News and Publications

A Merging of Chemistry and Biology

We explore the application of organic chemistry to the realm of biology. During the past year, our focus included the 3 research programs described: the ability of a nicotine metabolite to participate in the aberrant glycation of proteins, the development of human antibodies as biodefense agents against spores from the genus Bacillus, and the preparation of an active immunization vaccine for the treatment of septic shock.

Tobacco-Based Glycation of Proteins

Protein glycation, the process by which glucose covalently modifies the amino acid side chains of proteins, has been linked to a variety of pathologic disease states in the past 20 years. In these diseases, which include diabetes, cancer, atherosclerosis, and Alzheimer's disease, distinct chemical consequences occur in response to biological exposure to the products of protein glycation, termed advanced glycation end products.

Recently, we showed that nornicotine (compound 1 in Fig. 1), a minor tobacco alkaloid and psychoactive nicotine metabolite with an extended half-life, is a practical aqueous aldol catalyst. Our extended studies indicated that the reaction uses an enamine intermediate. This unique and heretofore undetected ability of a metabolite to form enamine structures under aqueous conditions led us to consider other physiologically relevant chemical reactions that make use of similar intermediates. The Amadori rearrangement fit these requirements, and we hypothesized that nornicotine would form the corresponding Amadori product (compound 2 in Fig. 1) when incubated with glucose under physiologically relevant conditions. Furthermore, this reaction scenario implied that this Amadori product could be an early intermediate in the glycation of proteins by nornicotine.

We discovered that a nornicotine-based glycation product can form in vitro, and we developed a sensitive immunoassay that facilitates the detection of this product. In the assay, the nornicotine alkaloid nucleus is used as the primary antigenic determinant and a monoclonal antibody, NIC6C12, raised against a hapten that resembles nicotine, is used to detect the end product. We based our reasoning on our knowledge that the glucose-derived cross-linking region may undergo a variety of chemical transformations over time, whereas the 3-pyrrolidine-2-yl-pyridine nucleus is expected to be conserved.

Analysis of a series of plasma samples from human smokers and nonsmokers revealed that the plasma of smokers contained a larger concentration of proteins that had become specifically modified by nornicotine. In additional studies, we found that nornicotine can also modify commonly prescribed steroids, such as cortisone, altering the chemical nature of steroidal drugs in a potentially adverse manner.

Our findings indicate a direct link between tobacco abuse and the development of advanced glycation end products. Furthermore, the fact that nornicotine can modify commonly used steroids indicates a potentially unrecognized pathway for drug interactions. We are assessing the pathologic role of these nornicotine-derived advanced glycation end products in specific disease states.

Human Antibodies Against Bacillus Spores: A Model Study For Detection of and Protection Against Anthrax

In the aftermath of September 11, 2001, anthrax spores were intentionally disseminated in the United States via the postal system, resulting in sickness, death, and societal disruption. This event greatly increased the public awareness of the threat of a biological warfare attack and illustrated that virtually any nation, terrorist organization, or individual can develop and deploy extremely deadly biological weapons.

Currently, techniques to detect anthrax require hours to days to give results, and although rapid diagnostic tests exist, none are widely available for use. The development of antibody reagents to detect the spores would be immensely useful. However, because of limitations in immunization protocols in which bacteria, viruses, and toxins must be inactivated before they are used to immunize animals, the antibodies obtained from these endeavors often lack affinity and specificity for the native target.

Using in vitro phage display technology developed in our laboratories, we searched for a way to produce human antibodies that bind viable, native spores from the aerobic, spore-forming genus Bacillus. This technique would allow the use of the native spore as the putative target and circumvent animal immunization and toxic effects. As a model system, we chose to use spores of Bacillus subtilis, an avirulent strain known to resemble Bacillus anthracis, the causative agent of anthrax.

Panning in vitro against a naive human antibody library yielded 9 clones of antibodies that recognized the spores, and DNA analysis indicated high diversity among the gene sequences. This lack of a dominant sequence suggests significant diversity in the epitopes available on the spore surface. Amazingly, some of the clones had very little cross-reactivity with the spores of other species, highlighting the ability of in vitro selection to provide human antibodies highly specific for a given spore. Additionally, this specificity was increased by using a Fab sublibrary prepared by using chain shuffling and then a subtractive panning procedure. Interestingly, during our assays, we observed that the epitopes displayed on the surface of immobilized spores differ from those displayed on the surfaces of free spores, reiterating the point that the spore coat is a complex antigenic environment, capable of eliciting multiple antibody responses.

Finally, a key advantage of using phage-displayed antibodies is that the phage particle can serve as an amplifier, yielding significantly increased sensitivity. For example, using our phage-displayed single-chain antibodies, we can obtain "single spore" resolution by using fluorescence microscopic techniques, because a single phage particle can be labeled with a large number of copies (>2000) of a given fluorophore. Thus, our assay is extremely sensitive and can be used as a stand-alone detection system or integrated further with other detection strategies.

Recent world events have drawn attention to significant weaknesses in our diagnostic and medicinal capabilities in the detection of anthrax. Our results give a model for the preparation of human antibodies that would be immensely useful both as intelligence-gathering agents and as therapeutic reagents for the treatment of military and civilian personnel exposed to anthrax spores.

Active Immunization With a Glycolipid Transition-State Analog to Protect Against Endotoxic Shock

Septic shock is 1 of the 10 leading causes of death among both infants and adults in the United States; it accounted for more than 300,000 deaths in 1999 alone. Many of the toxic effects associated with septic shock caused by gram-negative bacteria can be directly attributed to lipopolysaccharide released from the bacterial membrane after bacteriolysis. The glycolipid part of lipopolysaccharide known as lipid A is responsible for the endotoxic activity.

In an effort to prevent the serious consequences of exposure to lipopolysaccharide, we designed a series of lipid A mimics that when conjugated to a suitable carrier protein, offer significant protection in vivo against a sublethal challenge with lipid A. Our mimics incorporate 3 significant features: a glucosamine 4´-phosphate ß-O-butyl lipid X saccharide analog of lipid A; truncated 2´- and 3´-acyloxy chains that contain both the lipid A (R)-stereocenter and a phosphonate group; and a flexible linker and a rigid linker for conjugation to a carrier protein (Fig. 2).

We anticipated that active immunization with conjugates consisting of these haptens bound to keyhole limpet hemocyanin would provide antibodies that could either bind and neutralize lipid A or perhaps catalyze the hydrolysis of the critical 2´- and/or 3´-acyloxyacyl ester linkages, generating functionally in active lipid A derivatives. In order to obtain antibodies with catalytic activity, the phosphonate groups were incorporated as mimics of the presumed tetrahedral intermediate formed during hydroxide-catalyzed ester hydrolysis.

We used an in vivo mouse model to examine the protective effect of active immunization with our immunoconjugates against a lipid A challenge. All mice strains tested produced significant amounts of antibodies against the glycolipid hapten. Furthermore, immunization with the rigid linker 3b (in Fig. 2) yielded significantly higher amounts of antibody than did immunization with flexible linker 3a.

It is well documented that decreased serum levels of TNF-α during lipid A challenge correlate well with survival in mice with sepsis caused by gram-negative bacteria. Gratifyingly, analysis of the serum of mice immunized with the conjugates of our haptens indicated that significantly lower levels of TNF-α were present in all strains tested (Fig. 3). However, we do not know if this protection is due to antibody binding to lipid A or to antibody-catalyzed destruction of lipid A as a result of the phosphonate tetrahedral intermediate mimics installed in our haptens. We are investigating this aspect. Our eventual goal is to develop an immunopharamcotherapeutic approach for treating clinical septic shock syndrome.

Publications

Ahn, J.-M., Boyle, N.A., MacDonald, M.T., Janda, K.D. Peptidomimetics and peptide backbone modifications. Mini Rev. Med. Chem. 2:463, 2002.

Ahn, J.-M., Wentworth, P., Jr., Janda, K.D. Soluble polymer-supported convergent parallel library synthesis. Chem. Commun. (Camb.) 4:480, 2003.

Boyle, N.A., Janda, K.D. Formats for combinatorial synthesis: solid-phase, liquid-phase and surface. Curr. Opin. Chem. Biol. 6:339, 2002.

Cannizzaro, C.E., Ashley, J.A., Janda, K.D., Houk, K.N. Experimental determination of the absolute enantioselectivity of an antibody-catalyzed Diels-Alder reaction and theoretical explorations of the origins of stereoselectivity. J. Am. Chem. Soc. 125:2489, 2003.

Clapham, B., Lee, S.-H., Koch, G., Zimmermann, J., Janda, K.D. The preparation of polymer bound ß-ketoesters and their conversion into an array of oxazoles. Tetrahedron Lett. 43:5407, 2002.

Delgado, M., Janda, K.D. Polymeric supports for solid phase organic synthesis. Curr. Org. Chem. 6:1031, 2002.

Dickerson, T.J., Córdova, A., Chen, D.-W., Janda, K.D. The use of enzymes and catalytic antibodies in the asymmetric aldol reaction. In: Encyclopedia of Catalysis. Horvath, I.T. (Ed.). Wiley & Sons, New York, in press.

Dickerson, T.J., Janda, K.D. A previously undescribed chemical link between smoking and metabolic disease. Proc. Natl. Acad. Sci. U. S. A. 99:15084, 2002.

Dickerson, T.J., Reed, N.N., Janda, K.D. Soluble polymers as catalyst and reagent platforms: liquid-phase methodologies. In: Polymeric Materials in Organic Synthesis and Catalysis. Buchmeiser, M.R. (Ed.). Wiley & Sons, New York, in press.

Dickerson, T.J., Reed, N.N., Janda, K.D. Soluble polymers as scaffolds for recoverable catalysts and reagents. Chem. Rev. 102:3325, 2002.

Gambs, C., Dickerson, T.J., Mahajan, S., Pasternack, L.B., Janda, K.D. High-resolution diffusion-ordered spectroscopy to probe the microenvironment of JandaJel and Merrifield resins. J. Org. Chem. 68:3673, 2003.

Gao, C., Mao, S., Ditzel, H.J., Farnaes, L., Wirsching, P., Lerner, R.A., Janda, K.D. A cell-penetrating peptide from a novel pVII-pIX phage-displayed random peptide library. Bioorg. Med. Chem. 10:4057, 2002.

Gao, C., Mao, S., Kaufmann, G., Wirsching, P., Lerner, R.A., Janda, K.D. A method for the generation of combinatorial antibody libraries using pIX phage display. Proc. Natl. Acad. Sci. U. S. A. 99:12612, 2002.

Gao, C., Mao, S., Ronca, F., Zhuang, S., Quaranta, V., Wirsching, P., Janda, K.D. De novo identification of tumor-specific internalizing human antibody-receptor pairs by phage-display methods. J. Immunol. Methods 274:185, 2003.

Hasserodt, J., Janda, K.D. Catalytic antibodies. In: Encyclopedia of Catalysis. Horvath, I.T. (Ed.). Wiley & Sons, New York, in press.

Jones, L.H., Altobell, L.J. III, MacDonald, M.T., Boyle, N.A., Wentworth, P., Jr., Lerner, R.A., Janda, K.D. Active immunization with a glycolipid transition state analogue protects against endotoxic shock. Angew. Chem. Int. Ed. 41:4241, 2002.

Lee, K.J., Mao, S., Sun, C., Gao, C., Blixt, O., Arrues, S., Hom, L.G., Kaufmann, G.F., Hoffman, T.Z., Coyle, A.R., Paulson, J., Felding-Habermann, B., Janda, K.D. Phage-display selection of a human single-chain Fv antibody highly specific for melanoma and breast cancer cells using a chemoenzymatically synthesized G_M3-carbohydrate antigen. J. Am. Chem. Soc. 124:12439, 2002.

Lee, S.-H., Clapham, B., Koch, G., Zimmermann, J., Janda, K.D. Rhodium carbenoid N-H insertion reactions of primary ureas: solution and solid-phase synthesis of imidazolones. Org. Lett. 5:511, 2003.

Lee, S.-H., Clapham, B., Koch, G., Zimmermann, J., Janda, K.D. Solid-phase rhodium carbenoid N-H insertion reactions: the synthesis of a diverse array of indoles. J. Comb. Chem. 5:188, 2003.

McDunn, J.E., Dickerson, T.J., Janda, K.D. Antibody catalysis of disfavored chemical reactions. In: Catalytic Antibodies. Keinan, E. (Ed.). Wiley & Sons, New York, in press.

Reed, N.N., Delgado, M., Hereford, K., Clapham, B., Janda, K.D. Preparation of soluble and insoluble polymer supported IBX reagents. Bioorg. Med. Chem. Lett. 12:2047, 2002.

Shimomura, O., Clapham, B., Spanka, C., Mahajan, S., Janda, K.D. Application of microgels as polymer supports for organic synthesis: preparation of a small phthalide library, a scavenger, and a borohydride reagent. J. Comb. Chem. 4:436, 2002.

Spanka, C., Clapham, B., Janda, K.D. Preparation of new microgel polymers and their application as supports in organic synthesis. J. Org. Chem. 67:3045, 2002.

Spanka, C., Wentworth, P., Jr., Janda, K.D. Developing soluble polymers for high-throughput synthetic chemistry. Comb. Chem. High Throughput Screen. 5:233, 2002.

Sun, C., Wirsching, P., Janda, K.D. Synthesis of dendritic linkers containing chlorambucil residues for the preparation of antibody-multidrug immunoconjugates. Bioorg. Med. Chem. Lett. 12:2213, 2002.

Wang, Q., Raja, K.S., Janda, K.D., Lin, T., Finn, M.G. Blue fluorescent antibodies as reporters of steric accessibility in virus conjugates. Bioconjug. Chem. 14:38, 2003.

Wentworth, P., Jr., McDunn, J.E., Wentworth, A.D., Takeuchi, C., Nieva, J., Jones, T., Bautista, C., Ruedi, J.M., Gutierrez, A., Janda, K.D., Babior, B.M., Eschemoser, A., Lerner, R.A. Evidence for antibody-catalyzed ozone formation in bacterial killing and inflammation. Science 298:2195, 2002.

Wentworth, P., Jr., Wentworth, A.D., Zhu, X., Wilson, I.A., Janda, K.D., Eschenmoser, A., Lerner, R.A. Evidence for the production of trioxygen species during antibody-catalyzed chemical modification of antigens. Proc. Natl. Acad. Sci. U. S. A. 100:1490, 2003.

Zhou, B., Wirsching, P., Janda, K.D. Human antibodies against spores of the genus Bacillus: a model study for detection of and protection against anthrax and the bioterrorist threat. Proc. Natl. Acad. Sci. U. S. A. 99:5241, 2002.