger, B., Johnson, D.S., Cai, H., Goldberg, J., Turnbull, P. Duocarmycin SA shortened, simplified, and extended agents: A systematic examination of the role of the DNA binding subunit. J. Am. Chem. Soc. 119:4977, 1997.
Boger, D.L., Hikota, M., Lewis, B.M. Determination of the relative and absolute stereochemistry of fostriecin (CI-920). J. Org. Chem. 62:1748, 1997.
Boger, D.L., McKie, J.A., Boyce, C.W. Asymmetric synthesis of the CBI alkylation subunit of the CC-1065 and duocarmycin analogs. Synlett, April 1997, p. 515.
Boger, D.L., McKie, J.A., Nishi, T., Ogiku, T. Total synthesis of (+)-duocarmycin A, epi-(+)-duocarmycin A and their unnatural enantiomers: Assessment of chemical and biological properties. J. Am. Chem. Soc. 119:311, 1997.
Boger, D.L., Ozer, R.S., Andersson, C.-M. Generation of symmetrical compound libraries by solution-phase combinatorial chemistry. Bioorg. Med. Chem. Lett. 7:1903, 1997.
Boger, D.L., Teramoto, S., Cai, H. N-Methyl-l-threonine analogs of deglycobleomycin A2: Synthesis and evaluation. Bioorg. Med. Chem. 5:1577, 1997.
Boger, D.L., Turnbull, P. Synthesis and evaluation of CC-1065 and duocarmycin analogs incorporating the 1,2,3,4,11,11a-hexahydrocyclopropa[c]naphtho[2,1-b]azepin-6-one (CNA) alkylation subunit: Structural features that govern reactivity and reaction regioselectivity. J. Org. Chem. 62:5849, 1997.
Boger, D.L., Zhou, J., Borzilleri, R.M., Nukui, S., Castle, S.L. Synthesis of (9R,12S)- and (9S,12S)-cycloisodityrosine and their N-methyl derivatives. J. Org. Chem. 62:2059, 1997.
Cravatt, B.F., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A., Gilula, N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 384:83, 1996.
Eis, P.G., Smith, J.A., Rydzewski, J.M., Case, D.A., Boger, D.L., Chazin, W.J. High resolution solution structure of a DNA duplex alkylated by the antitumor agent duocarmycin SA. J. Mol. Biol. 272:237, 1997.
Sakya, S.M., Groskopf, K.K., Boger, D.L. Preparation and inverse electron demand Diels-Alder reactions of 3-methoxy-6-methylthio-1,2,4,5-tetrazine. Tetrahedron Lett. 38:3805, 1997.
B.F. Cravatt, D.K. Giang, M.P. Patricelli
Our laboratory is interested in understanding physiology and behavior at the level of chemistry and molecules. At the center of cross talk between different physiologic processes are endogenous small molecules that provide intersystem communication. However, many of these molecular messages remain unknown, and even in the cases in which the participating molecules have been defined, the mechanisms by which these compounds function are for the most part still a mystery.
Our current efforts focus on a family of chemical messengers termed the fatty acid amides, which affect many physiologic functions, including sleep, thermoregulation, sensitivity to pain, and angiogenesis. In particular, one member of this family, oleamide, accumulates selectively in the cerebrospinal fluid of tired animals. After the animals have rested, oleamide levels once more decrease. This finding suggests that oleamide may act as a molecular indicator of the organism's need for sleep. Indeed, rats treated with oleamide fall asleep. In addition, oleamide produces profound hypothermia (reduction in body temperature) in these rats. Therefore, oleamide may serve as a molecular messenger that couples thermoregulatory and sleep processes in vivo, two physiologic systems that have long been thought to interact intimately with each other.
The in vivo levels of chemical messengers such as the fatty acid amides must be tightly regulated to maintain proper control over their influence on brain and body physiology. Recently, we determined and characterized one mechanism by which the level of fatty acid amides can be regulated in vivo. The enzyme fatty acid amide hydrolase (FAAH) degrades fatty acid amides to inactive metabolites (Fig. 1). Thus, FAAH effectively terminates the signaling messages conveyed by fatty acid amides, possibly ensuring that these molecules do not induce physiologic responses in excess of their intended purpose.
We are interested in understanding what role FAAH may play in the dynamic regulation of fatty acid amide levels in vivo. Additionally, elements responsible for the biosynthesis of fatty acid amides likely are central to the regulation of these compounds in vivo, and we are studying such processes as well.
Cravatt, B.F., Boger, D.L., Lerner, R.A. Structure determination of an endogenous sleep-inducing lipid, cis-9-octadecenamide (oleamide): A synthetic approach to the chemical analysis of trace quantities of a natural product. J. Am. Chem. Soc. 118:580, 1996.
Cravatt, B.F., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A., Gilula, N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty acid amides. Nature 384:83, 1996.
Giang, D.K., Cravatt, B.F. Molecular characterization of human and mouse fatty acid amide hydrolases. Proc. Natl. Acad. Sci. U.S.A. 94:2238, 1997.
Regulation, Function, and Signaling Mechanisms of Cell Adhesion Molecules
The fundamental processes of embryogenesis have evolved to translate the linear genetic code reproducibly into a three-dimensional organism. Early in development, dividing cells derived from the fertilized egg adhere into specific aggregates. The gene program of each aggregate is then altered, moving the cells down differentiation pathways leading to specific tissues and organs. We have determined and characterized adhesion proteins on the surfaces of cells that allow aggregation, and we have shown that different tissues have different combinations of these cell adhesion molecules or CAMs. One of our current objectives is to describe the signals that regulate the expression of gene programs when cells aggregate via specific CAMs.
In addition to forming tissues and organs, development must proceed in such a way that each collective or aggregate of cells is in its proper place in the overall body plan. Transcription factors such as the protein products of genes called Hox and Pax are among the regulators of this process. These factors act by turning sets of other genes on or off at critical places along the axis of the embryo. We have shown that CAM genes are targets for these regulators, and our second major goal is to define what components, including Hox and Pax genes, are responsible for changing the expression of CAM genes during developmental processes.
Genes are controlled through regions of the DNA called promoters, and transcription factors can bind to specific parts of the promoter DNA to activate or repress gene expression. We found that the promoter of the gene for the neural CAM called N-CAM contains binding sites for both Hox and Pax proteins. In recent studies, we detected a binding site for the paired domain of Pax proteins called the Pax binding site (PBS). We showed that the Pax-6 protein binds to the PBS and greatly enhances expression of the gene for N-CAM.
Mutations in the PBS prevent activation of the N-CAM promoter by Pax-6 both in cells in culture and in mice expressing a reporter gene linked either to the N-CAM promoter or to the promoter with a mutated PBS. In mice, the native N-CAM promoter constructs showed high expression of the reporter throughout the nervous system in places where N-CAM is normally seen. Mutations in the PBS decreased expression of N-CAM in particular regions of the embryo, and after embryonic day 15, expression was no longer observed. These data indicate that the PBS in the N-CAM gene and Pax-6 are important regulators of the pattern of expression of the N-CAM gene during neural development.
In previous studies, we detected a regulatory region common to the promoter of two neural CAMs: Ng-CAM and L1. These CAMs are important for the outgrowth and bundling of neural processes during development. We used the DNA sequences in the promoter regions of these genes to determine new transcription factors that may contribute to the control of these important neural CAMs.
This analysis led to the discovery of a new transcription factor called Barx 2. Barx 2 stimulates the activity of the L1 promoter in vitro and is localized in the nervous system in regions that overlap the expression patterns of L1. It is also expressed in nonneural regions, particularly in facial structures. Thus, these studies suggest that Barx 2 is a new transcription factor that may be involved in the control of neural gene expression.
In addition to CAMs, proteins in the cellular substrate called the extracellular matrix influence important developmental processes such as cell migration. We have studied the promoter for tenascin, an extracellular matrix protein. Expression of tenascin coincides with important developmental events in the brain and is upregulated in tumors and during wound healing and nerve regeneration.
We isolated the promoter for the tenascin gene and determined critical regulatory elements within the promoter. At least four different DNA sequences in the promoter contribute to the expression of tenascin. Three of these elements bind known transcription factors. We also detected a novel control element in the tenascin promoter. Our studies showed that these four promoter elements together with the transcription factors bound to these sites are used combinatorially to produce different patterns of tenascin gene expression during development and disease.
To determine the signals that occur as a result of the aggregation of cells by CAMs, we have exploited the ability of brain glial cells (astrocytes) to divide in tissue culture. Previously, we showed that addition of soluble N-CAM to cultured astrocytes inhibited their proliferation and that N-CAM introduced at the site of a lesion in a rat's brain could prevent glial proliferation, or gliosis, a major impediment to neural regeneration in the central nervous system.
In recent studies, we determined particular gene targets that might be involved in N-CAM signaling to the astrocyte nucleus. Expression of N-CAM was decreased when astrocytes interacted with N-CAM, whereas expression of the proteins glutamine synthetase and calreticulin was increased. Glutamine synthetase and calreticulin are involved in signaling by corticosteroids, which are well-known hormones involved in stress responses. On the basis of these findings, we showed that corticosteroids can reduce glial proliferation and that agents that antagonize the effect of steroids prevent the effects of N-CAM on astrocyte proliferation and gene expression. The combined results of these experiments indicate that N-CAM signaling may involve glucocorticoid receptors.
In other studies, we found an astonishing correlation that was not previously noticed: a number of eukaryotic messenger RNAs (mRNAs) responsible for coding of proteins contain sequences that resemble segments of ribosomal RNAs. Ribosomal RNAs are a critical component of ribosomes, which are the cellular structures that translate mRNAs into the proteins encoded by these messengers. The extensive homology found in many mRNAs led to the hypotheses that ribosomal RNAlike sequences may have spread throughout the eukaryotic genome during evolution and that their presence in mRNA may differentially affect gene expression. This ongoing analysis may reveal a heretofore undetected mechanism by which protein expression can be regulated.
In the coming year, we plan to continue our studies on the promoters for N-CAM and L1 in transgenic mice. Specifically, we will focus on the control of these promoters by the products of a variety of Pax genes during development. We will also analyze the contributions of DNA elements called neural-restrictive silencer elements in restricting L1 expression to the nervous system. In our studies on astrocyte proliferation, we will investigate the contributions of various cellular signaling systems to the effects of N-CAM on proliferation and gene expression. We also plan to extend our studies to understand the functions of ribosomal sequences in particular mRNAs.
Copertino, D.W., Edelman, G.M., Jones, F.S. Multiple promoter elements differentially regulate the expression of the mouse tenascin gene. Proc. Natl. Acad. Sci. U.S.A. 94:1846, 1997.
Crossin, K.L., Krushel, L.A., Tai, M.-H., Mauro, V.P., Edelman, G.M. Glucocorticoid receptor pathways are involved in the inhibition of astrocyte proliferation. Proc. Natl. Acad. Sci. U.S.A. 94:2687, 1997.
Edelman, G.M., Jones, F.S. Gene regulation of cell adhesion: A key step in neural morphogenesis. Brain Res. Rev., in press.
Holst, B.D., Wang, Y., Jones, F.S., Edelman, G.M. A binding site for Pax proteins regulates expression of the gene for the neural cell adhesion molecule in the embryonic spinal cord. Proc. Natl. Acad. Sci. U.S.A. 94:1465, 1997.
Jones, F.S., Kioussi, C., Copertino, D.W., Kallunki, P., Holst, B.H., Edelman, G.M. Barx2, a new homeobox gene of the Bar class is expressed in neural and craniofacial structures during development. Proc. Natl. Acad. Sci. U.S.A. 94:2632, 1997.
Kallunki, P., Edelman, G.M., Jones, F.S. Tissue-specific expression of the L1 cell adhesion molecule is modulated by the neural restrictive silencer element. J. Cell Biol. 138:1343, 1997.
Mauro, V.P., Edelman, G.M. rRNA-like sequences occur in diverse primary transcripts: Implications for the control of gene expression. Proc. Natl. Acad. Sci. U.S.A. 94:422, 1997.
Phillips, G.R., Krushel, L.A., Crossin, K.L. Developmental expression of two rat sialyltransferases that modify the neural cell adhesion molecule, N-CAM. Dev. Brain Res. 102:143,1997.
Chemical Etiology of the Structure of Nucleic Acids
A. Eschenmoser, R. Krishnamurthy, T. Wagner, M. Beier, F. Reck, T. Mueller, P. Waldmeier, G. Ceulemans, M. Bolli
Our research programs are directed toward a chemical etiology of the structure of nucleic acids, a quest for a chemical rationalization of Nature's evolution of the structure of RNA. The research strategy is to study the chemical properties of potential alternatives to nucleic acids, alternatives with molecular structures similar to the structure of RNA that, according to chemical reasoning, could have, but did not, or may have only temporarily, become Nature's genetic system. Such alternatives are made by chemical synthesis, and their properties are systematically compared with those chemical properties of RNA that are fundamental to a genetic system's biological function, such as base pairing, replication, sequence-specific catalysis of peptide synthesis, and potential to evolve. The experimental approach is meant to mimic a hypothetical process of structural variation and functional selection that may have led to RNA. This approach is expected to provide insight into those structural features that are responsible for RNA's conjectured functional superiority to chemically conceivable evolutionary alternatives.
Members of the hexopyranosyl-(6´→4´)- and the pentopyranosyl-(4´→2´)-oligonucleotide families are conformationally promising potential alternatives to nucleic acids, having structures similar to that of RNA. Earlier work done at the Swiss Federal Institute of Technology showed that the pairing properties of (6´→4´)-hexopyranosyl alternatives were far inferior to those of RNA; the reason was steric hindrance in the pairing conformation ("too many atoms"). In sharp contrast, base pairing in the ribopyranosyl-(4´→2´)-system of the pentose series is both stronger and more selective (with respect to pairing modes) than in natural (i.e., furanosyl) RNA. Conformational and constellational criteria developed and refined in the course of this work led us to predict a unique position of ribose within the family of pentopyranosyl-(4´→2´)-oligonucleotides in the sense that lyxopyranosyl-, arabinopyranosyl-, and xylopyranosyl-(4´→2´)-oligonucleotides were expected to be (in the order given) weaker pairing systems than is pyranosyl-RNA (Fig. 1).
Our introductory work at the Skaggs Institute focused on the chemical synthesis of these three unknown pentopyranosyl systems.
We recently synthesized lyxopyranosyl oligonucleotides containing adenine and thymine bases, and we expect to achieve chemical synthesis of the xylopyranosyl oligonucleotides soon. First observations on base pairing in the (4´→2´)-lyxopyranosyl series indicate that these oligonucleotides are a functional pairing system. The pairing strength of the lyxopyranosyl oligonucleotides is comparable to (or even slightly greater than) that of pyranosyl-RNA; on the other hand, their pairing selectivity (with respect to pairing mode) is less.
Oligomers of the constitutional isomer of pyranosyl-RNA that contains the phosphodiester function between positions 4´ and 3´ (instead of 4´→2´) have been synthesized and tested (at the Skaggs Institute) for pairing capabilities. We confirmed that this alternative pyranosyl isomer of RNA is not a functional pairing system, as predicted by conformational criteria.
Properties of pyranosyl-RNA that have been compared (at the Swiss Federal Institute of Technology) with the properties of RNA include the replication of pyranosyl-RNA sequences by template-controlled ligation of oligomer 2´,3´-cyclophosphates and the chiroselective self-assembly of higher oligomers by oligomerization and cooligomerization of hemicomplementary tetramer 2´,3´-cyclophosphates. At the Skaggs Institute, we have initiated studies on the potential of pyranosyl-RNA to mediate and direct the formation of oligopeptides from activated /alpha/-amino acids. We have established a central element of the proposed mechanistic pathway of such a process, namely, the occurrence of a fast and unidirectional migration of an /alpha/-aminoacyl function from the 2´ to the 3´ position of a pyranosyl-RNA unit.
Further studies will focus on four areas: (1) continuation of efforts to synthesize xylopyranosyl- and arabinopyranosyl-(4´→2´)-oligonucleotides; (2) extension of the synthesis of the lyxopyranosyl-(4´→2´)-oligonucleotides to sequences containing guanine and cytosine, establishing the system's pairing behavior in comparison with pyranosyl-(4´→2´)-RNA, and rationalization of similarities and differences in the pairing behavior of these two closely related pairing systems in terms of constellational and conformational criteria; (3) systematic determination of duplex stabilities and analysis of those stabilities in terms of the ratio between interstrand and intrastrand base stacking in pyranosyl pairing systems such as homo-DNA, pyranosyl-RNA, and lyxopyranosyl-oligonucleotides, and extending the application of this parameter to the rationalization of differences in the pairing behavior of the natural systems RNA and DNA; (4) continuation of studies directed toward (noncoded) oligopeptide synthesis mediated by a pyranosyl-RNA template.
Bolli, M., Micura, R., Eschenmoser, A. Pyranosyl-RNA: Chiroselective self-assembly of base sequences by ligative oligomerization of tetranucleotide-2´,3´-cyclophosphates (with a commentary concerning the origin of biomolecular homochirality). Chem. Biol. 4:309, 1997.
Eschenmoser, A. Towards a chemical etiology of nucleic acid structure. Orig. Life Evol. Biosph. 27:535, 1997.
Bolli, M., Micura, R., Pitsch, S., Eschenmoser, A. Pyranosyl-RNA: Further observations on replication. Helv. Chim. Acta 80:1901, 1997.
Micura, R., Bolli, M., Windhab, N., Eschenmoser, A. Pyranosyl-RNA also forms hairpin structures. Angew. Chem. Int. Ed. Engl. 36:870, 1997.
Principles of Protein Structure for Chemical Recognition, Complementarity, and Catalysis
E.D. Getzoff, A.S. Arvai, Y. Bourne, R. Brudler, B.R. Crane, T. Cross, C.L. Fisher, K.T. Forest, U.K. Genick, T.P.K. Lo, S.E. Mylvaganam, J.L. Pellequer, M.E. Pique, S.M. Redford, M.M. Thayer
We study the structural chemistry underlying molecular recognition, complementarity, and catalysis in proteins at the atomic level. In these studies, we use molecular biology, x-ray crystallography, and spectroscopy together with computational and computer graphics analysis to characterize the structural chemistry and function of proteins. We then test our understanding by protein design. In our experimental work, we aim to use technological advances in x-ray diffraction methods and to stretch the limits of current technologies used to determine protein structure. State-of-the-art technologies we use include multiwavelength anomalous diffraction, time-resolved Laue crystallography, nanosecond laser initiation, and rapid spectroscopic characterization. Taken together, these methods are enabling us to realize the original promise of x-ray crystallography to provide a detailed understanding of how proteins work, rather than simply supplying a description of protein structures.
Biological Catalysis of Sulfur Transformations
Sulfite and nitrite reductases catalyze fundamental chemical transformations for biogeochemical cycling of sulfur and nitrogen. We used multiwavelength anomalous diffraction of the native siroheme and Fe4S4 cluster cofactors to solve the atomic resolution structure of sulfite reductase, which catalyzes the concerted six-electron reductions of sulfite to sulfide and nitrite to ammonia. Coupled crystallographic and spectroscopic studies of sulfite reductase benefit from the new technologies we have developed with funding from the Skaggs Institute to study single-crystal spectra (see later). The aim of this work is to develop descriptions at atomic resolution for intermediates at each step along the complex reaction pathway, and we have solved 12 of the key structures to date.
Oxygen Recognition and Reactive Oxygen Catalysis
Ongoing research in the Getzoff and Tainer laboratories aims to understand in atomic detail the unique structural metallobiochemistry of nitric oxide synthase (NOS) and Cu,Zn superoxide dismutase (SOD), which produce and regulate reactive oxygen species. We have also characterized the basis for oxygen recognition and release by the amazing hemoglobins in fish that can pump oxygen against 100 atm (1 X 107 Pa) of pressure. These chromatically active proteins are ideal for the coupled spectroscopic and crystallographic characterizations made possible by our new single-crystal microspectrophotometer and our laser systems under development.
SOD is a master eukaryotic regulator of reactive oxygen species, because most free radicals are scavenged by dioxygen to superoxide. SOD controls reactive oxygen species within cells by dismuting superoxide anions into oxygen and hydrogen peroxide. Biochemically, SOD is remarkable for its unusually great subunit and dimer stability; its enzyme-substrate recognition, which is faster than diffusion and is coupled to exquisite specificity; and its efficient catalysis, which requires alternate oxidation and reduction of superoxide. Biologically, SOD is important for decreasing aging and increasing life span by reducing oxidative stress from inflammation and injury. Medically, SOD is noteworthy because of the recent discovery that mutations in human SOD cause the fatal degenerative disease of motor neurons termed amyotrophic lateral sclerosis or Lou Gehrig disease.
Our new bacterial SOD structure is representative of SODs from bacterial pathogens and provides a potential basis for drug design. Whereas the fold and active-site geometry of bacterial SODs match those of human SODs, the elements recruited to form the dimer interface and the active-site channel are strikingly different. Our structures and redesign of SODs aim to elucidate the enzymes' structural metallobiochemistry and structure-function relationships. By controlling levels of superoxide that react rapidly with nitric oxide to form peroxynitrite, SODs affect the activity of nitric oxide, an important biological signal and defensive cytotoxin.
The research on SODs thus complements our new research on the structural and chemical biology of the enzyme NOS, which regulates the synthesis and thereby the biological activity of nitric oxide. Nitric oxide functions at low concentrations as a diffusible, biological messenger for neurotransmission, long-term potentiation, platelet aggregation, and regulation of blood pressure. At higher concentrations, nitric oxide serves as a cytotoxic agent for defense against tumor cells and intracellular parasites.
NOSs found in inducible, constitutive endothelial, and constitutive neuronal isoforms achieve their important biological function by adopting an intriguing calcium-regulated catalytic mechanism and incorporating a unique assembly of five cofactors: heme, tetrahydrobiopterin, FMN, FAD, and NADPH. NOSs generate nitric oxide by expending molecular oxygen and NADPH in each step of a two-step mechanism: l-arginine is first oxidized to the intermediate N/omega/-hydroxy-l-arginine by a monooxygenase-like reaction, and then this intermediate is oxidized to form citrulline and nitric oxide by an unprecedented reaction. Each NOS subunit is divided into two domains joined by a calmodulin-binding hinge region: (1) an oxygenase domain with binding sites for heme, tetrahydrobiopterin, and l-arginine that forms the catalytic center for production of nitric oxide and (2) a reductase domain with binding sites for NADPH, FAD, and FMN that supplies electrons to the heme. Our current research integrates crystallographic and biochemical studies for the inducible NOS oxygenase domain and the neuronal NOS reductase domain. We have successfully crystallized both these domains and have obtained diffraction data for structure determinations.
Chemical Biology of Per-Arnt-Sim Domains of Sensors and Clock Proteins
We aim to characterize the structural and chemical biology of Per-Arnt-Sim (PAS) domains (Fig. 1). PAS domains were originally identified as sequence repeats present in the Drosophila clock protein Per, and the basic-helix-loop-helix--containing transcription factors aryl hydrocarbon nuclear translocator (Arnt) in mammals and single-minded (Sim) in flies. PAS domain sequences, which mediate protein-protein interaction and in some cases bind small ligands, have also been found in sensor kinase proteins, the phytochrome photoreceptors of plants, and in the newly discovered clock proteins in the mouse and Neurospora, including the clock proteins that clearly couple photoreception to circadian rhythms (FRQ and WC-2). The PAS domain sequences show similarities to photoactive yellow protein, a bacterial blue-light photosensor whose structural molecular biology has been under study in this laboratory. We plan to use our knowledge of the high-resolution structure of photoactive yellow protein (now under refinement at 0.82-Å resolution) to determine structures for PAS domains and to characterize the roles of these structures in signal transduction.
Coupled Crystallographic and Spectroscopic Analysis of Protein Mechanisms
The purpose of our project on coupled crystallographic and spectroscopic analysis of protein mechanisms is to establish new state-of-the-art technology at The Scripps Research Institute and to provide leadership in a key area of chemical structural biology. The laser and spectroscopy facility we are establishing in this laboratory will enable us to initiate and spectroscopically monitor reactions in protein crystals on a timescale of nanoseconds. This facility offers many opportunities for structural and chemical biology. Many of the most interesting structural states of protein molecules responsible for catalysis and signaling occur only transiently and have typical lifetimes shorter than milliseconds.
The benefit of the new facility is threefold. First, the system will enable us to determine the exact state that protein molecules occupy in a crystal. This information is particularly important for a number of metalloenzymes for which knowledge of the precise oxidation state of metal centers is critical for the correct interpretation of structural data. Second, we will be able to determine if the proteins can perform the full spectrum of their functions in the crystalline state or if individual parameters are changed by crystal lattice contacts. The third and most exciting opportunity lies in the ability to establish conditions in which transient intermediates can be accumulated so that their structures can be determined by using the emerging technique of nanosecond time-resolved crystallography.
The first stage of the system consists of two main components: a microspectrophotometer and a tunable laser. The microspectrophotometer is designed for ultraviolet-visible spectroscopy of samples of 20-µm diameter (Fig. 2). The microspectrophotometer has two detector types: a CCD-array detector for recording of full spectra from the steady state to millisecond time resolution and a single-wavelength photomultiplier system for the millisecond-to-nanosecond timescale. The tunable laser system consists of an Nd:YAG Q-switched nanosecond laser that drives a tunable optical parametric oscillator for wavelength generation from the mid-ultraviolet to visible range. The power generated by a single pulse of the laser system is sufficient to deliver several photons for every molecule in a typically sized protein crystal, thus allowing initiation of the reaction for the entire sample.
The system is modular, and we will be able to adapt it to include low-temperature facilities for cryotrapping. We can also broaden the range of the spectroscopic capabilities to the fluorescence and resonance Raman techniques. We are completing the construction of this equipment and will use it for coupled spectroscopic and crystallographic analyses of important systems in chemical biology.
Boissinot, M., Karnas, S., Lepock, J.R., Cabelli, D.E., Tainer, J.A., Getzoff, E.D., Hallewell, R.A. Function of the Greek key connection analysed using circular permutants of superoxide dismutase. EMBO J. 16:2171, 1997.
Bourne, Y., Redford, S.M., Steinman, H.M., Lepock, J.R., Tainer, J.A., Getzoff, E.D. Novel dimeric interface and electrostatic recognition in bacterial Cu,Zn superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 93:12774, 1996.
Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K., Getzoff, E.D., Stuehr, D.J., Tainer, J.A. The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. Science, in press.
Crane, B.R., Siegel, L., Getzoff, E.D. Probing the catalytic mechanism of sulfite reductase by x-ray crystallography: Structure of the Escherichia coli hemoprotein in complex with substrates, inhibitors, intermediates and products. Biochemistry, in press.
Crane, B.R., Siegel, L., Getzoff, E.D. Structures of the siroheme and Fe4S4-containing active center of sulfite reductase in different states of oxidation: Heme activation via reduction-gated exogenous ligand exchange. Biochemistry, in press.
Devanathan, S., Genick, U.K., Getzoff, E.D., Meyer, T.E., Cusanovich, M.A., Tollin, G. Preparation and properties of a 3,4-dihydroxycinnamic acid chromophore variant of the photoactive yellow protein. Arch. Biochem. Biophys. 340:83, 1997.
Fisher, C.L., Cabelli, D.E., Hallewell, R.A., Beroza, P., Lo, T.P., Getzoff, E.D., Tainer, J.A. Computational, pulse-radiolytic, and structural investigations of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase. Proteins, in press.
Genick, U.K., Borgstahl, G.E.O., Ng, K., Ren, Z., Pradervand, C., Burke, P.M., Srajer, V., Teng, T.-T., Schildkamp, W., McRee, D.E., Moffat, K., Getzoff, E.D. Structure of a protein photocycle intermediate by millisecond time-resolved crystallography. Science 275:1471, 1997.
Genick, U.K., Devanathan, S., Meyer, T.E., Canestrelli, I.L., Williams, E., Cusanovich, M.A., Tollin, G., Getzoff, E.D. Active site mutants implicate key residues for control of color and light cycle kinetics of photoactive yellow protein. Biochemistry 36:8, 1997.
Self-Organized Autocatalytic Networks
M.R. Ghadiri, K. Broo, A.J. Kennan, K. Kumar, D.H. Lee, Y. Yokobayashi
What are the fundamental properties that distinguish the chemistry of living systems, which gives rise to animate characteristics, from inanimate in vitro chemical transformations? Recent advances in the mathematical understanding of complex nonlinear systems, chemistry, molecular biology, and analytical sciences are allowing a new, broad, and unique attack on the fundamental understanding of living processes.
The approach that we have undertaken in our laboratory is founded on the following premises. Living systems are viewed as autonomous self-reproducing entities that operate on the basis of "information." Information is originated at the molecular level by covalent chemistry, transferred and processed through noncovalent chemistry, expanded in complexity at the system level, and ultimately changed through reproduction and natural selection. In a living system, the complex blend of nonlinear molecular information-transfer processes is thought to bring about a coherent self-organized chemical system--a collective of interacting and interdependent molecular species, a "molecular ecosystem"--that as a whole can display emergent properties far greater than the simple sum of its chemical constituents. Therefore, to understand and ultimately mimic the properties of living systems, we must begin defining the basic forms of self-organized autocatalytic chemical networks, how the networks can be constructed, and how the interplay of information and nonlinear catalysis can lead to the expression of emergent properties (Fig. 1).
In the past year, within the context of de novo designed catalytic and autocatalytic peptides, we have constructed simple self-organized autocratic and hypercyclic networks. These systems begin to display some of the most basic properties often associated with living systems, such as selection, symbiosis, and error correction. Our current efforts focus on the design and characterization of reciprocal and parasitic networks.
Lee, D.H., Granja, J.R., Martinez, J.A., Severin, K., Ghadiri, M.R. A self-replicating peptide. Nature 382:525, 1996.
Lee, D.H., Severin, K., Ghadiri, M.R. Autocatalytic networks: The transition from molecular self-replication to ecosystems. Curr. Opin. Chem. Biol., in press.
Lee, D.H., Severin, K., Yokobayashi, Y., Ghadiri, M.R. Emergence of symbiosis in peptide self-replication through a hypercyclic network. Nature, in press.
Severin, K., Lee, D.H., Kennan, A.J., Ghadiri, M.R. A synthetic peptide ligase. Nature 389:706, 1997.
Severin, K., Lee, D., Martinez, J.A., Ghadiri, M.R. Peptide self-replication via template-directed fragment condensation. Chem. Eur. J. 3:1017, 1997.
Severin, K., Lee, D.H., Martinez, J.A., Vieth, M., Ghadiri, M.R. Dynamic error-correction in autocatalytic peptide networks. Angew. Chem. Int. Ed. Engl., in press.
Design of Biocatalysts
D. Hilvert, P. Kast, M. Asif-Ullah, G. Auditor-Dendle, R. Chavez, F. Deroose, C. Grisostomi, N. Jiang, K. Kikuchi, M. Lenz, G. Macbeath, L. Marshall, E. Peterson, R. Pulido, Y. Tang, S. Warren, S. Zhou
Our group is developing general strategies for creating protein molecules with tailored catalytic activities. The goal of this work is to understand, at the molecular level, the origins of the enormous catalytic rates and the exacting selectivities that enzymes achieve. Successful enzyme engineering may also provide researchers of the future with novel catalysts for a wide range of applications in research, medicine, and industry.
Although it is not yet practical to design and synthesize new proteins from their constituent amino acids, existing protein molecules can serve as convenient starting points for the construction of new active sites. Such molecules can be redesigned by using either recombinant techniques or site-selective chemical modification. For example, we have changed the serine protease subtilisin into an artificial selenoenzyme by chemically converting the active-site serine residue into a selenocysteine. The resulting protein, selenosubtilisin, has a number of remarkable properties. The modified enzyme catalyzes both the hydrolysis and the aminolysis of activated esters. Indeed, acyl transfer to amines is four orders of magnitude more efficient than with native subtilisin. This result, and the fact that selenosubtilisin does not hydrolyze peptides, suggests that the modified enzyme could be a practical peptide ligase, useful in the convergent synthesis of proteins. Because selenium has several accessible oxidation states, the redox chemistry of selenosubtilisin is also interesting. The artificial enzyme efficiently catalyzes the oxidation of thiols by alkyl hydroperoxides, mimicking the action of glutathione peroxidase, an important enzyme that protects mammalian cells from oxidative damage.
Site-directed mutagenesis, kinetic analyses, proton and selenium-77 nuclear magnetic resonance spectroscopy, and crystallography are integral to our effort to understand how the microenvironment of the active site influences the intrinsic reactivity of the selenium prosthetic group and to our attempts to optimize the chemical efficiency of selenosubtilisin. Extension of these studies to new protein templates and new prosthetic groups may make a wide range of tailored protein catalysts readily available.
In a complementary approach, we are exploiting the diversity and specificity of the mammalian immune system to produce monoclonal antibodies capable of catalysis. Using suitably designed transition-state analogs as haptens, we have prepared antibody catalysts (abzymes) for concerted reactions in which carbon-carbon bonds are made or broken. We have focused on these processes because of their intrinsic chemical interest and because they can illuminate the elementary mechanisms by which proteins catalyze reactions (e.g., the roles of strain, proximity, and desolvation). To that end, we have successfully generated antibody catalysts for a Claisen rearrangement, a bimolecular Diels-Alder reaction, and a decarboxylation. Like natural enzymes, our catalytic antibodies show substantial rate accelerations, substrate specificity, regioselectivity, and stereoselectivity. Moreover, because the selectivity and mechanism of action of these molecules are defined a priori by the structure of the immunizing hapten, this approach provides a versatile and general route to enzymelike molecules for a myriad of practical problems in chemistry and biology.
In addition to extending this technology through the preparation of improved transition-state analogs, incorporation of chemical cofactors, and the development of efficient activity assays, we are carrying out detailed investigations of our existing catalysts. In this regard, nuclear magnetic resonance and crystallographic studies on an immunoglobulin with chorismate mutase activity have provided the first insights into the structural basis of antibody catalysis.
Genetic Selection of Enzymes
Although it is now possible to create new enzyme active sites by using immunologic methods or by redesigning existing proteins, the chemical efficiency of these catalysts is typically considerably lower than that of naturally occurring enzymes. Genetic selection is a potentially general method for evolving the properties of these first-generation molecules. To test this notion, we have expressed the genes that encode abzymes with modest chorismate mutase activity in a strain of the yeast Saccharomyces cerevisiae that lacks the corresponding natural enzyme. The natural enzyme is required for the biosynthesis of the aromatic amino acids phenylalanine and tyrosine. Through random mutagenesis, we have identified a permissive host strain deficient in chorismate mutase that requires the activity of our antibody for efficient growth (Fig. 1). This work demonstrates the feasibility of using catalytic antibodies in vivo to effect vital biochemical reactions and establishes the growth-selection assay needed to improve the properties of the abzyme.
An analogous approach appears feasible in Escherichia coli. We have engineered an E. coli strain that lacks natural chorismate mutase and have used it to evaluate electrostatic effects in Bacillus subtilis chorismate mutase by combinatorial random mutagenesis and selection in vivo. We anticipate that genetic selection in living organisms will not only facilitate the redesign of protein structure and function but also provide novel catalysts for regulating cellular function, altering metabolism, and destroying toxins in a rational fashion.
Haynes, M.R., Lenz, M., Taussig, M.J., Wilson, I.A., Hilvert, D. Sequence similarity and cross-reactivity of a Diels-Alder catalyst and an anti-progesterone antibody. Isr. J. Chem. 36:151, 1996.
Hilvert, D., MacBeath, G., Shin, J.A. The structural basis of antibody catalysis. In: Peptides and Proteins. Hecht, S.M. (Ed.). Oxford University Press, New York, in press.
Kast, P., Asif-Ullah, M., Hilvert, D. Is chorismate mutase a prototypic entropy trap? Activation parameters for the Bacillus subtilis enzyme. Tetrahedron Lett. 37:2691, 1996.
Kast, P., Asif-Ullah, M., Jiang, N., Hilvert, D. Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: The importance of electrostatic catalysis. Proc. Natl. Acad. Sci. U.S.A. 93:5043, 1996.
Kast, P., Hartgerink, J.D., Asif-Ullah, M., Hilvert, D. Electrostatic catalysis of the Claisen rearrangement: Probing the role of Glu78 in Bacillus subtilis chorismate mutase by genetic selection. J. Am. Chem. Soc. 118:3069, 1996.
Kast, P., Hilvert, D. Genetic selection strategies for generating and characterizing catalysts. Pure Appl. Chem. 68:2017, 1996.
Kikuchi, K., Hilvert, D. Antibody catalysis via strategic use of haptenic charge. Acta Chem. Scand. 50:333, 1996.
Kikuchi, K., Thorn, S.N., Hilvert, D. Albumin-catalyzed proton transfer. J. Am. Chem. Soc. 118:8184, 1996.
MacBeath, G., Hilvert, D. Hydrolytic antibodies: Variations on a theme. Chem. Biol. 3:433, 1996.
Na, J., Houk, K.N., Hilvert, D. Transition state of the base-promoted ring-opening of isoxazoles: Theoretical prediction of catalytic functionalities and design of haptens for antibody production. J. Am. Chem. Soc. 118:6462, 1996.
O'Connor, M.J., Dunlap, R.B., Odom, J.D., Hilvert, D., Pusztai-Carey, M., Shenoy, B., Carey, P.R. Probing an acyl enzyme intermediate of selenosubtilisin by Raman spectroscopy. J. Am. Chem. Soc. 118:239, 1996.
Peterson, E.B., Hilvert, D. Selenosubtilisin's peroxidase activity does not require an intact oxyanion hole. Tetrahedron, in press.
Tang, Y., Jiang, N., Parakh, C., Hilvert, D. Selection of a functional linker for a catalytic single-chain antibody using phage display technology. J. Biol. Chem. 271:15682, 1996.
Zhou, Z.S., Jiang, N., Hilvert, D. An antibody-catalyzed selenoxide elimination. J. Am. Chem. Soc. 119:3623, 1997.
Antibody Catalysis and Combinatorial Chemistry
K.D. Janda, J.A. Ashley, T. Berg, S. Chen, A. Datta, C. Gao, D. Gravert, H. Han, C. Harwig, J. Hasserodt, T. Hoffman, M. Hori,* C.-H. Lin, C.-H. Lo, S. Mao, G.P. McElhaney, J. Shaw, D. Spivak, M. Taylor, D. Weiner, P. Wentworth, Jr., P. Wirsching, Y. Xie, J. Yoon, X. Zhao, B. Zhou, R.A. Lerner* Kanebo, Ltd., Osaka, Japan
Our group focuses on two areas of research: combinatorial chemistry and catalytic antibodies. Our work on catalytic antibodies is targeted toward catalysis involving reactive immunization and chemical selection for catalysis. The combination of these approaches has enabled us to isolate catalytic antibodies that rival enzymes in proficiency and to develop antibody catalysts that can be programmed to kinetically resolve racemic drugs into the drugs' therapeutically important enantiomers.
Inert antigens have been used exclusively to initiate immunization. We recently
developed a procedure in which the antigen used is so highly reactive that a
chemical reaction occurs at the antibody-combining site during immunization.
An organophosphorus diester hapten (Fig. 1) was used to illustrate this process,
termed "reactive immunization." The organophosphonate recruited chemical potential
from the immune response in a manner that resembled the way that organophosphonates
recruit the catalytic power of serine hydrolases. Reactive immunization can augment
traditional immunization and enhance the scope of catalytic antibody chemistry.
Profens are a widely prescribed class of nonsteroidal antiinflammatory drugs
used as analgesics for the treatment of rheumatoid arthritis. The most prominent
member of this class is naproxen (Fig. 2), which shows stereoselective activity
and disposition; the S-(+)-enantiomer of naproxen is 28 times more reactive
than its R-(-)-enantiomer. Using our reactive immunization strategy for
antibody production, we have designed a novel phosphonate diester as a hapten
(Fig. 2) to elicit antibody catalysts for the resolution of a racemic mixture
of naproxen esters. This approach has enabled us to generate a panel of stereoselective
biocatalysts that have the proficiency of natural enzymes. The programmable nature
and power of reactive immunization linked with the vast library of the immune
system means that kinetic resolution of any chiral therapeutic agent with a carboxylate
functionality can now be attempted.
Chemical Selection For Catalytic Antibodies
For more than a decade, our group has exploited the immune system as a rich
source of de novo catalysts. Catalytic antibodies are stereoselective and can
reroute chemical reactions. In many instances, catalysts have been made for reactions
for which no natural or man-made enzymes are known. However, the full power of
this combinatorial system can be exploited only if a system exists that allows
the direct selection of a particular function. Direct chemical selection for
catalysis from antibody libraries was made possible by devising a method whereby
the positive aspects of hybridoma technology were preserved and reformatted in
a filamentous phage system. This method is based on a selection process that
is purely chemical, making it more general than biologically based selection
systems because it is not limited to reaction products that perturb cellular
machinery. A mechanism-based panning strategy that applies our technique of direct
selection for catalysis to obtain antibody catalysts that cleave a terminal amide
bond in a peptide is shown in Figure 3.
We have used a multifaceted approach to study liquid-phase chemistry. The foundation of our studies encompasses aspects of polymer technology as applied to organic synthesis, combinatorial libraries, and materials science. In particular, we have developed and continue to expand on the chemistry of polyethylene glycol and similar polymers as soluble supports for catalysts, reagents, and combinatorial operations. Also, a method of "combinatorial polymer synthesis" has been introduced that will allow the rapid survey of a large number of new materials of potential value in chemistry and other fields (Fig. 4). Finally, we have used our liquid-phase combinatorial synthesis technology to procure novel peptide, peptidomimetics, and natural product libraries.
Chen, S., Janda, K.D. Synthesis of prostaglandin E2 methyl ester on a soluble-polymer support for the construction of prostanoid libraries. J. Am. Chem. Soc. 119:8724, 1997.
Gao, C., Lin, C.-H., Lo, C.-H.L., Mao, S., Wirsching, P., Lerner, R.A., Janda, K.D. Making chemistry selectable by linking it to infectivity. Proc. Natl. Acad. Sci. U.S.A. 94:11777, 1997.
Han, H., Janda, K.D. Azatides: Solution and liquid phase synthesis of a peptidomimetic. J. Am. Chem. Soc. 188:2539, 1996.
Han, H., Janda, K.D. Multipolymer-supported substrate and ligand approach to the Sharpless asymmetric dihydroxylation. Angew. Chem. Int. Ed. Engl. 36:1731, 1997.
Han, H., Janda, K.D. A soluble polymer-bound approach to the Sharpless catalytic asymmetric dihydroxylation (AD) reaction: Preparation and application of a [(DHQD)2PHAL-PEG-OMe] ligand. Tetrahedron Lett. 38:1527, 1997.
Han, H., Janda, K.D. Soluble polymer-bound ligand-accelerated catalysis: Asymmetric dihydroxylation. J. Am. Chem. Soc. 188:7632, 1996.
Hasserodt, J., Janda, K.D., Lerner, R.A. Antibody catalyzed terpenoid cyclization. J. Am. Chem. Soc. 188:11654, 1996.
Hasserodt, J., Janda, K.D., Lerner, R.A. Formation of bridge-methylated decalins by antibody-catalyzed tandem cationic cyclization. J. Am. Chem. Soc. 119:5993, 1997.
Janda, K.D., Han, H. Combinatorial chemistry: A liquid phase approach. Methods Enzymol. 267:234, 1996.
Janda, K.D., Lo, L.-C., Lo, C.-H.L., Sim, M.M., Wang, R., Wong, C.-H., Lerner, R.A. Chemical selection for catalysis in combinatorial antibody libraries. Science 275:945, 1997.
Jung, K.W., Janda, K.D., Sanfilippo, P.J., Wachter, M. Synthesis and biological evaluation of two new naproxen analogs. Bioorg. Med. Chem. Lett. 6:2281, 1996.
Jung, K.W., Zhao, X.-Y., Janda, K.D. Development of new linkers for the formation of aliphatic C-H bonds on polymeric supports. Tetrahedron 53:6645, 1997.
Jung, K.W., Zhao, X.-Y., Janda, K.D. A linker that allows efficient formation of aliphatic C-H bonds on polymeric supports. Tetrahedron Lett. 37:6491, 1996.
Lavey, B.J., Janda, K.D. Antibody catalyzed hydrolysis of a phosphotriester. Bioorg. Med. Chem. Lett. 6:1523, 1996.
Li, T., Janda, K.D., Lerner, R.A. Cationic cyclopropanation: Controlling the reaction routes of carbocations by antibody catalysis. Nature 379:326, 1996.
Lin, C.-H., Hoffman, T.Z., Wirsching, P., Barbas, C.F., Janda, K.D., Lerner, R.A. On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use an enamine mechanism. Proc. Natl. Acad. Sci. U.S.A. 94:11773, 1997.
Lo, C.-H.L., Gao, C., Mao, S., Matsui, K., Janda, K.D. Chain shuffling: Investigations into the specificity and selectivity of antibody catalysis. Isr. J. Chem. 36:195, 1996.
Lo, C.-H.L., Wentworth, P., Jung, K.W., Yoon, J., Ashley, J.A., Janda, K.D. Reactive immunization strategy generates antibodies with high catalytic proficiencies. J. Am. Chem. Soc. 119:10251, 1997.
Lo, L.-C., Lo, C.-H., Janda, K.D., Kassel, D.B., Raushel, F.M. A versatile mechanism based reaction probe for the direct selection of biocatalysts. Bioorg. Med. Chem. Lett. 6:2217, 1996.
Sakurai, M., Wirsching, P., Janda, K.D. Design and synthesis of a cocaine-diamide hapten for vaccine development. Tetrahedron Lett. 37:5479, 1996.
Sakurai, M., Wirsching, P., Janda, K.D. Synthesis of a nucleoside hapten with a [P(O)-O-N] linkage to elicit catalytic antibodies with phosphodiesterase activity. Bioorg. Med. Chem. Lett. 6:1055, 1996.
Vandersteen, A.M., Han, H., Janda, K.D. Liquid-phase combinatorial synthesis: In search of small-molecule enzyme mimics. Mol. Divers. 2:89, 1996.
Vandersteen, A.M., Janda, K.D. A re-examination of two linear pentapeptides claimed to be serine protease mimics. J. Am. Chem. Soc. 118:8787, 1996.
Weiner, D.P., Wiemann, T., Wolfe, M.M., Wentworth, P., Janda, K.D. A pentacoordinate oxorhenium (V) metallochelate elicits antibody catalysis for phosphodiester cleavage. J. Am. Chem. Soc. 119:4088, 1997.
Wentworth, P., Janda, K.D. A facile and efficient route to 3´,5´-diamino-3´,5´ dideoxynucleosides. J. Chem. Soc. Chem. Commun., 1996, p. 2097.
Wentworth, P., Vandersteen, A.M., Janda, K.D. Poly(ethylene glycol) (PEG) as a reagent support: The preparation and utility of a PEG-triarylphosphine conjugate in liquid-phase synthesis (LPOS). Chem. Commun., 1997, p. 759.
Wentworth, P., Wiemann, T., Janda, K.D. A new class of pentacoordinate ribonuclease inhibitors: Synthesis, characterization, and inhibition studies of ribonucleoside and anhydropentofuranose oxorhenium (V) complexes. J. Am. Chem. Soc. 118:12521, 1996.
Yli-Kauhaluoma, J.T., Ashley, J.A., Lo, C.-H.L., Coakley, J., Wirsching, P., Janda, K.D. Catalytic antibodies with peptidyl-prolyl cis-trans isomerase activity. J. Am. Chem.. Soc. 118:5496, 1996.
Zhao, X.-Y., Janda, K.D. Synthesis of alkylated malonates on a traceless linker derived soluble polymer support. Tetrahedron Lett. 38:5437, 1997.
Zhao, X.-Y., Jung, K.W., Janda, K.D. Soluble polymer synthesis: An improved traceless linker methodology for aliphatic C-H bond formation. Tetrahedron Lett. 38:977, 1997.
Catalytic Nucleic Acids for Treating the Molecular Basis of Disease
G.F. Joyce, T.L. Sheppard, J. Nowakowski, R.K. Bruick, S.W. Santoro, M. Anderson
In recent years, antisense technology has emerged as a promising approach for the treatment of cancer and viral diseases. The antisense strategy uses short oligonucleotides that bind particular cellular RNAs, leading to inactivation of the RNAs. The discovery and subsequent development of catalytic nucleic acids have enhanced antisense technology by providing agents that both recognize and inactivate the target.
Our laboratory has used in vitro evolution techniques to produce two novel classes of nucleic acid catalysts: RNA enzymes that cleave DNA and DNA enzymes that cleave RNA. If these molecules are to be effective as therapeutic agents, their catalytic properties must be applicable to biologically relevant targets in living cells. As a test case, we are targeting human type 1 and feline immunodeficiency viruses (HIV-1 and FIV, respectively).
The HIV-1 and FIV genomes are single-stranded RNA molecules that are copied to DNA when the molecules enter a cell. Before the DNA is integrated into the host-cell genome, it exists transiently in a form that can be attacked by the DNA-cleaving RNA enzyme. The enzyme contains a substrate recognition domain of 12 subunits that can be configured to target different DNA substrates.
We prepared synthetic substrates corresponding to critical regions within HIV-1 DNA. Each substrate was cleaved efficiently by the appropriately configured RNA enzyme. We analyzed the sequence specificity of the DNA-cleavage reaction and found that the enzyme discriminates with high stringency between matched and mismatched substrates.
Three years ago, using in vitro selection, our laboratory developed the first example of a DNA enzyme. Since then, we have produced several additional examples, including a DNA enzyme that can be made to cleave almost any targeted RNA under simulated cellular conditions. Compared with synthetic RNA enzymes, DNA enzymes are easier to prepare and are more stable in biological tissues.
Our preferred DNA enzyme contains a catalytic domain of 15 subunits flanked by RNA recognition domains of 7 subunits each (Fig. 1). The enzyme binds the RNA substrate in a predictable way, allowing us to target different RNAs simply by altering the recognition domains. Despite its small size, the DNA enzyme is the most efficient nucleic acid catalyst known. We directed it to cleave substrates that correspond to regions within HIV-1 and FIV RNA. Each substrate was cleaved efficiently by the corresponding DNA enzyme. In a collaboration with J. Elder at The Scripps Research Institute, these DNA enzymes are being applied to cells infected with FIV. We will determine the extent to which the DNA enzymes inhibit viral replication.
Use of oligonucleotide therapeutics may be limited by the poor ability of these compounds to penetrate the cell membrane and become localized within the appropriate cellular compartment. One possible solution to this problem involves attaching a peptide to the end of the oligonucleotide. The peptide can direct the conjugate molecule to a particular cell-surface receptor, where the molecule then becomes internalized. The peptide can also provide a signal sequence that causes the conjugate to become localized within a particular cellular compartment.
We developed a method for the chemical ligation of unprotected peptides and oligonucleotides in aqueous solution. The two components are joined via a highly stable amide bond in a template-directed reaction. As a test case, we attached two different peptides, dynorphin A, which binds tightly to the ß-endorphin receptor, and nuclear localization signal, which directs importation to the cell nucleus.
Because DNA enzymes were discovered so recently, little is known about their mechanism of action. It is not known, for example, what parts of our enzyme constitute its active site, what interactions stabilize its active conformation, and how it uses metal ions to carry out a chemical reaction. To answer these questions, we are attempting, in collaboration with W. Chazin at The Scripps Research Institute, to use nuclear magnetic resonance spectroscopy to solve the three-dimensional structure of a small DNA enzyme. Knowledge of the three-dimensional structure will provide us with information about the enzyme's mechanism and will enhance our ability to control its function.
Bruick, R.K., Koppitz, M., Joyce, G.F., Orgel, L.E. A simple procedure for constructing 5´-amino-terminated oligodeoxynucleotides in aqueous solution. Nucleic Acids Res. 25:1309, 1997.
Hendrix, M., Priestley, E.S., Joyce, G.F., Wong, C.-H. Direct observation of aminoglycoside-RNA interactions by surface plasmon resonance. J. Am. Chem. Soc. 119:3641, 1997.
Joyce, G.F. Evolutionary chemistry: Getting there from here. Science 276:1658, 1997.
Joyce, G.F., Still, W.C., Chapman, K.T. Combinatorial chemistry: Searching for a winning combination. Curr. Opin. Chem. Biol. 1:3, 1997.
Raillard, S.-A., Joyce, G.F. Targeting sites within HIV-1 cDNA with a DNA-cleaving ribozyme. Biochemistry 35:11693, 1996.
Santoro, S.W., Joyce, G.F. A general-purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. U.S.A. 94:4262, 1997.
Tsang, J., Joyce, G.F. Specialization of the DNA-cleaving activity of a group I ribozyme through in vitro evolution. J. Mol. Biol. 262:31, 1996.
Antibody-Catalyzed Organic and Organometallic Transformations
E. Keinan, S.C. Sinha, F. Grynszpan, D. Shabat, H. Itzhaky, A. Haskel, S. Nimri, O. Levy, S. Saphier, H. Shulman, A. Shulman
Catalytic antibodies catalyze a wide variety of organic reactions with high levels of chemoselectivity, regioselectivity, and stereoselectivity. These reactions include difficult and disfavored transformations. We focus our attention on the following issues: catalysis of nonnatural reactions and organometallic reactions, in particular, catalysis of reactions that are otherwise disfavored in an aqueous environment, use of antibodies as cost-effective catalysts in the total synthesis of natural products, and use of antibodies to model enzymes and other functional proteins.
Organometallic transformations are of special interest in antibody catalysis because many of them have no enzymatic counterparts. Furthermore, the large coordination numbers and variable coordination geometries of the transition metals allow the creative design of haptens closely related to the postulated structure of the transition state of a given organometallic transformation.
Metal ions in the active sites of many metalloproteins have distinctive spectral and chemical features that differ from those of small inorganic complexes of the same metal ions. According to the "entatic state" hypothesis, the enzyme imposes steric and electronic constraints on the metal ion within the active site. These constraints enhance the enzyme's catalytic activity. Modeling this behavior to prove or disprove the hypothesis has been difficult.
We used antibodies to alter the geometry and spectroscopic properties of an organometallic complex. Specifically, antibodies designed to bind an organosilicon compound (1 in Fig. 1) also bind the geometrically similar but larger Cu(I) complex (2 in Fig. 1). Upon binding, the absorption spectrum of the Cu(I) complex is red shifted. This shift is consistent with the idea that the coordination sphere of the copper complex is slightly compressed to fit into the antibody's binding site. In addition, cyclic voltammetry measurements of the complex in the presence of the antibodies suggest that the initial oxidation product is a high-energy tetrahedral Cu(II) complex. These findings strongly support the entatic state hypothesis and are an important step toward antibody-catalyzed organometallic transformations.
We are using antibodies elicited against the platinum complex (3 in Fig. 1) to catalyze several fundamental reactions of organotransition metals, such as ligand exchange, oxidative addition, and migratory insertion.
Total Synthesis With Catalytic Antibodies
The synthesis of natural products is the ultimate testing ground for new concepts in organic chemistry. We have achieved the total synthesis of (-)-/alpha/-multistriatin (11 in Fig. 1), an essential component of the aggregation pheromone of the European elm bark beetle. The key step in our synthetic strategy is the antibody-catalyzed enantioselective protonolysis of an enol ether (9 in Fig. 1) to produce a ketone (10 in Fig. 1). The synthesis of other biologically active target molecules, such as pravastatin and lovastatin, by means of antibody catalysis is under way.
One obvious need in antibody catalysis, particularly for practical applications in organic synthesis, is to enhance the antibodies' cost-effectiveness. We reported the first successful noncovalent entrapment of catalytic antibodies in a sol-gel matrix. Antibodies entrapped directly within a tetramethoxysilane-derived glass retained their activity for long periods. This continuous-flow-reactor approach seems to be the method of choice for preparative-scale organic synthesis using catalytic antibodies.
Emulating Enzymes and Other Functional Proteins
In the past, we showed that catalytic antibodies elicited against metalloporphyrin haptens mimic some of the enzymatic features of cytochrome P-450. Our improved haptens are designed to elicit substrate-selective catalytic antibodies that will function as analogs of cytochrome P-450. For example, a water-stable porphyrin hapten (5 in Fig. 1) presents the metalloporphyrin element together with the organic substrate in the correct orientation to mimic the transition state of several oxygenation reactions.
The intensive research on vitamin A and its related compounds (e.g., retinal and retinoic acid) within the past few years indicates the special importance of retinoid substrates in a plethora of biological processes. For example, 11-cis-retinal covalently binds to an opsin receptor. Its isomerization to all trans within the protein triggers a cascade of biochemical events resulting in vision. Another example is the important role that such compounds play in transcription. All-trans retinoic acid binds specifically to RAR retinoic acid receptors, whereas the 9-cis isomer binds directly to both RAR and RXR receptors. Ligand-activated heterodimeric receptors bind to DNA response elements that regulate the transcription of different genes involved in a variety of biological functions. We designed three haptens (6, 7, and 8 in Fig. 1) on the basis of reported agonist activities and chemical reactivities to elicit (via homologous and heterologous immunization) antibodies that will mimic both the binding and chemical features of the different retinoid receptors.
Ghosh, P., Shabat, D., Kumar, S., Sinha, S.C., Grynszpan, F., Li, J., Noodleman, L., Keinan, E. Using antibodies to perturb the coordination sphere of a transition metal complex. Nature 382:339, 1996.
Keinan, E., Lerner, R.A. The first decade of antibody catalysis: Perspective and prospects. Isr. J. Chem. 36:113, 1996.
Keinan, E., Sinha, S.C., Shabat, D., Itzhaky, H., Reymond, J.-L. Asymmetric organic synthesis with catalytic antibodies. Acta Chem. Scand. 50:679, 1996.
Shabat, D., Grynszpan, F., Saphier, S., Turniansky, A., Avnir D., Keinan, E. An efficient sol-gel reactor for antibody catalyzed transformations. Chem. Mater. 9:2258, 1997.
Shabat, D., Sinha, S.C., Reymond, J.-L, Keinan, E. Catalytic antibodies as evolutionary probes: Modeling of a primordial glycosidase. Angew. Chem. Int. Ed. Engl. 35:2628, 1996.
Sinha, S.C., Keinan, E. /alpha/-Multistriatin: The first total synthesis of a natural product via antibody catalysis. Isr. J. Chem. 36:185, 1996.
Chemical Libraries of Annonaceous Acetogenins
S.C. Sinha, A. Sinha, A. Yazbak, T. Doundoulakis, S.C. Sinha, E. Keinan
Annonaceous acetogenins, particularly those with adjacent bis-tetrahydofuran (THF) rings, have remarkable cytotoxic, antitumor, antimalarial, immunosuppressive, pesticidal, and antifeedant activities. Many of these fatty acid derivatives have similar carbon skeletons; their striking diversity originates mainly from the relative and absolute configuration of their various stereogenic oxygen functions. More than 300 different acetogenins have been isolated, and thousands of these compounds probably occur naturally.
We have used two different approaches to generate chemical libraries of adjacent and nonadjacent bis-THF acetogenins. One approach is based on the enantioselective oxidation of a "naked carbon skeleton." It uses enantioselective olefin oxidation methods in combination with ligand-assisted chirality transfer techniques based on the chemistry of rhenium(VII) and vanadium(V) oxides.
For example, we have developed an efficient method of synthesizing a polytetrahydrofuran compound by oxidative cyclization of a polyalkene bis-homoallylic alcohol. Highly stereoselective tandem oxidative cyclizations have been achieved with rhenium(VII) reagents. This approach has been used for the total synthesis of several naturally occurring acetogenins (Fig. 1), including mono-THF structures (e.g., solamin and reticulatacin), bis-THF compounds (e.g., asimicin, bullatacin, trilobacin, trilobin, rolliniastatin), and even tris-THF molecules (e.g., goniocin and its isomers).
With the second approach, a complete 64-member library of the adjacent bis-THF acetogenins is produced by rapidly synthesizing sublibraries of a mixture of four diastereomers and then separating the products chromatographically. Each set of diastereomeric building blocks is prepared by epoxidation of an enantiomerically pure dien-diol skeleton followed by acid-catalyzed cyclization. Exploratory work into more complex, newly discovered acetogenins is rapidly leading to the completion of the asymmetric synthesis of mucocin and many stereoisomers of these cytotoxic molecules.
Keinan, E., Sinha, A., Yazbak, A., Sinha, S.C., Sinha, S.C. Towards chemical libraries of annonaceous acetogenins. Pure Appl. Chem. 69:423, 1997.
Sinha, S.C, Keinan, E. Total synthesis of (+)-aspicilin: The naked carbon skeleton strategy vs the bioorganic approach. J. Org. Chem. 62:377, 1997.
Sinha, S.C., Sinha, A., Sinha, S.C., Keinan, E. Tandem oxidative cyclization with rhenium oxide: Total synthesis of 17,18-bisepi-goniocin. J. Am. Chem. Soc., in press.
Sinha, S.C., Sinha, A., Yazbak, A., Keinan, E. Towards chemical libraries of annonaceous acetogenins: Total synthesis of trilobacin. J. Org. Chem. 61:7640, 1996.
Expression of Antibodies and Novel Enzymatic Functions in Algae
S. Mayfield, A. Cohen, J. Kim, R. Bruick, A. Lentz, P. Choi, J. Allen
We have chosen to express antibodies in algae for several reasons. First, because of the advent of bacteria resistant to antibiotics, alternative treatments are essential to maintain our standards of health care. Treatment of bacterial infections with antibodies is effective, but the cost of producing antibodies makes such treatments prohibitively expensive. Production of antibodies in algae should reduce these costs significantly and make antibody therapy a practical alternative to treatment with antibiotics. In addition, antibodies are protein molecules that can bind other molecules, both complex and simple, with high specificity and affinity. This attribute has made antibodies ideal molecules for a number of biotechnologic uses, and this specific binding of antibodies has been exploited by research groups to engineer antibody catalysts, including catalysts that are not normally found in nature.
As antibodies are increasingly used as therapeutic and research tools, the need to produce these proteins in pure form and in large quantities is obvious. Plants or algae can be used to produce these pharmacologically important proteins and enzymes on a large scale and in relatively pure form. Expression of antibody catalysts in plants and algae will enable us to introduce novel enzymatic functions into the algae to alter metabolites to produce new compounds.
Microalgae have several unique characteristics that make them ideal organisms for the production of antibodies. First, unlike most organisms and cells currently used to produce transgenic proteins, algae can be grown on a large scale in minimal media (inorganic salts) with sunlight as the energy source. Second, plants and algae have two distinct compartments, the cytoplasm and the chloroplast, in which proteins can be expressed. The cytoplasm of algae is similar to that of other eukaryotic organisms used for protein expression, such as yeast and insect cells. Chloroplasts are unique to plants and algae, and proteins expressed in this environment most likely will have properties different from those of cytoplasmically expressed proteins.
We have engineered a strain of the green alga Chlamydomonas reinhardtii to express the gene for a single-chain antibody in the chloroplast. This transgenically expressed antibody has properties that seem identical to those of antibodies expressed in mouse cell cultures, from which this antibody was originally derived. In addition, the antibody accumulates to about 1% of cellular protein and appears to be stable within the chloroplast. Studies indicate that pharmacologically important antibodies can be produced in algae at high levels and that these antibodies act exactly as do antibodies produced in more traditional and expensive systems. The stability of chloroplast-produced antibodies should allow easy purification, and algae-produced antibodies should lack many of the undesirable toxins that often contaminate antibodies expressed in bacterial cells.
Chloroplasts are the site of synthesis of many key components of plants, including lipids, amino acids, and carbohydrates. The main carbohydrate produced by chloroplasts is starch, a storage compound produced at high levels that we use for a variety of purposes, including animal food and as a starting material in organic synthesis. A number of carbohydrates with significant pharmacologic and commercial importance have starting material similar to that used in starch biosynthesis.
We are designing gene constructs to introduce nonplant enzymes into chloroplasts to determine if novel carbohydrates can be produced in C. reinhardtii chloroplasts. Production of these unique carbohydrates in microalgae could provide an important source for these scarce compounds and for novel carbohydrates that may be difficult to produce by standard synthetic means.
Cohen, A., Mayfield, S.P. Translational regulation of gene expression in plants. Curr. Opin. Biotechnol. 8:189, 1997.
Cohen, A., Yohn, C.B., Bruick, R., Mayfield, S.P. Translational regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Methods Enzymol., in press.
Cravatt, B.F., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A., Gilula, N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty acid amides. Nature 384:83, 1996.
Mayfield, S.P. Double agent: Translational regulation by a transcription factor. Chem. Biol. 3:415, 1996.
Mayfield, S.P., Cohen A. Translational regulation in the chloroplast. In: A Look Beyond Transcription: Mechanisms Controlling mRNA Stability and Translation in Plants. Gallie, D., Bailey-Serres, J. (Eds.). ASPP Press, Rockville, MD, in press.
Yohn, C., Cohen, A., Danon, A., Mayfield, S.P. Altered mRNA binding activity and decreased translation initiation in a nuclear mutant lacking translation of the psbA mRNA. Mol. Cell Biol. 16:3560, 1996.
Chemical Synthesis and Chemical Biology
K.C. Nicolaou, C. Agrios, C. Boddy, L. Boulton, S. Bräse, X.-J. Chu, S. Conley, E. Couladouros, M. Finlay, P. Gärtner, J. Gross, J. Gunzner, M. Härter, Y. He, D. Hepworth, S. Hosokawa, R. Huber, M. Izraelewicz, B. Jandeleit, Z. Jin, H. Khatuya, S. Kim, N. King, K. Koide, H. Li, J. Li, T. Li, S. McComb, N. Miller, H. Mitchell, S. Natarajan, S. Ninkovic, J. Pastor, M. Postema, J. Ramanjulu, R. Rodriguez, F. Roschangar, F. Rubsam, F. Sarabia, G. Shi, B. Smith, J. Trujillo, H. Vallberg, F. van Delft, D. Vourloumis, N. Wanatabe, D. Weinstein, N. Winssinger, J. Xu, G. Yang, Z. Yang, W.-H. Yoon, E. Yue, T.-Y. Yue
Our laboratory focuses on research in chemistry, biology, and medicine. Specifically, we are involved in the chemical synthesis and chemical biology of naturally occurring and designed substances. Nature has been amply generous to us as synthetic chemists, biologists, and clinicians by providing us with wondrous molecules that have interesting biological activities and that often can cure disease and provide challenges and opportunities in research. Aspirin, penicillin, and Taxol (paclitaxel) are natural products well known for their biological effects on the human body and for their beneficial value in treating pain, infectious diseases, and cancer, respectively. As chemists, we can synthesize such natural compounds and redesign their molecular structures to produce new substances with improved pharmacologic properties.
Such naturally occurring target molecules provide unique opportunities for us to make discoveries and inventions in new synthetic technology and synthetic strategies, total synthesis, chemical biology, and medicine. Currently, targets in our group include the anticancer agents Taxol, maduropeptin, and the epothilones; the antibiotics vancomycin and everninomycin; the cholesterol-lowering agents zaragozic acid and CP-225,917; a number of angiogenesis inhibitors and potential gene therapy agents; and a number of marine neurotoxins associated with the "red tide" phenomenon (Fig. 1).
Particularly exciting is our program in cancer chemotherapy involving the molecular design, chemical synthesis, and biological screening of a new class of compounds called epothilones. Aided by principles of organic chemistry and computer modeling, we design new molecules, synthesize them in the laboratory, and then test them against cancer cells in a search for anticancer agents that may be superior to the known therapeutic agents such as Taxol. Progress on this and other fronts during the past year has been both exciting and highly rewarding.
Bunnage, M.E., Nicolaou, K.C. The oxide anion accelerated retro-Diels-Alder reaction. Chem. Eur. J. 3:187, 1997.
Nicolaou, K.C., Boddy, C.N., Natarajan, S., Yue, T.-Y., Li, H., Bräse, S. New synthetic technology for the synthesis of aryl ethers: Construction of C-O-D and D-O-E ring model systems of vancomycin. J. Am. Chem. Soc. 119:3421, 1997.
Nicolaou, K.C., Bräse, S., Pastor, J., Sarabia, F.R., Rodriguez, R.M. Total synthesis of naturally occurring substances. In: Proceedings of the 26 Reunión Bienal de la Real Sociedad Española de Qumíca. University of Cadiz, Cadiz, Spain, in press.
Nicolaou, K.C., Chu, X.-J., Ramanjulu, J.M., Natarajan, S., Bräse, S., Rübsam, F., Boddy, C.N.C. New synthetic technology for the construction of vancomycin-type biaryl ring systems. Angew. Chem. Int. Ed. Engl. 36:1539, 1997.
Nicolaou, K.C., Claiborne, C.F., Paulvannan, K., Postema, M.H.D., Guy, R.K. The chemical synthesis of C-ring aryl taxoids. Chem. Eur. J. 3:339, 1997.
Nicolaou, K.C., Guy, R.K., Gunzner, J.L. Intelligent drug discovery from nature. MedChem News 7:12, 1997.
Nicolaou, K.C., Härter, M.W., Boulton, L., Jandeleit, B. Synthesis of the bicyclic core of CP-225,917 and CP-263,114 by an intramolecular Diels-Alder strategy. Angew. Chem. Int. Ed. Engl. 36:1194, 1997.
Nicolaou, K.C., Härter, W.M., Gunzner, J.L., Nadin, A. The Wittig and related reactions in natural product synthesis. Liebigs Ann. July 1997, p. 1283.
Nicolaou, K.C., He, Y., Vourloumis, D., Vallberg, H., Roschangar, F., Sarabia, F., Ninkovic, S., Yang, Z. The olefin metathesis approach to epothilone A and its analogues. J. Am. Chem. Soc. 119:7960, 1997.
Nicolaou, K.C., He, Y., Vourloumis, D., Vallberg, H., Yang, Z. An approach to epothilones based on olefin metathesis. Angew. Chem. Int. Ed. Engl. 35:2399, 1996.
Nicolaou, K.C., He, Y., Vourloumis, D., Vallberg, H., Yang, Z. Total synthesis of epothilone A: The olefin metathesis approach. Angew. Chem. Int. Ed. Engl. 36:166, 1997.
Nicolaou, K.C., Koide, K. Synthetic studies on maduropeptin chromophore 1: Construction of the aryl ether and attempted synthesis of the [7.3.0] bicyclic system. Tetrahedron Lett. 38:3667, 1997.
Nicolaou, K.C., Koide, K., Xu, J., Izraelewicz, M.H. Synthetic studies on maduropeptin chromophore 2: Synthesis of the madurosamine aryl amide and the C1´-C9´ fragments. Tetrahedron Lett. 38:3671, 1997.
Nicolaou, K.C., Ninkovic, S., Sarabia, F., Vourloumis, D., He, Y., Vallberg, H., Yang, Z. Total syntheses of epothilones A and B via a macrolactonization based strategy. J. Am. Chem. Soc. 119:7974, 1997.
Nicolaou, K.C., Sarabia, F., Finlay, M.R.V., Ninkovic, S., King, N.P., Vourloumis, D., He, Y. Total synthesis of oxazole- and cyclopropane-containing epothilone B analogs by the macrolactonization approach. Chem. Eur. J., in press.
Nicolaou, K.C., Sarabia, F., Ninkovic, S., Yang, Z. Total synthesis of epothilone A: The macrolactonization approach. Angew. Chem. Int. Ed. Engl. 36:525, 1997.
Nicolaou, K.C., Shi, G.-Q., Gunzner, J.L., Gärtner, P., Yang, Z. Palladium-catalyzed functionalization of lactones via their cyclic ketene acetal phosphates: Efficient new synthetic technology for the construction of medium and large cyclic ethers. J. Am. Chem. Soc. 119:5467, 1997.
Nicolaou, K.C., Smith, B.M., Pastor, J., Watanabe, Y., Weinstein, D.S. Synthesis of DNA-binding oligosaccharides. Synlett May 1997, p. 401.
Nicolaou, K.C., Theodorakis, E.A. Total synthesis of natural products and designed molecules: Brevetoxin B. In: Medicinal Chemistry: Today and Tomorrow. Yamazaki, M. (Ed.). Blackwell Science, Cambridge, England, 1997, p. 49.
Nicolaou, K.C., Trujillo, J.I., Chibale, K. Design, synthesis and biological evaluation of carbohydrate-based mimetics of RGDFV. Tetrahedron 53:8751, 1997.
Nicolaou, K.C., Vallberg, H., King, N.P., Roschangar, F., He, Y., Vourloumis, D. Total synthesis of oxazole- and cyclopropane-containing epothilone A analogs by the olefin metathesis approach. Chem. Eur. J., in press.
Nicolaou, K.C., Winssinger, N., Pastor, J., DeRoose, F. A general and highly efficient solid phase synthesis of oligosaccharides: Total synthesis of heptasaccharide phytoalexin elicitor (HPE). J. Am. Chem. Soc. 119:449, 1997.
Nicolaou, K.C., Winssinger, N., Pastor, J., Ninkovic, S., Sarabia, F., He, Y., Vourloumis, D., Yang, Z., Li, T., Giannakakou, P., Hamel, E. Synthesis of epothilones A and B in solid and solution phase. Nature 387:268, 1997.
Nicolaou, K.C., Yang, Z., Ouellette, M., Shi, G.-Q., Gärtner, P., Gunzner, J.L., Agrios, C., Huber, R., Huang, D.H. New synthetic technology for the construction of 9-membered ring cyclic ethers: Construction of the EFGH ring skeleton of brevetoxin A. J. Am. Chem. Soc. 119:8105, 1997.
Nicolaou, K.C., Yue, E.W. New roads to molecular complexity. In: The New Chemistry. Cambridge University Press, Cambridge, England, in press.
Nicolaou, K.C., Yue, E.W. Total synthesis of selected natural products. Pure Appl. Chem. 69:413, 1997.
Weinstein, D.S., Li, T., Nicolaou, K.C. The chemical end-ligation of homopyrimidine oligodeoxyribonucleotides within a DNA triple helix. Chem. Biol. 4:209, 1997.
Intermolecular Forces and Interactions
J. Rebek, Jr., C. Boss, C.G. Hilmersson, C. Horn, B.-H. Kim, T. Martin, S. Mecozzi, D. Mink, D. Nelson, U. Obst, C. Rojas, D. Rudkevich, J. Santamaria, T. Szabo, Y. Tokunaga, B. Vauzeilles, S. Waldvogel, S. Wallbaum
Being complementary and self-complementary is the universal feature of self-assembling
systems. In Nature, multiple copies of a single entity, such as a viral coat
protein or an allosteric enzyme, give rise to superstructures with functions
that emerge only in the assembled state. We have been exploring small, completely
synthetic molecules that also have these properties. In particular, we have been
able to generate capsule structures, closed-shell surfaces that can encapsulate
smaller molecules (Fig. 1). With these structures, we have seen how release of
solvent can drive encapsulation, how more than one molecule can be encapsulated,
how the concentrations of an encapsulated species resemble the concentrations
in the liquid state, and how accelerations in the rate of bimolecular reactions
can be observed inside the capsule. Control of the uptake and release of encapsulated
small molecules is the current goal.
Molecular Diversity and Combinatorial Chemistry
One of the most rapidly developing areas in the chemical sciences is molecular
diversity. In bioorganic chemistry, the research centers on the development of
synthetic combinatorial libraries. Recent innovations, many from the Department
of Chemistry, make use of solution-phase approaches in addition to solid-phase
synthesis more attractive. We have developed methods of synthesizing and analyzing
tetraurea-based libraries. The urea function is more biologically available and
stable than is the secondary amide (peptide) bond, and we have generated several
libraries of more than 2000 molecules, each with a xanthene skeleton (Fig. 2).
A screening protocol (deconvolution) was used to detect and characterize molecules
that inhibit binding of transcription factors to DNA. These molecules show activity
toward DNA as intercalators.
Studies In Molecular Recognition
How molecules fit together is often determined by their surface shapes and how well the molecules fill space. Many biologically interesting macromolecule targets present a concave surface to their small-molecule substrates and messengers. We are exploring these complementary relationships with synthetic structures that have large concave surfaces and functional groups that are directed inward. Figure 3 shows such a scaffold in which the carboxylic function is directed at an asymmetric microenvironment. These concave molecules can be used to distinguish between small molecules and mirror images of the small molecules and can also be used to measure the strength of hydrogen bonds.
Catalysis and Autocatalysis
Molecules that self-assemble and are self-complementary are also prime candidates for acting as templates for their own construction (Fig. 4). We have observed autocatalysis during the formation of some unusual receptors, in which recognition at one end of the molecule positions reactive functionalities at another end that accelerate the reaction. The product is an exact copy of the template. These systems may represent a new generation of self-replicating molecules that show function.
We have devised a new kind of capsule in which the two pieces are attached to each other in such a way that they must assemble in polymeric form. These polymeric capsules or "polycaps" can bind simple organic and bioorganic molecules such as camphor in a reversible fashion and alow uptake and release of the molecules on time cales that range from seconds to hours (Fig. 5). A second generation of these systems that involves three components lead to a highly cross-linked structure that is also formed reversibly. We are exploring the applications of polycaps in slow-release devices for delivery of small organic structures and as sensors.
Boumendjel, A., Roberts, J., Hu, E., Pallai, P., Rebek, J., Jr. Design and asymmetric synthesis of ß-strand peptidomimetics. J. Org. Chem. 61:4434, 1996.
Castellano, R., Rudkevich, D., Rebek, J., Jr. Tetramethoxy calixarenes revisited: Conformational control through self-assembly. J. Am. Chem. Soc. 118:10002, 1996.
Castellano, R.K., Rudkevich, D.M., Rebek, J., Jr. Polycaps: Reversibly formed polymeric capsules. Proc. Natl. Acad. Sci. U.S.A., in press.
Conn, M.M., Rebek, J., Jr. Self-assembling capsules. Chem. Rev., in press.
Kang, J., Rebek, J., Jr. Acceleration of a Diels-Alder reaction by a self-assembled molecular capsule. Nature 385:50, 1997.
Kang, J., Rebek, J., Jr. Entropically-driven binding in a self-assembling molecular capsule. Nature 382:239, 1996.
Kato, Y., Toledo, L.M., Rebek, J., Jr. Energetics of a low barrier hydrogen bond in nonpolar solvents. J. Am. Chem. Soc. 118:8575, 1996.
Meissner, R., Garcias, X., Mecozzi, S., Rebek, J., Jr. Synthesis and assembly of new molecular hosts: Solvation and the energetics of encapsulation. J. Am. Chem. Soc. 119:77, 1997.
Rebek, J., Jr. Assembly and encapsulation with self-complementary molecules. Chem. Soc. Rev., 1996, p. 255.
Rebek, J., Jr. Molecular assembly and encapsulation. Pure Appl. Chem. 68:1261, 1996.
Rojas, C., Rebek, J., Jr. Functional groups positioned in unusual asymmetric microenvironments. Bioorg. Med. Chem. Lett. 6:3013, 1996.
Rudkevich, D.M., Rebek, J., Jr. Chemical selection and self-assembly in a cyclization reaction. Angew. Chem. Int. Ed. Engl. 36:846, 1997.
Shipps, G.W., Pryor, K.E., Xian, J., Skyler, D.A., Davidson, E.H., Rebek, J., Jr. Synthesis and screening of small molecule libraries active in binding to DNA. Proc. Natl. Acad. Sci. U.S.A., in press.
Valdés, C., Toledo, L., Spitz, U., Rebek, J., Jr. Structure and selectivity of a small dimeric encapsulating assembly. Chem. Eur. J. 2:989, 1996.
Wintner, E.A., Rebek, J., Jr. Combinatorial libraries in solution: Polyfunctionalized core molecules. In: Combinatorial Chemistry Synthesis and Application. Wilson, S.R., Czarnik, A.W. (Eds.). Wiley, New York, 1997, p. 95.
Wintner, E.A., Rebek, J., Jr. Recent developments in the design of self-replicating systems. In: Supramolecular Control of Structure and Reactivity. Hamilton, A.D. (Ed.). Wiley, New York, 1996, p. 225.
K.B. Sharpless, H. Adolfsson, M. Bruncko, J. Chiang, H.-T. Chang, C. Copéret, R. Dress, A. Gypser, P.T. Ho, S. Immel, J. Jeong, L. Kolla, G. Li, D. Michel, D. Nirschl, W. Pringle, J. Rudolph, E. Rubin, G. Schlingloff, E. Stevens, B. Tao, A. Thomas, C. Vlaar, A. Yudin
Discovery of new reactivity is our research mission. Past discoveries include metal-catalyzed oxidations of organic molecules, particularly olefins; examples include the asymmetric epoxidation, dihydroxylation, and aminohydroxylation reactions. Our current goals include the development of practical, metal-catalyzed olefin epoxidation and aziridination reactions (Fig. 1).
Epoxides are among the most useful organic intermediates; their chemistry often combines the disparate qualities of great reactivity and excellent selectivity (Fig. 2). Although many chemists consider epoxidation a "solved" problem, existing methods often fail because of the epoxides' greatest weakness: acid sensitivity, both Lewis and Brønsted. The most common epoxidation reagents, peracids, are Brønsted acids, whereas the catalysts in metal-catalyzed systems are strong Lewis acids. The most innocuous reagents, the dioxiranes, are also the most impractical. Our recent work suggests that rhenium catalysts may offer the ideal combination of reactivity and mildness.
The chemistry of aziridines, the nitrogen analogs of epoxides, is far less developed than the chemistry of epoxides, largely because of the lack of direct procedures for the aziridination of olefins. The need for aziridination methods is evidenced in part by the myriad laboratories (our own included) engaged in the search for them.
Much effort in our previous searches for new reactions has involved screening: running a reaction on a particular substrate over and over while varying the catalyst, oxidant, solvent, temperature, and so on. Although this strategy has been successful, it does have drawbacks. It is boring and repetitive, the large number of failed reactions tends to be discouraging, and negative results tend not to be publishable. Our new strategy is to use robotics.
Automating the manual-labor part of this process enables researchers to spend more time on the interesting and creative aspects of chemistry. In addition, the automated system can screen more substrates, catalysts, and conditions than can be screened manually; hence, the reaction variables and conditions can be mapped more completely. Finally, researchers' fatigue is lessened, reducing the likelihood that significant results might be overlooked.
The direction of our future research hinges largely on the success of the automation program. Although many laboratories use robots to generate combinatorial libraries, reactivity screening is a new area for automation. Success with the aziridination project could lead to a new basis for conducting our research, and use of the findings in future reactivity prospecting should accelerate the discovery process.
Bruncko, M., Schlingloff, G., Sharpless, K.B. N-Bromoacetamide: A new nitrogen source for the catalytic asymmetric aminohydroxylation. Angew. Chem. Int. Ed. Engl. 36:1483, 1997.
Chang, H.-T., Sharpless, K.B. Molar scale synthesis of enantiopure stilbene oxide. J. Org. Chem. 61:6456, 1996.
Copéret, C., Adolfsson, H., Sharpless, K.B. A simple and efficient method for epoxidation of terminal alkenes. Chem. Commun. 16:1565, 1997.
Li, G., Angert, H.H., Sharpless, K.B. N-Halocarbamate salts provide a more useful catalytic-asymmetric-aminohydroxylation process. Angew. Chem. Int. Ed. Engl. 35:2813, 1996.
Nelson, D.W., Gypser, A., Ho, P.T., Kolb, H.C., Kondo, T., Kwong, H.-L., McGrath, D.V., Rubin, A.E., Norrby, P.-O., Gable, K.P., Sharpless, K.B. Toward an understanding of the high enantioselectivity in the osmium-catalyzed asymmetric dihydroxylation. 4. Electronic effects in amine-accelerated osmylations. J. Am. Chem. Soc. 119:1840, 1997.
Rudolph, J., Reddy, K.L., Chiang, J.P., Sharpless, K.B. Highly efficient epoxidation of olefins using aqueous H2O2 and catalytic methyltrioxorhenium/pyridine: Pyridine-mediated ligand acceleration. J. Am. Chem. Soc. 119:6189, 1997.
Rudolph, J., Sennhenn, P.C., Vlaar, C.P., Sharpless, K.B. Smaller substituents on nitrogen facilitate the osmium catalyzed asymmetric aminohydroxylation process. Angew. Chem. Int. Ed. Engl. 35:2810, 1996.
Vanhessche, K.P.M., Sharpless, K.B. Catalytic asymmetric synthesis of new halogenated chiral synthons. Chem. Eur. J. 3:517, 1997.
Vanhessche, K.P.M., Sharpless, K.B. Ligand-dependent reversal of facial selectivity in the asymmetric dihydroxylation. J. Org. Chem. 61:7978, 1996.
Studies in Organic Synthesis and Bioorganic Chemistry
Novel molecular structures found in nature continue to foster advances in synthetic organic chemistry because these structures challenge the capabilities of the science, stimulate the development of new chemical reactions, and inspire solutions to long-standing problems in organic chemistry. My group seeks efficient and innovative pathways for the chemical synthesis of novel organic molecules.
From the realm of natural products, fumagillin, a fungal metabolite that inhibits angiogenesis; dysiherbaine, a novel marine-derived amino acid with neurotoxic properties; and hispidospermidin, a cell growth inhibitor with an interesting cagelike structure, are three representative biologically active natural products for which total syntheses will be sought (Fig. 1). In each case, an effort will be made to develop concepts that are of a more general interest to the science of organic chemistry.
We are also applying principles of mechanistic organic chemistry to the design and synthesis of novel molecules that could potentially serve as mechanism-based inhibitors of ß-lactamase enzymes. In this research, we are attempting to engage a part of the normal catalytic cycle of a lactamase enzyme to generate highly reactive chemical entities that subsequently cause irreversible inhibition of the lactamase enzyme through covalent modification of an active-site residue. Our involvement in this area is motivated by the general problem of resistance in bacteria.
Master Keys for Chemical Molecular Biology From Macromolecular Structures
J.A. Tainer, A.S. Arvai, Y. Bourne, C. Bruns, B. Crane, D. Daniels, K. Forest, D. Hosfield, T.P. Lo, C. Mol, S. Parikh, C. Putnam, G. Slupphaug, M.M. Thayer
We use experimental and computational structural analysis to address central structural questions at the interface of cellular and molecular biology with chemistry. We wish to determine protein structures to develop chemical regulators as master keys for controlling fundamental processes associated with biological signaling, DNA repair and genetic variation, and the cell cycle. The Skaggs Institute has transformed our basic structural studies by enabling us to pursue in-depth biological understanding and long-term goals for the development of new treatments for high blood pressure, stroke, degenerative and infectious diseases, and cancer.
Regulation of Genome Stability and Variation
Proteins involved in DNA repair and genome stability are of great fundamental biological and medical importance because they balance genome fidelity and variability. For example, cancer research supports the hypothesis that specific mutations leading to decreased genome stability are critical early events in tumorigenesis, and DNA-repair enzymes are key components in maintaining genome stability. We have expanded our studies of DNA-repair enzymes in Escherichia coli to obtain structures for human repair enzymes that excise DNA bases and for crystals of key enzymes that excise nucleotides. As part of this work, we discovered a new model for enzyme-DNA recognition that involves flipping of nucleotides to expose a damaged DNA base for chemical recognition of specific damage.
We have now solved several structures of human uracil DNA glycosylase complexes with different duplex DNA substrates. We have also solved structures of the human dUTP pyrophosphatase, which breaks down uracil nucleotide triphosphates to keep the RNA base uracil out of DNA and to provide material for the biosynthesis of the DNA building block dTTP. These functions prevent a cycle of misincorporation and removal of uracil that causes DNA strand breaks and cell death. This cycle, which is termed thymineless cell death, is the target of several current anticancer drugs, but none of these act directly on dUTP pyrophosphatase.
We have determined a series of atomic structures of dUTP pyrophosphatase with bound nucleotides that reveal how uracil binds within a groove that is then capped when the flexible tail region closes over the bound dUTP substrate. In addition to the general implications for understanding primordial interactions between nucleic acids and peptides, establishing how dUTP pyrophosphatase recognizes and reacts with its substrate provides a basis for the design of inhibitors as future anticancer drugs.
Damage of DNA bases is directly reversed by the alkyltransferases, which are directly relevant to chemotherapy with alkylating agents. With funding from the Skaggs Institute, we have obtained crystals and high-resolution diffraction data for the human alkylguanine DNA alkyltransferase, begun to design inhibitors, and crystallized complexes with bound synthetic inhibitors for drug-design studies.
Repair involving excision of nucleotides, which is essential for the repair of bulky lesions, requires enzymes such as hexameric DNA helicases. These hexameric helicases are amazing molecular motors that unwind DNA for replication, recombination, and repair. Funding from the Skaggs Institute has enabled us to obtain crystals and diffraction data for RuvB, one of the key helicases involved in recombination repair (Fig. 1).
We have also begun studies on another protein key to the integrity of the eukaryotic genome, the multifunctional enzyme flap endonuclease (FEN-1). Mutations in FEN-1 result in defects in DNA duplication seen in human tumors and inherited human diseases. FEN-1, which cleaves unpaired, overhanging flaps in double-stranded DNA during repair and the terminal priming RNA base during DNA replication, is a structure-specific nuclease essential for DNA repair and for processing the 5´ ends of Okazaki fragments during the synthesis of lagging strands of DNA. Current research focuses on the structure and chemistry of FEN-1, including its complexes with DNA and with the processivity factor for DNA polymerase, termed proliferating cell nuclear antigen.
Stress activation of transposable elements produces genomic variation when variation is most needed. Transposition provides an effective pathway for duplication and movement of exons (exon shuffling) to create new genes. The most common result of these movements of transposable elements, however, is the duplication and rearrangement of regulatory DNA sequences called enhancers, which in turn affect the transcription of nearby genes. We have crystallized and collected diffraction data for the L1 transposase that constitutes 4% of the human genome DNA. The L1 transposase, which is unique to primates, can cause huge changes in genomes by bursts of transposition that can be activated by severe environmental stress. L1 is therefore expected to play a key role in variation in the human genome and may have directed changes key to human evolution.
DNA repair and genome fidelity are tightly controlled by checkpoints in the cell cycle that link DNA repair and replication. Our structures of human Cks proteins, which are essential to cell-cycle progression, suggest novel mechanisms for regulation of the cell cycle that involve a unique conformational switch that controls two distinct folds and assemblies. The crystal structure and mutational analysis of the human kinase CDK2 complex with Cks show how conformationally triggered domain swapping by Cks may regulate kinase binding. Only the single domain fold of Cks with the closed or bent ß-hinge conformation can bind the cell-cycle kinase. Experiments with S. Reed's group, Department of Molecular Biology, The Scripps Research Institute, show that blocking expression of Cks causes death in several types of cancer cells, so structure-based development of drugs is planned.
Genomic variation of human pathogens creates new threats to health in the form of emerging infectious diseases. Support from the Skaggs Institute has allowed us to broaden our studies on the pili of Neisseria gonorrhoeae to studies of cholera and type IV pili from E. coli. Type IV pili are hairlike fibers on many bacterial pathogens that are required for attachment, mobility, and uptake of DNA. The uptake of new pathogenicity genes by pili allows alterations in existing pathogens, such as those found in new forms of the organism that causes cholera, and the emergence of new pathogens, such as the strain of E. coli in beef that has killed children. Because bacterial attachment by pili is the first step in many bacterial infections, blocking this process is especially attractive for preventing and treating these infections.
We have determined the atomic structure of the protein pilin that forms pili in pathogenic Neisseria responsible for meningitis and gonorrhea. The purpose of this research is to redesign pilin to test its function and assembly and ultimately to develop both drugs that can block attachment and assembly of pili and pilin-based vaccines. Recently, we discovered that phosphorylated pilin residues may modulate assembly and disassembly processes that allow pathogenic bacteria to crawl across host cells and to take in DNA containing new pathogenicity genes.
Regulation of Reactive Oxygen
We have discovered new mutations in superoxide dismutase that cause the fatal degenerative disease of motor neurons termed amyotrophic lateral sclerosis or Lou Gehrig disease (Fig. 2). We have found an unexpected link between superoxide dismutase and the sporadic form of Lou Gehrig disease. Finally, we have crystallized and solved structures of several mutant superoxide dismutases that cause Lou Gehrig disease, and we expect these structures to reveal much about the defects underlying this fatal disease.
A major focus for our research on the regulation of reactive oxygen is determination of the x-ray structure and structure-based drug design for the enzyme nitric oxide synthase (NOS), in collaboration with E. Getzoff, the Skaggs Institute. NOS regulates neurotransmission, blood clotting, and blood pressure and participates in killing of tumor cells and intracellular parasites by the immune system. NOS enzymes, found in inducible, constitutive endothelial, and constitutive neuronal isoforms, achieve their important biological function by adopting an intriguing calcium-regulated catalytic mechanism and incorporating a unique assembly of five cofactors: heme, tetrahydrobiopterin, FMN, FAD, and NADPH. Each NOS subunit is divided into two domains joined by a calmodulin-binding hinge region: an oxygenase domain with binding sites for heme, tetrahydrobiopterin, and l-arginine forms the catalytic center for production of nitric oxide; and a reductase domain with binding sites for NADPH, FAD, and FMN supplies electrons to the heme. Electron transfer from the flavins to the heme is controlled by calmodulin, which fulfills a novel role for a calcium-binding protein.
We aim to determine the structures of all three isoforms of NOS. Current crystallographic research is focused on the inducible NOS oxygenase domain and the neuronal NOS reductase domain, which have been overexpressed and crystallized. After completing these characterizations, we will use the results to determine structures of full-length NOSs and of different analogous isozyme domains. We hope to use the results from these coupled biochemical and crystallographic experiments to increase understanding of NOS catalysis and ultimately to design isozyme-selective NOS inhibitors, which will be valuable tools for discovering isoform functions in vivo and are desirable as therapeutic agents for controlling blood pressure, septic shock, and inflammatory damage.
Boissinot, M., Karnas, S., Lepock, J.R., Cabelli, D.E., Tainer, J.A., Getzoff, E.D., Hallewell, R.A. Function of the Greek key connection analysed using circular permutants of superoxide dismutase. EMBO J., in press.
Bourne, Y., Watson, M.H., Hickey, M.J., Holmes, W., Rocque, W., Reed, S.I., Tainer, J.A. Crystal structure and mutational analysis of the human CDK2 complex with cell cycle-regulatory protein CksHs1. Cell 84:863, 1996.
Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K., Getzoff, E.D., Stuehr, D.J., Tainer, J.A. The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. Science, in press.
Fisher, C.L., Cabelli, D.E., Tainer, J.A., Hallewell, R.A., Getzoff, E.D. Computational, pulse-radiolytic and structural investigations of lysine 136 and its role in the electrostatic triad of human of Cu,Zn superoxide dismutase. Proteins, in press.
Forest, K.T., Tainer, J.A. Type IV pilin structure, assembly, and immunodominance: Applications to vaccine design. In: Vaccines. Brown, F., et al. (Eds.). Cold Spring Harbor Press, Cold Spring Harbor, NY, 1997, p. 167.
Forest, K.T., Tainer, J.A. Type IV pilus structure: Outside to inside and top to bottom. Gene, in press.
Kavli, B., Slupphaug, G., Mol, C.D., Arvai, A.S., Peterson, S.B., Tainer, J.A., Krokan H.E. Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase. EMBO J. 15:3442, 1996.
Mol, C.D., Harris, J.M., McIntosh, E.M., Tainer, J.A. Crystal structures of free and nucleotide-bound complexes of human dUTP pyrophosphatase: Uracil recognition by a ß-hairpin and active sites formed by three separate subunits. Structure 4:1077, 1996.
Ogihara, N.L., Parge, H.E., Hart, P.J., Weiss, M.S., Goto, J.J., Crane B.R., Tsang, J., Slater, K., Roe, J.A., Valentine, J.S., Eisenberg, D., Tainer, J.A. Unusual trigonal-planar copper configuration revealed in the atomic structure of yeast copper-zinc superoxide dismutase. Biochemistry 35:2316, 1996.
Slupphaug, G., Mol, C.D., Kavli, B., Arvai, A.S., Krokan H.E., Tainer, J.A. A nucleotide flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. Nature 384:87, 1996.
Zu, J.S., Deng, H.-X., Lo, T.P., Mitsumoto, H., Ahmed, M.S., Hung, W.-Y., Cai, Z.-J., Tainer, J.A., Siddique, T. Exon 5 is not required for the toxic function of mutant SOD1 but is essential for dismutase activity: Identification and characterization of two new SOD1 mutations associated with familial amyotrophic lateral sclerosis. Neurogenetics, in press.
Structural Studies of Immune Recognition of Human Pathogens and the Design of Anticancer and Erythropoietin-Mimetic Agents
I.A. Wilson, K.C. Garcia, E.A. Stura, M. Degano, S.E. Greasley, O. Livnah, V.M. Reyes, R.S. Stefanko, Y. Su, M.M. Yamashita
Our research supported by the Skaggs Institute centers on x-ray structural studies of proteins that are potential targets for therapeutic drugs. These targets include proteins important in cancer therapy, proteins involved in immune recognition, and cytokine receptors such as those critical in hematopoiesis.
The T-cell receptor (TCR) is a heterodimeric glycoprotein whose structure was determined in our laboratory last year. The TCR is found on the cell surface of T lymphocytes and recognizes foreign peptide antigens bound to the MHC molecules on the surface of cells. Our recently determined structure for the TCR-MHC-peptide ternary complex (Fig. 1) has greatly enhanced our understanding of the fine interactions in the TCR-MHC complex.
Other TCR-MHC complexes with different affinities are under study to provide information on the molecular principles that govern discrimination between foreign antigens and self-antigens and positive and negative selection in the immune system. Of particular interest is the phenomenon known as alloreactivity, in which TCRs react with foreign MHC molecules to which the receptors have not been previously exposed. This allorecognition is important, for example, in transplantation, in which mismatching of the MHC alleles between donor and recipient can cause graft rejection. Alloreactivity is also responsible for the adverse immune responses that occur in many autoimmune disorders. Currently, suppression of the immune response is achieved therapeutically with relatively toxic, nonspecific drugs. Elucidation of the structural basis of alloreactivity will facilitate the design of exquisitely specific immunosuppressive reagents that act at the level of an individual haplotype. The specificity of this type of drug would presumably reduce the extensive adverse side effects of the relatively nonspecific immunosuppressive regimens used today.
The entire TCR signaling complex comprises not only the TCR and MHC, but also the accessory molecules CD8 and CD3. These molecules are responsible for the propagation of the activating signal when the TCR engages the MHC. We recently showed that CD8 can have a dramatic effect on the affinity of the interaction between the TCR and the MHC. Therefore, we must rationalize the roles of CD8 and CD3 in the T-cell recognition event. We are producing bioactive recombinant forms of CD8 and CD3 as the initial stage in the eventual determination of the structures of individual molecules and intermolecular complexes. Because CD8 and CD3 facilitate the signal transduction and enzymatic functions of T-cell activation, these molecules are attractive targets for immunosuppressive and immunostimulating drugs.
In summary, the TCR, MHC, CD8, and CD3 are a dynamic ensemble of interacting components. The spectrum of intermolecular interactions between these molecules presents exciting opportunities for the discovery of highly specific inhibitory compounds. Three-dimensional models are not currently available for most of the complexes that these molecules make, so our goal is to produce high-resolution structures of the critical interacting surfaces so that structure-based efforts to design drugs can be applied to the problem of immune regulation.
Glycinamide Ribonucleotide Transformylase
Glycinamide ribonucleotide transformylase (GAR Tfase) catalyzes the first of two steps in de novo purine biosynthesis that require reduced folate cofactors. The enzyme catalyzes the transfer of the formyl group of 10-formyltetrahydrofolate to the amino acid group of glycinamide ribonucleotide to form formyl glycinamide ribonucleotide and tetrahydrofolate. The discovery that inhibitors of GAR Tfase, derivatives of 5,10-dideazatetrahydrofolate, can stop proliferation of tumor cells in culture suggested this enzyme as a target for antineoplastic agents. In collaboration with D. Boger, The Scripps Research Institute, and S. Benkovic, Pennsylvania State University, we are studying the crystal structures of GAR Tfase from Escherichia coli in complex with several substrate cofactors and inhibitors.
The structures of apo GAR Tfase and of a complex consisting of GAR Tfase and an extremely high-affinity multisubstrate adduct have been determined and were used to define a phamacophore model for interactions between the drug and GAR Tfase. Comparisons of this model with structurally diverse chemical databases has yielded novel motifs that can be modified to inhibit GAR Tfase with greater selectivity and thus fewer toxic effects than the multisubstrate adduct inhibitor has. In addition to studies on the wild-type protein, a number of single-site mutations based on these structures have been made by Dr. Benkovic, including two mutants of the catalytic residue His108 (Fig. 2). Crystal structures have been determined at high resolution for these mutants in complex with folate-derived inhibitors to determine the precise role of these residues in conjunction with kinetic studies. Thus, these studies guide rational modification of current drug candidates by providing an atomic picture of the binding site and other regions that can be exploited for potential interaction with small-molecule inhibitors.
Receptor For Erythropoietin
Erythropoietin is the sole regulator of the proliferation, differentiation, and maturation of erythroid cells into red blood cells. Erythropoietin is produced by the kidney in response to hypoxia and binds to the erythropoietin receptor, a member of the cytokine receptor superfamily, on committed erythroid cells. Human recombinant erythropoietin is used in therapies for anemia associated with renal disease, cancer chemotherapy, and treatment with azidothymidine. Human recombinant erythropoietin is currently the most successful therapeutic drug in the pharmaceutical industry, generating sales of almost $3 billion in 1996. Erythropoietin is expensive and is available only for intravenous administration. The search for a small-molecule mimetic of erythropoietin that is less costly and orally available is under way.
The three-dimensional x-ray structure of the extracellular domain of the receptor for erythropoietin (termed EBP, for erythropoietin-binding protein) complexed with a 20 amino acid agonist peptide called EMP1 (for erythropoietin-mimetic peptide) that was discovered through phage-display combinatorial methods was determined to a resolution of 2.8 Å (Fig. 3A). The structure shows that two EMP1 molecules bind two EBP molecules to form a symmetric 2:2 complex. The receptor dimerization induced by the ligands most likely differs from that induced by the natural, asymmetric hormone erythropoietin, indicating that more than one receptor dimer configuration may promote signaling.
The structure also highlights the residues on the receptor surface that are in contact with the small ligand and provide the chemical basis for this interaction. The contact surface of EBP with EMP1 is analogous to the minimized "functional epitope" of the growth hormone receptor molecule, a member of the cytokine receptor superfamily, indicating that a small synthetic peptide can recognize a smaller and essential binding part of the natural epitope. This highly detailed information on the mode of dimerization and binding epitope is being used, in collaboration with D. Boger, The Scripps Research Institute, and L. Joliffe, R.W. Johnson Pharmaceutical Research Institute, to design small-molecule nonpeptidic potential mimetics of erythropoietin.
We recently found another EBP complex that sheds light on the importance and role of dimerization of the erythropoietin receptor. The peptide, EMP33, is a modification of EMP1 that has a single amino acid replacement of a tyrosine with a 3,5-dibromotyrosine. This modification reduces the binding affinity to the erythropoietin receptor by a factor of 50 and abolishes the activation properties, so that the peptide is now an antagonist. The structure of the EBP-EMP33 complex was determined to a resolution of 2.8 Å and reveals a different mode of dimerization (Fig. 3A). The almost perfect twofold dimerization for the EMP1 agonist-peptide complex is shifted now by a rotation of 15° to an asymmetric EBP dimer (Fig. 3B). This alteration in mode of receptor dimerization most likely is the reason for the loss in activity. This structural information combined with similar information on the agonist complex emphasizes the importance of the dimer configuration of the receptor in the initial, extracellular signaling process and provides additional conceptual data for the design of small-molecule agonists and antagonists of the erythropoietin receptor and other cytokine receptors.
Garcia, K.C., Degano, M., Stanfield, R.L., Brunmark, A., Jackson, M.R., Peterson, P.A., Teyton, L., Wilson, I.A. The structure of an /alpha/ß T-cell receptor at 2.5 Å and its orientation in the TCR-MHC complex. Science 274:209, 1996.
Garcia, K.C., Scott, C.A., Brunmark, A., Carbone, F.R., Peterson, P.A., Wilson, I.A., Teyton, L. CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes. Nature 384:577, 1996.
Livnah, O., Stura, E.A., Johnson, D.L., Middleton, S.A., Mulcahy, L.S., Wrighton, N.C., Dower, W.J., Jolliffe, L.K., Wilson, I.A. Functional mimicry of a protein hormone by a peptide agonist: The EPO receptor complex at 2.8 Å. Science 273:464, 1996.
Wilson, I.A., Garcia, K.C., Degano, M., Stanfield, R.L., Zeng, Z., Segelke, B., Stura, E.A., Castano, A.R., Jewell, D.A., Brunmark, A., Jackson, M.R., Peterson, P.A., Teyton, L. Structural basis of cellular immune recognition. In: Alfred Benzon Symposium 40. HLA and Disease: The Molecular Basis. Svejaard, A., Fugger, L., Buus, S. (Eds.). Munksgaard Publishers, Copenhagen, 1997, p. 46.
Wu, S.-K., Zeng, K., Wilson, I.A., Balch, W.E. Structural insights into the function of the Rab GDI superfamily. Trends Biochem. Sci. 21:472, 1996.
Zeng, Z.-H., Castaño, A.R., Segelke, B.W., Stura, E.A., Peterson, P.A., Wilson,I.A. Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove. Science 277:339, 1997.
Chemical-Biological Approach to Drug Discovery
C.-H. Wong, G.-J. Shen, P.S. Sears, P. Alper, M. Burkart, M. Hendrix, J.H. Hogg, S.-C. Hung, C. Huwe, J. Jablonowski, J. Jiricek, M.-J. Kim, T. Kimura, K. Koeller, T. Lampe, K. Laslo, T. Lee, C.-C. Lin, R. Martin, G. McGarvey, W. Moree, F. Moris-Varas, B. Murray, I. Ollmann, P. Pachlatko, S. Priestley, X. Qian, O. Seitz, M. Shelton, E. Simanek, M. Smith, S. Takayama, V. Vassilev, K. Witte, T. Wittmann, X.-S. Ye, J. Yun, Z. Zhang
Our research programs focus on the development of new chemistry and new strategies for the study of and intervention in important biological recognition processes. Our current interests are directed toward (1) development of new chemoenzymatic strategies for the synthesis of biologically active compounds and chiral intermediates, (2) combinatorial and rational synthesis of mechanism-based inhibitors of enzymes and receptors, and (3) investigation of reaction mechanisms.
Our work on chemoenzymatic organic synthesis includes the design of substrates and the exploitation of native, recombinant, and rationally modified enzymes for organic synthesis. Our synthetic strategy emphasizes a combination of chemical and enzymatic methods, with particular focus on the use of enzymes for stereocontrolled processes. Our goal is to develop effective and environmentally friendly procedures for the large-scale synthesis of biomedically important compounds. In the past year, we have developed several new synthetic methods. These include novel aldol addition reactions catalyzed by recombinant aldolases and chemoenzymatic synthesis of glycopeptides and glycoproteins that involves use of engineered subtilisins and recombinant glycosyltransferases in combination with solid-phase methods developed in this laboratory.
Our goals in enzyme and receptor inhibition are to develop new strategies and discover new therapeutic agents that have high selectivity. Our efforts focus on the design and synthesis of structure- or mechanism-based inhibitors of enzymes or receptors associated with metabolic disorders or diseases. Current targets for investigation include carbohydrate receptors (e.g., selectins and nucleic acids); viral proteases and RNA; and the enzymes involved in the cell cycle, the processing of glycoproteins, and the hydrolysis of oleamide (an endogenous sleep-inducing molecule). We have developed new tight-binding inhibitors of leukotriene A4 hydrolase, certain viral and bacterial RNA, and HIV proteases from drug-resistant mutants. We have also discovered a new RNA recognition motif based on cyclic 1,3-hydroxyamines, and we are developing new sequence-specific RNA inhibitors that contain this motif as novel antibiotics and antiviral agents (Fig. 1).
Alper, P.B., Hendrix, M., Wong, C.-H. Probing the specificity of aminoglycoside-RNA interactions with designed synthetic analogs. J. Am. Chem. Soc., in press.
Burkart, M.D., Hung, S.-C., Zhang, Z., Wong, C.-H. A new method for the synthesis of fluorocarbohydrates and glycosides using Selectfluor. J. Am. Chem. Soc. 119:11743, 1997.
Cappi, M.W., Moree, W.J., Qiao, L., Marron, T.G., Weitz-Schmidt, G., Wong, C.H. Synthesis of novel 6-amino-6-deoxy-l-galactose derivatives as potent sialyl Lewis X mimetics. Bioorg. Med. Chem. 5:283, 1997.
Fitz, W., Wong, C.-H. Oligosaccharide synthesis by enzymatic glycosidation. In: Preparative Carbohydrate Chemistry. Hanessian, S. (Ed.). Marcel Dekker, New York, 1997, p. 485.
Hayashi, T., Murray, B.W., Wang, R., Wong, C.-H. A chemo-enzymatic synthesis of UDP-(2-deoxy-2-fluoro)galactose and evaluation of its interaction with galactosyltransferase. Bioorg. Med. Chem. 5:497, 1997.
Hendrix, M., Alper, P.B., Priestley, E.S., Wong, C.-H. Hydroxyamines as a new motif for the molecular recognition of phosphodiesters: Implications for aminoglycoside RNA interactions. Angew. Chem. Int. Ed. Engl. 36:95, 1997.
Hendrix, M., Priestley, E.S., Joyce, G., Wong, C.-H. Direct observation of aminoglycoside-RNA interactions by surface plasmon resonance. J. Am. Chem. Soc. 119:3641, 1997.
Hendrix, M., Wong, C.-H. A chemo-enzymatic approach to the study of carbohydrate recognition in biological systems. Enantiomer 1:305, 1996.
Hendrix, M., Wong, C.-H. A chemoenzymatic approach to carbohydrate-mediated cell adhesion. Pure Appl. Chem. 68:2081, 1996.
Hung, S.-C., Wong, C.-H. Samarium diiodide-mediated coupling of glycosyl phosphates with carbon radical or carbon anion acceptors for the synthesis of C-glycosides. Angew. Chem. Int. Ed. Engl. 35:2671, 1996.
Ikeda, T., Kajimoto, T., Kondo, H., Wong, C.-H. Design and synthesis of an /alpha/-mannosyl terpenoid as a selective inhibitor of p-selectin. Bioorg. Med. Chem. Lett. 7:2485, 1997.
Janda, K.D., Lo, L.-C., Lo, C.-H., Sim, M.-M., Wang, R., Wong, C.-H., Lerner, R.A. Chemical selection for catalysis in combinatorial antibody libraries. Science 275:945, 1997.
Kanebo, M., Kanie, O., Kajimoto, T., Wong, C.-H. Toward a transition state analog inhibitor of N-acetylglucosaminyl transferase V. Bioorg. Med. Chem. Lett. 7:2809, 1997.
Kim, M.J., Lim, I.T., Kim, H.-J., Wong, C.-H. Enzymatic single aldol reactions of remote dialdehydes. Tetrahedron Asymmetry 8:1507, 1997.
Kimura, T., Vassilev, V.P., Shen, G.-J., Wong, C.-H. Enzymatic synthesis of ß-hydroxy-/alpha/-amino acids based on d- and l-threonine aldolases. J. Am. Chem. Soc. 119:11734, 1997.
Koketsu, M., Nitoda, T., Sugino, H., Juneja, L.R., Kim, M., Yamamoto, T., Abe, N., Kajimoto, T., Wong, C.-H. Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent. J. Med. Chem. 40:3332, 1997.
Lin, C.-C., Lin, C.-H., Wong, C.-H. Sialic acid aldolase-catalyzed condensation of pyruvate and N-substituted mannosamine: A useful method for the synthesis of N-substituted sialic acids. Tetrahedron Lett. 38:2649, 1997.
Lin, C.-H., Murray, B.W., Ollmann, I.R., Wong, C.-H. Why is CMP-KDO highly unstable? Biochemistry 36:780, 1997.
Marron, T.G., Woltering, T.J., Weitz-Schmidt, G., Wong, C.-H. C-Mannose derivatives as potent mimics of sialyl Lewis X. Tetrahedron Lett. 37:9037, 1996.
McGarvey, G.J., Wong, C.-H. Chemical, enzymatic and structural studies in molecular glycobiology. Liebigs Ann. 6:1059, 1997.
Moree, W.J., Sears, P.S., Kawashiro, K., Witte, K., Wong, C.-H. Exploitation of subtilisin BPN´ as catalysts for the synthesis of peptides containing noncoded amino acids, peptide mimetics and peptide conjugates. J. Am. Chem. Soc. 16:3641, 1997.
Moris-Varas, F., Lin, C.-C., Weitz-Schmidt, G., Wong, C.-H. Small molecules as structural and functional mimics of the tetrasaccharide sialyl Lewis X in selectin inhibition. J. Am. Chem. Soc., in press.
Murray, B.W., Wittmann, V., Burkart, M.D., Hung, S.-C., Wong, C.-H. Mechanism of human /alpha/-1,3-fucosyltransferase V: Glycosidic cleavage occurs prior to nucleophilic attack. Biochemistry 36:823, 1997.
Seitz, O., Wong, C.-H. Chemo-enzymatic solution and solid phase synthesis of an O-sialyl-Lewis X-octapeptide of the mucin domain of MAdCAM-1. J. Am. Chem. Soc. 119:8766, 1997.
Takayama, S., Martin, R., Wu, J., Laslo, K., Siuzdak, G., Wong, C.-H. Chemoenzymatic preparation of novel cyclic imine sugars and rapid biological activity evaluation using electrospray mass spectrometry and kinetic analysis. J. Am. Chem. Soc. 119:8146, 1997.
Takayama, S., McGarvey, G.J., Wong, C.-H. Microbial aldolases and transketolases: New biocatalytic approaches to simple and complex sugars. Annu. Rev. Microbiol. 51:285, 1997.
Takayama, S., Wong, C.-H. Chemo-enzymatic approach to carbohydrate recognition. Curr. Org. Chem. 1:109, 1997.
Wang, R., Steensma, D.H., Takaoka, Y., Wong, C.-H. A search of pyrophosphate mimetics for the development of substrates and inhibitors of glycosyltransferases. Bioorg. Med. Chem. 5:641, 1997.
Witte, K., Sears, P.S, Wong, C.-H. Enzymatic glycoprotein synthesis: Preparation of ribonuclease glycoforms via enzymatic glycopeptide condensation and glycosylation. J. Am. Chem. Soc. 119:2114, 1997.
Wittmann, V., Wong, C.-H. 1H-Tetrazole as catalyst in phosphoromorpholidate coupling reactions: Efficient synthesis of GDP-fucose, GDP-mannose and UDP-galactose. J. Org. Chem. 63:2144, 1997.
Wong, C.-H. A chemo-enzymatic approach to the study of carbohydrate-based biological recognitions. Pure Appl. Chem. 69:419, 1997.
Wong, C.-H., Moris-Varas, F., Lin, C.-C., Weitz-Schmidt, G. Small molecules as structural and functional mimics of the tetrasaccharide sialyl Lewis X in selectin inhibition. J. Am. Chem. Soc. 119:8152, 1997.
Wu, J., Takayama, S., Wong, C.-H., Siuzdak, G. Quantitative electrospray mass spectrometry for the rapid assay of enzyme inhibitors. Chem. Biol. 4:653, 1997.
Studies of Macromolecular Recognition by Multidimensional Nuclear Magnetic Resonance
P.E. Wright, H.J. Dyson, M. Foster, S. Holmbeck, R. Kriwacki, G. Legge, M. Osborne, G. Perez-Alvarado, I. Radhakrishnan
Specific interactions between molecules are of fundamental importance in all biological processes. Knowing how biological macromolecules such as proteins and nucleic acids recognize each other is essential for understanding the basic molecular events of life. Knowledge of the three-dimensional structures of biological macromolecules is key to understanding the molecules' interactions and functions and is the basis for rational design of new drugs. A particularly powerful method for determining the three-dimensional structures and interactions of biological macromolecules in the solution state is multidimensional nuclear magnetic resonance (NMR) spectroscopy. We are using this method to study a number of protein-protein and proteinnucleic acid interactions of fundamental importance in health and disease.
Knowledge of the molecular interactions through which proteins recognize specific DNA sequences is essential for understanding the regulation of genes during the growth and development of all living organisms. Understanding the principles of sequence-specific DNA recognition could ultimately lead to the development of novel therapeutic agents for a wide variety of diseases.
The polyomavirus enhancer-binding protein 2 participates in the normal functioning of T cells and in the onset of certain types of leukemia. The protein contains a small domain, termed the runt domain, that recognizes a specific DNA sequence. To gain insights into the mechanism by which the protein participates in both normal T-cell regulation and the onset of leukemia, we are determining the structure of the runt domain both free in solution and bound to its DNA recognition site. Current work focuses on production of a fragment of the protein suitable for NMR structural studies. Knowledge of the detailed three-dimensional structure of the runt-DNA complex could form the basis for design of new therapeutic agents.
Other projects address the structural basis for sequence-specific DNA recognition by nuclear hormone receptors. The solution structure of the DNA-binding domain of the 9-cis retinoic acid receptor has been refined to high resolution. Ongoing studies are aimed at determining structural changes that take place when DNA binding occurs. The DNA-binding domain of the human estrogen-related receptor has been expressed and labeled with carbon and nitrogen isotopes for NMR structural studies. This protein differs from the 9-cis retinoic acid receptor and many other hormone receptors in that it binds DNA as a monomer. Determination of the structure and dynamics of the domain of the human estrogen-related receptor bound to DNA is in progress.
Other major projects in the laboratory involve elucidation of the structural basis for key protein-protein interactions involved in regulation of gene expression and in cellular adhesion. We recently determined the three-dimensional structure of a domain of the transcriptional coactivator CREB-binding protein bound to the kinase-inducible activation domain of the transcription factor CREB. The domain of the CREB-binding protein adopts a novel helical fold (Fig. 1). CREB-binding protein mediates interactions between a number of gene regulatory proteins and viral proteins, including proteins from human T-cell leukemia virus and hepatitis B virus. Because of the central role played by the CREB-binding protein in cell growth and development, understanding the molecular mechanisms by which it recognizes its various target proteins is of fundamental biomedical importance.
Organization of cells into multicellular assemblies is mediated by proteins called cell adhesion molecules. The interactions between these proteins are generally weak and are poorly understood at the structural level. We are using NMR methods to determine the structures of key domains of several important cell adhesion proteins, including the neural cell adhesion molecule and the integrins. NMR provides an especially sensitive tool for mapping the surface sites through which cell adhesion proteins interact. An understanding at the molecular level of the specific protein-protein interactions involved in cell adhesion should allow design of small molecules that enhance or inhibit binding; such molecules could find important applications in the treatment of a number of diseases.
Bai, Y., Karimi, A., Dyson, H.J., Wright, P.E. Absence of a stable intermediate on the folding pathway of protein A. Protein Sci. 6:1449, 1997.
Chen, Y., Case, D.A., Reizer, J., Saier, M.H., Jr., Wright, P.E. High-resolution solution structure of Bacillus subtilis IIA glc. Proteins, in press.
Foster, M.P., Wuttke, D.S., Radhakrishnan, I., Case, D.A., Gottesfeld, J.M., Wright, P.E. Domain packing and dynamics in the DNA complex of the aminoterminal zinc fingers of transcription factor IIIA. Nature Struct. Biol. 4:605, 1997.
Kriwacki, R.W., Wu, J., Tennant, L., Wright P.E., Siuzdak, G. Probing protein structure using biochemical and biophysical methods: Proteolysis, MALDI mass spectrometry, HPLC, and size-exclusion chromatography of p21 Wafl/Cipl/Sdi11. J. Chromatogr. 777:23, 1997.
Prytulla, S., Dyson, H.J., Wright, P.E. Gene synthesis, high level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin. FEBS Lett. 399:283, 1996.
Reymond, M.T., Huo, S., Duggan, B., Wright, P.E., Dyson, H.J. Contribution of increased length and intact capping sequences to the conformational preference for helix in a 31-residue peptide from the C-terminus of myohemerythrin. Biochemistry 36:5234, 1997.
Reymond, M.T., Merutka, G., Dyson, H.J., Wright, P.E. Folding propensities of peptide fragments of myoglobin. Protein Sci. 6:706, 1997.
Sem, D.S., Casimiro, D.R., Kliewer, S.A., Provencal, J., Evans, R.M., Wright, P.E. NMR spectroscopic studies of the DNA-binding domain of the monomer-binding nuclear orphan receptor, human ERR2: The carboxy-terminal extension to the zinc-finger region is unstructured in the free form of the protein. J. Biol. Chem. 272:18038, 1997.
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