The Skaggs Institute
for Chemical Biology

Scientific Report 2005

The Development of New Tools for Investigations in Drug Addiction and Environmental Contamination

K.D. Janda, J. Ashley, M. Atsumi, C. Berndt, G. Boldt, A. Brogan, T. Dickerson, L. Eubanks, M. Hixon, J. Liu, Y. Liu, G. Kaufmann, J. Kennedy, Y.-S. Kim, C. Lowery, H. Ma, S. Mahajan, L. McAllister, G. McElhaney, K. McKenzie, J. Mee, M. Meijler, J. Moss, S. Steiniger, S. Stokols, Y. Xu, B. Zhou, H. Zhou

Altered Retinoid Homeostasis by a Tobacco Metabolite

Retinoids (vitamin A) serve 2 distinct functions in higher animals: light absorption for vision and gene regulation for growth and development. In the visual cycle, all-E-retinol is converted to 11-Z-retinal via a multistep enzymatic process. Disruption of this tightly controlled process has distinct pathologic implications. For example, accumulation of all-E-retinal feeds the pathway for synthesis of N-retinylidene-N-retinylethanolamine, a molecule that is formed by sequential condensation of phosphatidylethanolamine with 2 molecules of all-E-retinal and is an undigestible byproduct of the visual cycle and characteristic of the pathologic changes in age-related macular degeneration. Cigarette smoking is the primary environmental factor that contributes to age-related macular degeneration, and smoking increases the risk of birth defects; however, altered retinoid homeostasis has received little attention as a potential mechanism for smoking-associated toxic effects.

Despite the recent increase in the use of organic compounds as chemical catalysts, minimal effort has been devoted to biologically relevant organocatalysis. During the course of recent studies on the chemistry of long-lived nicotine metabolites, we found that nornicotine, a metabolite of nicotine and a component of tobacco and cigarette smoke, can cause aberrant protein glycation in vivo via iminium ion–based intermediates. We speculated that nornicotine may also serve as a catalyst in the alkene isomerization of unsaturated aldehydes and ketones, including the retinals, in a biomimetic fashion by an energy-lowering mechanism (Fig. 1), thus providing an intriguing mechanism for the pathologic aspects of key smoking-associated diseases that may stem from disrupted retinoid metabolism.

Fig. 1. Mechanism of nornicotine-catalyzed Z-to-E isomerization of retinoids. Shown is the isomerization of 9-Z-retinal to all-E-retinal.

Using a combination of model systems and related retinals, we conclusively showed that nornicotine does indeed catalyze the Z-to-E isomerization of unsaturated compounds at rates that may have biological significance in the context of the development of tobacco-associated diseases. Nornicotine-catalyzed retinal isomerization implies an underlying molecular mechanism for age-related macular degeneration, the birth defects associated with smoking, and other smoking-associated abnormalities that stem from disruption of retinoid metabolism.

Detection of Environmental Contaminants with a Field-Adaptable Precipitator

The introduction of heavy metals into the environment has occurred in tandem with the emergence of the tools of modern society, including automobiles, industrial activities, and the burning of coal. Because of the varied deleterious biological activities of these metals, their detection has attained significant prominence within the scientific community and the public consciousness. In addition, concerns about heavy metals in the environment have been furthered by the discovery that aquatic organisms convert elemental mercury to methylmercury, which subsequently is concentrated through the food chain in the tissues of fish and marine mammals. Unquestionably, the development of easy and efficient methods for detecting heavy metals in a wide range of settings is paramount in the effort to minimize mortality attributable to the toxic effects of the metals.

Although many indicators for the detection of heavy metals have been developed, the majority of the assays are solution based, and thus the response is highly dependent on the assay environment. Recently, we developed a crystalline assay with fluorescent dyes that offers excellent metal selectivity and provides detection comparable to that of conventional solution-based ligands used for the spectrofluorometric analysis of thiophilic heavy metal ions (Fig. 2).

Fig. 2. Complexation of heavy metals by dithiopthalide ligands. The solid in the background of the product on the right is a fluorescent dye–containing crystalline complex of ligand and heavy metal.

Although our method is comparable in analytical performance to known methods, the formation of crystalline analytes in our assay provides signal amplification and, consequently, a powerful platform that can be directly used with high-throughput video systems to acquire data. Because the product of the detection reaction (i.e., the heavy metal–ligand complex) is insoluble in all tested solvents and buffers, our assay has little sensitivity to environmental factors, including pH, ionic strength, and impurities. Also, we were surprised to discover that ligand binding quantitatively removes heavy metals from solution, even when 1:1 mixtures of ligand to metal are used in the initial reactions.

With this assay, we were able to detect as little as 0.3 parts per billion of heavy metal, well below the accepted limit of the U.S. Environmental Protection Agency. We envision that our precipitation strategy for detecting heavy metals will be valuable in the development of field test kits for measuring the amount of a heavy metal in environmental samples (e.g., soil, water), food (e.g., vegetable matter, fish), and biological samples (e.g., fluids, tissue).

Viruses For The Treatment of Cocaine Addiction

Cocaine is a powerful stimulant and may be the most reinforcing of all drugs. Consequently, the abuse of cocaine continues to be a major societal and health problem. Despite intensive efforts, no effective pharmacotherapy for cocaine abuse is available. Using an approach termed immunopharmacotherapy, we have spent considerable effort during the past 15 years developing antibodies to cocaine that can sequester the drug, retarding its ability to enter the CNS. In a parallel strategy, we have used catalytic antibodies specific for the hydrolysis of the benzoyl ester of cocaine to give the nonpsychoactive products benzoate and methyl ecgonine (Fig. 3). The efficacy of any protein-based cocaine treatment is limited by the inability of the protein to access the CNS. Thus, the success of these treatments depends on contact between the protein and ingested cocaine outside the brain.

Fig. 3. Hydrolysis products of cocaine ester cleavage. Uncatalyzed hydrolysis (path a) yields the psychoactive product benzoyl ecgonine, whereas hydrolysis catalyzed by cocaine esterase (path b) forms the nonpsychoactive compounds methyl ecgonine and benzoic acid.

In an improved treatment, protein would come in contact with cocaine both in circulation and in the CNS. Filamentous bacteriophage, a bacterial virus, with foreign proteins displayed on its surface can penetrate the CNS of mice after various routes of administration (e.g., intravenous, intraperitoneal, intramuscular, intranasal) and can be administered multiple times without visible toxic effects. Furthermore, bacteriophage can also diffuse into a wide variety of peripheral organs, including the lung, kidney, spleen, liver, and intestine.

We have developed an approach in which cocaine esterase, a globular bacterial enzyme that is the most efficient protein catalyst for cocaine hydrolysis reported to date, is displayed on the surface of filamentous bacteriophage. The kinetic profiles of cocaine esterase–phage constructs were determined by using high-performance liquid chromatography. We found that the catalytic efficiency of the enzyme in the constructs, although reduced relative to that of the native enzyme in solution, was a therapeutically relevant value. Importantly, the catalytic efficiency of the construct is greater than the efficiencies of any known catalytic antibodies to cocaine and similar to that of a designed mutant of the enzyme butyrylcholinesterase. In total, our results indicate a potential method for catalytic degradation of cocaine in both the CNS and the circulation that has both suitable kinetic parameters and a pharmacologic profile that make it suitable for use in mammals.

Publications

Brogan, A.P., Dickerson, T.J., Boldt, G.E., Janda, K.D. Altered retinoid homeostasis catalyzed by a nicotine metabolite: implications in macular degeneration and normal development. Proc. Natl. Acad. Sci. U. S. A. 102:10433, 2005.

Carrera, M.R.A., Meijler, M.M., Janda, K.D. Cocaine pharmacology and current pharmacotherapies for its abuse. Bioorg. Med. Chem. 12:5019, 2004.

Carrera, M.R.A., Trigo, J.M., Wirsching, P., Roberts, A.J., Janda, K.D. Evaluation of the anticocaine monoclonal antibody GNC92H2 as an immunotherapy for cocaine overdose. Pharmacol. Biochem. Behav. 81:709, 2005.

Dickerson, T.J., Janda, K.D. Recent advances for the treatment of cocaine abuse: central nervous system immunopharmacotherapy. AAPS J. 7:E579, 2005.

Dickerson, T.J., Kaufmann, G.F., Janda, K.D. Bacteriophage-mediated protein delivery into the central nervous system and its application in immunopharmacotherapy. Expert Opin. Biol. Ther. 5:773, 2005.

Dickerson, T.J., Lovell, T., Meijler, M.M., Noodleman, L., Janda, K.D. Nornicotine aqueous aldol reactions: synthetic and theoretical investigations into the origins of catalysis. J. Org. Chem. 69:6603, 2004.

Dickerson, T.J., Reed, N.N., La Clair, J.J., Janda, K.D. A precipitator for the detection of thiophilic metals in aqua. J. Am. Chem. Soc. 126:16582, 2004.

Dickerson, T.J., Yamamoto, N., Janda, K.D. Antibody-catalyzed oxidative degradation of nicotine using riboflavin. Bioorg. Med. Chem. 12:4981, 2004.

Dickerson, T.J., Yamamoto, N., Ruiz, D.I., Janda, K.D. Immunological consequences of methamphetamine protein glycation. J. Am. Chem. Soc. 126:11446, 2004.

Eubanks, L.M., Dickerson, T.J., Janda, K.D. Vitamin B₂-mediated cellular photoinhibition of botulinum neurotoxin A. FEBS Lett. 579:5361, 2005.

Felding-Habermann, B., Lerner, R.A., Lillo, A., Zhuang, S., Weber, M.R., Arrues, S., Gao, C., Mao, S., Saven, A., Janda, K.D. Combinatorial antibody libraries from cancer patients yield ligand-mimetic Arg-Gly-Asp-containing immunoglobulins that inhibit breast cancer metastasis. Proc. Natl. Acad. Sci. U. S. A. 101:17210, 2004.

Kaufmann, G.F., Meijler, M.M., Sun, C., Chen, D.W., Kujawa, D.P., Mee, J.M., Hoffman, T.Z., Wirsching, P., Lerner, R.A., Janda, K.D. Enzymatic incorporation of an antibody-activated blue fluorophore into DNA. Angew. Chem. Int. Ed. 44:2144, 2005.

Kaufmann, G.F., Sartorio, R., Lee, S.H., Rogers, C.J., Meijler, M.M., Moss, J.A., Clapham, B., Brogan, A.P., Dickerson, T.J., Janda, K.D. Revisiting quorum sensing: discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones. Proc. Natl. Acad. Sci. U. S. A. 102:309, 2005.

Kim, Y.S., Lillo, A., Moss, J.A., Janda, K.D. A contiguous stretch of methionine residues mediates the energy-dependent internalization mechanism of a cell-penetrating peptide. Mol. Pharmacol., in press.

Kim, Y.S., Moss, J.A., Janda, K.D. Biological tuning of synthetic tactics in solid-phase synthesis: application to Aβ(1-42). J. Org. Chem. 69:7776, 2004.

Lee, B.S., Mahajan, S., Janda, K.D. Cross-linked poly(4-vinylpyridine/styrene) copolymers as a support for immobilization of ytterbium triflate. Tetrahedron 61:3081, 2005.

Lee, B.S., Mahajan, S., Janda, K.D. Molecular iodine-catalyzed imine activation for three-component nucleophilic addition reactions. Synlett 8:1325, 2005.

Lee, B.S., Mahajan, S., Janda, K.D. Novel method for catalyst immobilization using an ionic polymer: a case study using recyclable ytterbium triflate. Tetrahedron Lett. 46:807, 2005.

Lee, S.H., Matsushita, H., Koch, G., Zimmermann, J., Clapham, B., Janda, K.D. Smart cleavage reactions: the synthesis of an array of ureas from polymer-bound carbamates. J. Comb. Chem. 6:822, 2004.

Lee, S.H., Yoshida, K., Matsushita, H., Clapham, B., Koch, G., Zimmermann, J., Janda, K.D. N-H insertion reactions of primary ureas: the synthesis of highly substituted imidazolones and imidazoles from diazocarbonyls. J. Org. Chem. 69:8829, 2004.

Lowery, C.A., McKenzie, K.M., Qi, L., Meijler, M.M., Janda, K.D. Quorum sensing in Vibrio harveyi: probing the specificity of the LuxP binding site. Bioorg. Med. Chem. Lett. 15:2395, 2005.

Matsushita, H., Lee, S.H., Yoshida, K., Clapham, B., Koch, G., Zimmermann, J., Janda, K.D. N-H insertion reactions of Boc-amino acid amides: solution- and solid-phase synthesis of pyrazinones and pyrazines. Org. Lett. 6:4627, 2004.

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, 2005, p. 184.

McKenzie, K.M., Meijler, M.M., Lowery, C.A., Boldt, G.E., Janda, K.D. A furanosyl-carbonate autoinducer in cell-to-cell communication of V harveyi. Chem. Commun. (Camb.) 4863, 2005, Issue 38.

Meijler, M.M., Kaufmann, G.F., Qi, L., Mee, J.M., Coyle, A.R., Moss, J.A., Wirsching, P., Matsushita, M., Janda, K.D. Fluorescent cocaine probes: a tool for the selection and engineering of therapeutic antibodies. J. Am. Chem. Soc. 127:2477, 2005.

Moss, J.A., Lillo, A., Kim, Y.S., Gao, C., Ditzel, H., Janda, K.D. A dimerization “switch” in the internalization mechanism of a cell-penetrating peptide. J. Am. Chem. Soc. 127:538, 2005.

Qi, L., Yamamoto, N., Meijler, M.M., Altobell, L.J. III, Koob, G.F., Wirsching, P., Janda, K.D. Δ⁹-Tetrahydrocannabinol immunochemical studies: haptens, monoclonal antibodies, and a convenient synthesis of radiolabeled Δ⁹-tetrahydrocannabinol. J. Med. Chem. 48:7389, 2005.

Reed, N.N., Dickerson, T.J., Boldt, G.E., Janda, K.D. Enantioreversal in the Sharpless asymmetric epoxidation reaction controlled by the molecular weight of a covalently appended achiral polymer. J. Org. Chem. 70:1728, 2005.

Rogers, C.J., Dickerson, T.J., Brogan, A.P., Janda, K.D. Hammett correlation of nornicotine analogues in the aqueous aldol reaction: implications for green organocatalysis. J. Org. Chem. 70:3705, 2005.

Rogers, C.J., Dickerson, T.J., Janda, K.D. Kinetic isotope and thermodynamic analysis of the nornicotine catalyzed aqueous aldol reaction. Tetrahedron 62:352, 2006.

Rogers, C.J., Dickerson, T.J., Wentworth, P., Jr., Janda, K.D. A high-swelling reagent scaffold suitable for use in aqueous and organic media. Tetrahedron 61:12140, 2005.

Rogers, C.J., Mee, J.M., Kaufmann, G.F., Dickerson, T.J., Janda, K.D. Toward cocaine esterase therapeutics. J. Am. Chem. Soc. 127:10016, 2005.

Shimomura, O., Lee, B.S., Meth, S., Suzuki, H., Mahajan, S., Nomura, R., Janda, K.D. Synthesis and application of polytetrahydrofuran-grafted polystyrene (PS-PTHF) resin supports for organic synthesis. Tetrahedron 61:12160, 2005.

Shute, T.S., Matsushita, M., Dickerson, T.J., LaClair, J.J., Janda, K.D., Burkart, M.D. A site-specific bifunctional protein labeling system for affinity and fluorescent analysis. Bioconjug. Chem. 16:1352, 2005.

Xu, Y., Yamamoto, N., Janda, K.D. Catalytic antibodies: hapten design strategies and screening methods. Bioorg. Med. Chem. 12:5247, 2004.

Xu, Y., Yamamoto, N., Ruiz, D.I., Kubitz, D.S., Janda, K.D. Squaric monoamide monoester as a new class of reactive immunization hapten for catalytic antibodies. Bioorg. Med. Chem. Lett. 15:4304, 2005.