Scientific Report 2006

Chemistry

A Merging of Chemistry and Biology

K.D. Janda, J. Ashley, C. Berndt, G. Boldt, A. Brogan, C. Chung, S. De Lamo Marin, T. Dickerson, L. Eubanks, M. Hixon, A. Ino, G. Kaufmann, J. Kennedy, Y. Kim, J. Liu, Y. Liu, C. Lowery, H. Ma, S. Mahajan, L. McAllister, G. McElhaney, K. McKenzie, J. Mee, M. Meijler, J. Park, S. Steiniger, J. Treweek, A. Willis, Y. Xu, B. Zhou, H. Zhou

During the past year, we explored various applications of organic chemistry at the interface of chemistry and biology. Representative examples of our results were obtained in 3 research programs: catalysis of retinal isomerization by the nicotine metabolite nornicotine, “superactivation” of botulinum neurotoxin by small molecules, and the development of a cocaine esterase–bacteriophage construct with suitable kinetics for the degradation of cocaine in humans.

Altered Retinoid Homeostasis Catalyzed by a Nicotine Metabolite

In recent years, we have been studying the role of long-lived drugs of abuse and their metabolites in drug-related diseases. Much of this effort has centered on the Maillard reaction, a process by which amine-containing molecules irreversibly react with proteins through the intermediacy of glucose, the dominant serum monosaccharide. The mechanism of the Maillard reaction parallels that of amine organocatalysts; iminium and/or enamine intermediates are necessary for rate enhancement. In the past year, we expanded our studies beyond the Maillard reaction to other biological processes in which iminium ion intermediates are critical.

Retinoids (vitamin A) play 2 major roles in higher animals: light absorption in vision and gene regulation in growth and development. Specifically, these processes are regulated by the conformation of the double bonds in the polyunsaturated hydrocarbon chain. For example, in the visual cycle, 11-Z-retinal is converted to all-E-retinal by a photon of light, ultimately leading to the perception of vision. Much of the biosynthetic pathways leading up to this reaction are controlled by the formation of iminium ions between the retinal terminal aldehyde group and a lysine side chain from an appropriate enzyme.

We hypothesized that nornicotine, a metabolite of nicotine, could also perform this type of chemistry (Fig. 1) and thus alter the concentrations of retinal intermediates. This reaction would provide an intriguing mechanism for the pathologic changes in key smoking-related diseases, because the accumulation of compounds such as all-E-retinal feeds the N-retinylidene-N-retinylethanolamine biosynthetic pathway, forming an undigestible byproduct of the visual cycle and a fluorescent chromophore characteristic of the pathologic changes in age-related macular degeneration, a leading cause of blindness. Smoking is accepted as the primary environmental factor contributing to age-related macular degeneration, and thus elucidating the molecular mechanism of this contribution is clinidand retinal compounds, we conclusively showed that nornicotine can indeed catalyze the Z-to-E isomerization of unsaturated compounds at rates that could have biological significance in the context of disease.

Fig. 1. Mechanism of nornicotine-catalyzed Z-to-E isomerization.

Superactivation of Botulinum Neurotoxin Serotype a Light-Chain Metalloprotease

The 7 neurotoxins (A–G) of the bacterium Clostridium botulinum are the most lethal poisons known. Exposure to these toxins leads to progressive flaccid paralysis resulting from cleavage of proteins critical for proper release of neurotransmitters from peripheral nerve cells. Despite their potent toxicity, botulinum neurotoxins are widely used in medicine, as well as cosmetically for treating facial wrinkles. Conditions including multiple sclerosis, stroke, cerebral palsy, migraine, and backache can all be treated with the neurotoxins. Yet, repeated exposure to the toxins can result in the development of a marked immune response to them, thereby compromising their efficacy. Tolerance develops most rapidly when patients are treated frequently with high doses of the toxins. We speculated that the coadministration of a botulinum neurotoxin with a molecule that can “activate” the catalytic activity of the toxin would lead to lower doses, thus reducing the unintended immune response.

In recent investigations of light-chain metalloprotease inhibitors of botulinum neurotoxin A, we discovered that the molecule arginine hydroxamic acid is a modest inhibitor. Using this compound as a guide, we prepared a small collection of compounds containing a zinc-binding motif (2-acylthiophene) combined with an arginine-side-chain mimetic (acylguanidine). To our surprise, although no inhibition occurred, one compound (compound 1 in Fig. 2) consistently produced a 2-fold enhancement of activity. Further structure-activity relationship studies revealed that specific features of this compound were critical for activation, such as the thiophene sulfur atom and the acylguanidine group. When these initial screening efforts were completed, compound 2 (Fig. 2) was the most potent activator.

Fig. 2. Chemical structures of molecules that can superactivate botulinum neurotoxin serotype A. The specific motifs used in the design of these compounds are highlighted.

Because of the clinical promise of an activator of botulinum neurotoxin, we further examined the mechanism of this phenomenon. Extensive kinetic characterization indicated that these compounds operate primarily by reducing the Michaelis constant (K_m), not by altering the turnover number (k_cat). In this context, compound 2 is the most potent small-molecule activator of a protease reported to date, with up to 14-fold rate enhancement at limiting concentrations of substrate. Indeed, as little as 2-fold enzyme activation has previously been reported as a state of superactivation.

In total, the activation profile and structure-activity relationship for activation suggests the presence of a specific “activation domain” on the enzyme. Because the importance of botulinum neurotoxins continues to expand, methods such as this may ultimately provide a method for minimizing dosage of the toxins and thereby increase the clinical efficacy of the molecules.

Degrading Cocaine with Viruses

Cocaine is a powerful stimulant and among the most reinforcing of all drugs. Consequently, abuse of cocaine continues to be a major problem. Cocaine acts as an indirect dopamine agonist by blocking the dopamine transporter in the pleasure-reward center of the brain. This obstruction leads to an excess of dopamine in the synapses, amplifying the sensation of pleasure. Despite intensive efforts, no effective pharmacotherapy for cocaine abuse exists. The inherent difficulties in antagonizing a blocker have led to the development of protein-based therapeutics designed to treat cocaine abuse. In an approach termed immunopharmacotherapy, we have devoted extensive efforts to the use of antibodies to cocaine that can sequester cocaine, retarding its ability to enter the CNS. We have also developed a parallel strategy that involves use of catalytic antibodies specific for the hydrolysis of the benzoyl ester of cocaine to give the nonpsychoactive products benzoate and methyl ecgonine (Fig. 3).

Although the potential of this method has been demonstrated in rodent models of cocaine overdose and reinforcement, the kinetic constants of these antibodies must be improved before the method will be practical as a clinical treatment. Furthermore, these approaches are only effective in the periphery, whereas a pharmacotherapy that could act in both the CNS and the periphery is desirable.

Fig. 3. Hydrolysis products resulting from cleavage of cocaine esters. Both the uncatalyzed reaction (path a) and the cocaine esterase–catalyzed hydrolysis (path b) pathways are shown.

Bacteriophages are viruses that infect bacteria yet lack intrinsic tropism for eukaryotic cells. Because of the genetic flexibility of bacteriophages, a wide range of proteins and peptides can be expressed on the phage coat in an approach termed phage display. Furthermore, phage molecules can penetrate virtually all tissues, including the CNS.

We recently reported that cocaine-binding antibodies displayed on the surface of bacteriophages can be administered intranasally and that the treated animals are protected from the locomotor stimulation associated with exposure to cocaine. However, because of the requisite 1:1 stoichiometry of any traditional antibody pharmacotherapy, obtaining a meaningful concentration of the therapeutic agent in vivo is difficult. We envisioned that this limitation could be overcome by displaying a catalyst on the phage surface that can degrade cocaine, yielding a therapeutically practical approach for treating cocaine abuse.

For these studies, we used cocaine esterase, a globular bacterial enzyme that is the most efficient protein catalyst for cocaine hydrolysis reported to date. We displayed this enzyme on the phage coat and then used high-performance liquid chromatography to determine the kinetic parameters. We found that the catalytic efficiency of cocaine esterase–phage constructs was reduced relative to the efficiency of the native enzyme yet exceeded the postulated therapeutically relevant threshold. No reported catalytic antibody capable of cocaine hydrolysis achieves this value, and indeed, only recently described “designer” mutants of the enzyme butyrylcholinesterase are comparable to our cocaine esterase–phage constructs.

These results indicate that phage display of clinically relevant enzymes can be achieved without compromising the catalytic efficacy of the desired enzyme. We envision that this new technology will stimulate further development of other protein-based treatments for CNS-related disorders and will lead to powerful tools to combat drug abuse.

Publications

Boldt, G.E., Dickerson, T.J., Janda, K.D. Emerging chemical and biological approaches for the preparation of discovery libraries. Drug Discov. Today 11:143, 2006.

Boldt, G.E., Eubanks, L.M., Janda, K.D. Identification of a botulinum neurotoxin A protease inhibitor displaying efficacy in a cellular model. Chem. Commun. (Camb.) 3063, 2006, Issue 29.

Boldt, G.E., Kennedy, J.P., Hixon, M.S., McAllister, L.A., Barbieri, J.T., Tzipori, S., Janda, K.D. Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin A inhibitors. J. Comb. Chem. 8:513, 2006.

Boldt, G.E., Kennedy, J.P., Janda, K.D. Identification of a potent botulinum neurotoxin A protease inhibitor using in situ lead identification chemistry. Org. Lett. 8:1729, 2006.

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., 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., Beuscher, A.E. IV, Rogers, C.J., Hixon, M.S., Yamamoto, N., Xu, Y., Olson, A.J., Janda, K.D. Discovery of acetylcholinesterase peripheral anionic site ligands through computational refinement of a directed library. Biochemistry 44:14845, 2005.

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

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

Kaufmann, G.F., Sartorio, R., Lee, S.H., Mee, J.M., Altobell, L.J. III, Kujawa, D.P., Jeffries, E., Clapham, B., Meijler, M.M., Janda, K.D. Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing. J. Am. Chem. Soc. 128:2802, 2006.

Kim, Y., 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. Pharm. 2:528, 2005.

Lee, B.S., Mahajan, S., Janda, K.D. Asymmetric dihydroxylation catalyzed by ionic polymer-supported osmium tetroxide. Tetrahedron Lett. 46:4491, 2005.

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

Lillo, A.M., McKenzie, K.M., Janda, K.D. Phage-displayed antibody libraries. In: Cell Biology: A Laboratory Handbook, 3rd ed. Celis, J., et al. (Eds.). Academic Press, San Diego, 2006, p. 491.

Ma, H., Zhou, B., Kim, Y., Janda, K.D. A cyclic peptide-polymer probe for the detection of Clostridium botulinum neurotoxin serotype A. Toxicon 47:401, 2006.

Matsushita, M., Meijler, M.M., Wirsching, P., Lerner, R.A., Janda, K.D. A blue fluorescent antibody-cofactor sensor for mercury. Org. Lett. 7:4943, 2005.

McAllister, L.A., Hixon, M.S., Kennedy, J.P., Dickerson, T.J., Janda, K.D. Superactivation of the botulinum neurotoxin serotype A light chain metalloprotease: a new wrinkle in botulinum neurotoxin. J. Am. Chem. Soc. 128:4176, 2006.

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.

Moss, J.A, Stokols, S., Hixon, M.S., Ashley, F.T., Chang, J.Y., Janda, K.D. Solid-phase synthesis and kinetic characterization of fluorogenic enzyme-degradable hydrogel cross-linkers. Biomacromolecules 7:1011, 2006.

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.

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 solvents. 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., La Clair, 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.

Toker, J.D., Tremblay, M.R., Yli-Kauhaluoma, J., Wentworth, A.D., Zhou, B., Wentworth, P., Jr., Janda, K.D. Exploring the scope of the 29G12 antibody catalyzed 1,3-dipolar cycloaddition reaction. J. Org. Chem. 70:7810, 2005.

Wu, W., Luo, Y., Sun, C., Liu, Y., Kuo, P., Varga, J., Xiang, R., Reisfeld, R., Janda, K.D., Edgington, T.S., Liu, C. Targeting cell-impermeable prodrug activation to tumor microenvironment eradicates multiple drug-resistant neoplasms. Cancer Res. 66:970, 2006.

Xu, Y., Shi, J., Yamamoto, N., Moss, J.A., Vogt, P.K., Janda, K.D. A credit-card library approach for disrupting protein-protein interactions. Bioorg. Med. Chem. 14:2660, 2006.

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.

Yamashita, M., Lee, S.-H., Koch, G., Zimmermann, J., Clapham, B., Janda, K.D. Solid-phase synthesis of oxazolones and other heterocycles via Wang resin-bound diazocarbonyls. Tetrahedron Lett. 46:5495, 2005.

Yao, Y., Martinez-Yamout, M., Dickerson, T.J., Brogan, A.P., Wright, P.E., Dyson, H.J. Structure of the Escherichia coli quorum sensing protein SdiA: activation of the folding switch by acyl homoserine lactones. J. Mol. Biol. 355:262, 2006.

Zhang, L., Long, H., Boldt, G.E., Janda, K.D., Schatz, G.C., Lewis, F.D. α- and β-Stilbenosides as base-pair surrogates in DNA hairpins. Org. Biomol. Chem. 4:314, 2006.

Zhu, X., Dickerson, T.J., Rogers, C.J., Kaufmann, G.F., Mee, J.M., McKenzie, K.M., Janda, K.D., Wilson, I.A. Complete reaction cycle of a cocaine catalytic antibody at atomic resolution. Structure 14:205, 2006.