Scientific Report 2007

Chemistry

A Merging of Chemistry and Biology

K.D. Janda, J. Ashley, A. Brogan, C. Carney, K. Capkova, C. Chung, S. De Lamo Marin, J. Denery, T. Dickerson, L. Eubanks, K. Fukuchi, C. Hernandez, A. Hoyt, A. Ino, G. Kaufmann, J. Liu, Y. Liu, C. Lowery, S. Mahajan, A. Mayorov, G. McElhaney, K. McKenzie, J. Mee, A. Moreno, Y. Nakai, J. Park, N. Salzameda, S. Steiniger, J. Treweek, A. Willis, Y. Xu, Y. Yoneda, B. Zhou, H. Zhou

During the past year, we used various applications of organic chemistry to address biological problems. The results of 3 research programs—in vivo identification of botulinum neurotoxin antagonists, development of a cyclic peptide as a drug delivery module, and discovery of antibodies capable of degrading the active component of marijuana—are highlighted in the following sections.

Identification of Antagonists of Botulinum Neurotoxin A

The neurotoxins produced by the bacteria Clostridium botulinum, Clostridium butyricum, and Clostridium barati are the most toxic proteins known; the toxins are classified as 1 of the 6 highest-risk agents for bioterrorism (category A agent). These proteins (serotypes A–G) are the agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis. Botulinum neurotoxins cause the paralysis by entering neuronal cells and cleaving soluble N-ethylmaleimide-sensitive–factor attachment protein receptors, thereby blocking fusion of synaptic vesicles to the plasma membrane and the release of the neurotransmitter acetylcholine at neuromuscular junctions. Interestingly, this same property has led to the use of the neurotoxins for therapeutic and cosmetic purposes, such as treatment of strabismus, hemifacial spasms, and wrinkles.

Although no approved pharmacologic treatments exist, current strategies to combat the effects of the neurotoxins rely mainly on vaccination or passive administration of antibodies derived from either immunized personnel or equine sources. Both vaccines and antibodies are effective only if administered before the neurotoxins enter neurons. Because of their potential to enter cells, small molecules provide an opportunity to combat botulism both before and after entry of the toxins into neurons. Published studies on small-molecule antagonists of the botulinum neurotoxins have relied on in vitro methods; therefore, to establish a model for predicting in vivo efficacy, we recently identified several compounds that extend the time of death in mice exposed to lethal doses of botulinum neurotoxin A.

The mechanism of action of botulinum neurotoxin A is a series of protein-protein interactions that culminate in a catalytic event. Accordingly, we hypothesized that screening a library of compounds known to disrupt protein-protein interactions would yield antagonists of the neurotoxin. Initially, we used a high-throughput screening method with purified neurotoxin A metalloprotease to detect possible antagonists, and then we verified the effects of the putative antagonists in a neuroblastoma cell–based assay. On the basis of the data obtained from the 2 initial screens, several compounds were advanced to animal trials. Two of the compounds (Fig. 1) significantly extended the time of death in mice given botulinum neurotoxin A. These 2 compounds were unlikely candidates after the first
2 rounds of screening; NA-A1B2C10 showed poor protection in cellular assays, and 2,4-dichlorocinnamic hydroxamic acid, originally included simply for comparison, was cytotoxic in the cellular assays. The emergence of these 2 antagonists only after 3 rounds of screening highlights the importance of a multidisciplinary approach involving both in vivo and in vitro approaches in the development of therapies for botulism.

Fig. 1. Chemical structures of molecules that can inhibit botulinum neurotoxin serotype A in animal models.

Cyclic Peptides as Ligands of Cancer Cell-Surface Receptors

Currently available therapeutic agents for cancer often are not specific for cancer cells and are associated with severe side effects and distress for patients. Peptidic ligands of cellular receptors have gained attention for their ability to recognize specific receptors and thus penetrate cells to deliver a 'payload.' With this information in mind, we used whole-cell phage-panning technology to screen a library of cyclic peptides against 2 melanoma cell lines, the highly metastatic Me6652/4 and the low metastatic clone Me5552/56, to identify cyclic peptides with specificity for highly metastatic cells.

DNA sequencing of the peptides specific for melanoma cells in the panning revealed a cyclic 13mer peptide, Pep42, with a novel sequence (Fig. 2A). For further biological analysis, a synthetic analog of Pep42, which had the same activity as the originally isolated peptide, was designed and prepared for future conjugation with a desired payload. Using fluorescein-labeled Pep42 and confocal microscopy, we found that internalization of the peptide by the highly metastatic Me6652/4 cell line was more efficient than internalization by the control Me6652/56 cell line (Fig. 2B), validating our phage-panning approach.

Fig. 2. A, Structure of the cyclic peptide Pep42 with conjugated payload. B, Localization of Pep42 to highly metastatic cancer cells Me6652/4.

To elucidate the mechanism of Pep42 internalization, we constructed a Pep42 variant incorporating a photoaffinity label. Incubation of this peptide with cells revealed that the molecular target of Pep42 is glucose-regulated protein 78 (GRP78). Expression of GRP78 increases in response to stressful conditions, and upregulation of GRP78 has been correlated with an increase in malignancy of cancer cells, making this protein an attractive target as a drug delivery receptor. To demonstrate the usefulness of GRP78-targeted peptides, we conjugated Pep42 to the anticancer drug paclitaxel (Taxol) and tested the conjugate for the ability to induce apoptosis in melanoma cells. The Pep42-paclitaxel conjugate was 2-fold more potent than paclitaxel alone because of the efficient delivery of the drug to tumor cells by Pep42. Importantly, incubation of cells with Pep42 resulted in minimal cell death, paralleling the amount of death in the presence of growth medium alone. Taken together, these results establish Pep42 as a candidate for the construction of drug conjugates to deliver anticancer agents to malignant cells while reducing undesirable side effects.

Catalytic Antibodies Against Δ⁹-Tetrahydrocannabinol

Marijuana is the most commonly abused illicit drug in the United States and often leads to the abuse of other illicit drugs, as postulated by the 'cannabis gateway hypothesis.' Recently, it was shown that Δ⁹-tetrahydrocannabinol (Δ⁹-THC) increases opiate self-administration in mice by altering the endogenous opioid system, providing a molecular basis for the gateway hypothesis. Despite the dangers posed by marijuana abuse, currently no clinical treatments for abuse of this drug are available.

During the past several years, researchers in our laboratory have advanced a tactic known as immunopharmacotherapy to combat drugs of abuse. This technique hinges on the administration of an antibody to bind a drug before the drug reaches its cognate receptor. Using this strategy, we have developed approaches to treat cocaine, nicotine, and methamphetamine abuse. One drawback to this approach is the need for stoichiometric amounts of antibody to bind the target drug; alternatively, we envisioned a catalytic antibody capable of decomposing Δ⁹-THC as a promising approach for therapies that require substoichiometric amounts of antibody.

Despite the absence of any obvious candidate bonds for antibody-promoted degradation, we reasoned that the C9-C10 olefin would be susceptible to oxidation. Additionally, recent research has shown that antibodies have the intrinsic ability to perform oxidative degradations by generating reactive oxygen species from singlet oxygen.

We examined 2 previously generated panels of Δ⁹-THC–binding antibodies for the capacity to degrade Δ⁹-THC. In the presence of singlet oxygen, generated by riboflavin and sunlight, all tested antibodies oxidatively degraded Δ⁹-THC to form a single major product and several minor products. Exhaustive spectroscopic analyses indicated that the major product of the antibody-catalyzed reaction was cannabitriol, in which the C9-C10 olefin of Δ⁹-THC has essentially undergone dihydroxylation (Fig. 3). On the basis of the regiochemistry and stereochemistry of the product and our knowledge of antibody-catalyzed reactions, we have proposed a mechanism for this conversion that involves oxidation, epoxide opening, and elimination.

3. Antibody-catalyzed conversion of Δ9-THC to cannabitriol.

Pharmacologically, the cytotoxicity of cannabitriol is similar to that of Δ⁹-THC, but the addition of 2 hydroxyl groups leads us to expect that the increase in polarity will aid in the elimination of cannabitriol through natural metabolic pathways. These findings not only reveal the usefulness of a catalytic antibody–based therapy for drugs of abuse but also illustrate the inherent potential of all antibodies to catalyze complex chemical reactions.

Publications

Brogan, A.P., Dickerson, T.J., Janda, K.D. Catalytic antibodies: past, present, and future. In: Wiley Encyclopedia of Chemical Biology. Wiley, New York, in press.

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.

Brogan, A.P., Eubanks, L.M., Koob, G.F., Dickerson, T.J., Janda, K.D. Antibody-catalyzed oxidation of Δ⁹-tetrahydrocannabinol. J. Am. Chem. Soc. 129:3698, 2007.

De Lamo Marin, S., Xu, Y., Meijler, M.M., Janda, K.D. Antibody catalyzed hydrolysis of a quorum sensing signal found in gram-negative bacteria. Bioorg. Med. Chem. Lett. 17:1549, 2007.

Debler, E.W., Kaufmann, G.F., Kirchdoerfer, R.N., Mee, J.M., Janda, K.D., Wilson, I.A. Crystal structures of a quorum-quenching antibody. J. Mol. Biol. 368:1392, 2007.

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. ACS Chem. Biol. 2:359, 2007.

Eubanks, L.M., Dickerson, T.J., Janda, K.D. Technological advancements for the detection of and protection against biological and chemical warfare agents. Chem. Soc. Rev. 36:458, 2007.

Eubanks, L.M., Hixon, M.S., Jin, W., Hong, S., Clancy, C.M., Tepp, W.H., Baldwin, M.R., Malizio, C.J., Goodnough, M.C., Barbieri, J.T., Johnson, E.A., Boger, D.L., Dickerson, T.J., Janda, K.D. An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists. Proc. Natl. Acad. Sci. U. S. A. 104:2602, 2007.

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.

Kim, Y., Lillo, A.M., Steiniger, S.C., Liu, Y., Ballatore, 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.

Liu, Y., Steiniger, S.C., Kim, Y., Kaufmann, G.F., Felding-Habermann, B., Janda, K.D. Mechanistic studies of a peptidic GRP78 ligand for cancer cell-specific drug delivery. Mol. Pharm. 4:435, 2007.

McAllister, L.A., Hixon, M.S., Schwartz, R., Kubitz, D.S., Janda, K.D. Synthesis and application of a novel ligand for affinity chromatography based removal of endotoxin from antibodies. Bioconjug. Chem. 18:559, 2007.

McKenzie, K.M., Mee, J.M., Rogers, C.J., Hixon, M.S., Kaufmann, G.F., Janda, K.D. Identification and characterization of single chain anti-cocaine catalytic antibodies. J. Mol. Biol. 365:722, 2007.

Rogers, C.J., Eubanks, L.M., Dickerson, T.J., Janda, K.D. Unexpected acetylcholinesterase activity of cocaine esterases. J. Am. Chem. Soc. 128:15364, 2006.

Shigenaga, A., Moss, J.A., Janda, K.D. New synthetic methodology for the synthesis of cyclic depsipeptides employing acylphenyldiazene activation. In: Peptide Science 2005: Proceedings of the 42nd Japanese Peptide Symposium. Wakamiya, T. (Ed.). Protein Research Foundation, Osaska, Japan, 2006, p. 137.

Silvaggi, N.R., Boldt, G.E., Hixon, M.S., Kennedy, J.P., Tzipori, S., Janda, K.D., Allen, K.N. Structures of Clostridium botulinum neurotoxin serotype A light chain complexed with small-molecule inhibitors highlight active-site flexibility. Chem. Biol. 14:533, 2007.

Steiniger, S.C., Altobell, L.J. III, Zhou, B., Janda, K.D. Selection of human antibodies against cell surface-associated oligomeric anthrax protective antigen. Mol. Immunol. 44:2749, 2007.

Xu, Y., Hixon, M.S., Yamamoto, N., McAllister, L.A., Wentworth, A.D., Wentworth, P., Jr., Janda, K.D. Antibody-catalyzed anaerobic destruction of methamphetamine. Proc. Natl. Acad. Sci. U. S. A. 104:3681, 2007.

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.

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.