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The Skaggs Institute
for Chemical Biology

Scientific Report 2006

Chemical Tools for Improving Human Health

K.D. Janda, A. Accurso, J. Ashley, G. Boldt, A. Brogan, C. Carney, C. Chung, S. De Lamo Marin, T. Dickerson, L. Eubanks, M. Hixon, A. Hoyt, 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, A. Moreno, Y. Nakai, J. Park, S. Steiniger, J. Treweek, A. Willis, Y. Xu, B. Zhou, H. Zhou

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. Indeed, serotype A has an LD50 for a 70-kg human of a mere 0.8 μg by inhalation. Exposure to these toxins leads to progressive flaccid paralysis resulting from cleavage of proteins that are critical for the proper release of neurotransmitter-containing vesicles from peripheral nerve cells. Because of their extreme toxicity, botulinum neurotoxins have been classified by the Centers for Disease Control and Prevention as one of the greatest threats in the context of bioterrorism agents. Despite this potent toxicity, botulinum toxins 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 inhibitors of the light-chain metalloprotease of botulinum neurotoxin A, we discovered that the simple amino acid derivative arginine hydroxamic acid is a modest inhibitor of this enzyme. 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. 1) 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. 1) was the most potent activator.

Fig. 1. Chemical structures of molecules that can superactivate botulinum neurotoxin serotype A.

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 (Km), not by altering the turnover number (kcat). 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 suggest the presence of a specific “activation domain” on the enzyme. Because the importance of botulinum neurotoxins continues to expand, methods such as this one may ultimately provide a way to minimize dosage of the toxins, and thereby increase the clinical efficacy of the molecules.

Vaccination Against Weight Gain

Approximately 1 billion persons worldwide are overweight or obese (body mass index 25–29.9 or 30 or greater, respectively), with disproportionately higher prevalence in affluent countries. For example, according to the National Health and Nutrition Examination Survey, in 2003–2004, 66% of all adults in the United States 20 years or older were overweight or obese. Almost 4 of every 5 adult men 40–59 years old were so classified. Among children and adolescents between the ages of 6–11 and 12–19, 19% and 17%, respectively, were overweight. Alarmingly, the prevalence of obesity has tripled for adolescents during the past 2 decades.

The increase in the number of persons who are overweight or obese cuts across all ages, all racial and ethnic groups, and both sexes and is increasingly global. For example, the prevalence of obesity in urban preschoolers in China increased 8-fold between 1989 and 1997, and the rate of obesity in British adults increased almost 3-fold from 1980 to 2002. In 2000, a total of 110,000 deaths in the United States were associated with obesity, as shown by confound-adjusted analysis of the National Health and Nutrition Examination Survey cohorts, and the economic cost of obesity in the United States was estimated to be $117 billion.

Unfortunately, no nonsurgical treatments are currently available for treating obesity. Indeed, although several purported drug treatments exist, when treatment is discontinued, weight gain inevitably results. For any obesity therapy to show efficacy, it must affect energy intake, absorption, expenditure, or storage. Fortunately, research during the past 15 years has revolutionized understanding of the molecular mechanisms that homeostatically control body weight and fat. Accumulated findings support a lipostatic hypothesis of energy homeostasis in which the brain seeks to keep stored energy constant as adipose tissue during long periods. In this context, ghrelin was identified in 1999 as an endogenous ligand for the growth hormone secretagogue receptor (ghrelin receptor), previously localized to peripheral tissues and hypothalamic nuclei that control energy homeostasis.

Ghrelin stimulates or increases appetite when administered to humans or sated rodents and most likely has a central site of action. Ghrelin also promotes weight gain and adiposity through its metabolic actions, decreasing both energy expenditure and fat catabolism. Relevant to a possible role for ghrelin in longer term energy homeostasis, circulating ghrelin levels are persistently increased during weight loss and are suppressed in obese states.

Ghrelin is the first peptide isolated from animal sources with the posttranslational modification of octanoylation. Specifically, the hydroxyl group of a serine residue (Ser-3) is acylated by n-octanoic acid (Fig. 2). Octanoylation is essential for the growth hormone–releasing activity of ghrelin. Moreover, short peptides that encompass the first 4–5 residues of ghrelin activate the growth hormone secretagogue receptor as efficiently as does full-length ghrelin, suggesting that the N-terminal glycine-serine-serine(n-octanoyl)-phenylalanine segment constitutes the requisite core for receptor binding and activation.

Fig. 2. Structures of ghrelin haptens used in obesity vaccine studies. The asterisks denote the sites of conjugation with the carrier proteins keyhole limpet hemocyanin or bovine serum albumin.

During the past 20 years, we have developed a technique termed immunopharmacotherapy for the development of vaccines against drugs of abuse, including cocaine, nicotine, and marijuana. We hypothesized that this approach could be extended to the development of a vaccine that would bind circulating ghrelin before the ghrelin reached its cognate target, thereby inactivating this peptide and leading to weight loss. To develop these vaccines, we prepared a series of immunoconjugates that recapitulate the critical structural features of ghrelin, namely, the lipophilic Ser-3 ester and native peptide sequence. These peptides were then linked to carrier proteins to generate immunoconjugates suitable for immunization.

We found that the efficacy of a vaccine was related to the ability of the conjugate to induce high, specific plasma binding affinity for n-octanoyl ghrelin, as opposed to des-octanoyl ghrelin. Immunized rats ate normally, but once antibody titers to the conjugate increased, accrued less body weight and fat, indicating reduced energy thrift. Furthermore, the ratio of brain to serum levels of ghrelin was lower in rats with strong antighrelin immune responses. The observed effects were not attributable to nonspecific inflammatory responses, and in total, our data indicate that vaccination against the endogenous hormone ghrelin can slow weight gain in rats by decreasing feed efficiency.


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., Janda, K.D. Enamine-based aldol organocatalysis in water: are they really “all wet”? Angew. Chem. Int. Ed. 45:8100, 2006.

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. The use of small molecules to investigate molecular mechanisms and therapeutic targets for treatment of botulinum neurotoxin A intoxication [published correction appears in ACS Chem. Biol. 1:470, 2006]. ACS Chem. Biol. 1:359, 2006.

Eubanks, L.M., Rogers, C.J., Beuscher, A.E. IV, Koob, G.F., Olson, A.J., Dickerson, T.J., Janda, K.D. A molecular link between the active component of marijuana and Alzheimer’s disease pathology. Mol. Pharm. 3:773, 2006.

Fu, Z., Chen, S., Baldwin, M.R., Boldt, G.E., Crawford, A., Janda, K.D., Barbieri, J.T., Kim J.J. Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate. Biochemistry 45:8903, 2006.

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.

Kim, Y., Lillo, A.M., Steiniger, S.C., Liu, Y., Bellatore, C., Anichini, A., Mortarini, R., Kaufmann, G.F., Zhou, B., Felding-Habermann, B., Janda, K.D. Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand. Biochemistry 45:9434, 2006.

Kravchenko, V.V., Kaufmann, G.F., Mathison, J.C., Scott, D.A., Katz, A.Z., Wood, M.R., Brogan, A.P., Lehmann, M., Mee, J.M., Iwata, K., Pan, Q., Fearns, C., Knaus, U.G., Meijler, M.M., Janda, K.D., Ulevitch, R.J. N-(3-oxo-acyl)-homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways. J. Biol. Chem. 281:28822, 2006.

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

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 47:901, 2006.

Matsushita, H., Yamamoto, N., Meijler, M.M., Wirsching, P., Lerner, R.A., Matsushita, M., Janda, K.D. Chiral sensing using a blue fluorescent antibody. Mol. Biosyst. 1:303, 2005.

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.

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.

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., Lu, H., Kennedy, J.P., Yan, X., McAllister, L.A., Yamamoto, N., Moss, J.A., Boldt, G.E., Jiang, S., Janda, K.D. Evaluation of “credit card” libraries for inhibition of HIV-1 gp41 fusogenic core formation. J. Comb. Chem. 8:531, 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.

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.

Zorrilla, E.P., Iwasaki, S., Moss, J.A., Chang, J., Otsuji, J., Inoue, K., Meijler, M.M., Janda K.D. Vaccination against weight gain. Proc. Natl. Acad. Sci. U. S. A. 103:13226, 2006.


Kim D. Janda, Ph.D.
Ely R. Callaway, Jr., Chair in Chemistry

Janda Web Site