To further examine the catalytic activity of XYD-11G2, we measured the ability of the hapten shown in Figure 1 to inhibit XYD-11G2–mediated hydrolysis of the homoserine lactone. The hapten inhibited the enzymatic activity of XYD-11G2, and kinetic analysis confirmed the hapten's moderate catalytic efficiency. To examine the potential of XYD-11G2 to disrupt AHL-based quorum sensing, we assayed cultures of P aeruginosa to determine concentrations of the virulence factor pyocyanin, whose production by this bacterium is regulated by AHL-based signaling. When applied to cultures of P aeruginosa for 12 hours, XYD-11G2 did not alter growth of the bacteria but did suppress synthesis of pyocyanin. In conclusion, using our reactive immunization strategy with the squaric monoester monoamide hapten (Fig. 1), we successfully generated catalytic antibodies specific to AHLs, and these antibodies were able to curtail bacterial infectivity by disrupting quorum sensing.
Molecular Link Between the Active Component of Marijuana and Pathologic Changes in Alzheimer's Disease
Alzheimer's disease is the leading cause of dementia among the elderly, and cases of this disease are expected to triple during the next 50 years as the baby boomer population matures. Consequently, development of treatments that slow or halt the disease progression have become imperative to both improve the quality of life for patients and reduce the health care costs attributable to Alzheimer's disease.
The primary neuropathologic features of Alzheimer's disease include accumulation of abnormally hyperphosphorylated tau to form intracellular neurofibrillary tangles, degeneration of cholinergic neurons in the basal forebrain, and deposition of amyloid β-peptide (Aβ) into amyloid plaques in areas of the brain important for memory and cognition. However, the exact factors that give rise to these neuropathologic changes and the extent to which these features are related remain elusive. As a result, current pharmacotherapy provides symptomatic relief; acetylcholinesterase inhibitors provide the greatest benefit.
Inhibition of acetylcholinesterase appears to address both the cholinergic hypothesis and the amyloid hypothesis for neurodegeneration in Alzheimer's disease. Whereas acetylcholinesterase inhibitors improve cholinergic transmission by increasing the synaptic content of acetylcholine, the inhibitors also seem to stabilize cognitive function by affecting the processing and deposition of Aβ. This latter function may be related to the ability of acetylcholinesterase inhibitors to block the peripheral anionic site (PAS) of the enzyme rather than bind the catalytic triad of the active site. Thus, research continues on the discovery and mechanism of action of novel drugs that mediate acetylcholinesterase inhibition by interacting with key residues of either site.
We hypothesized that Δ9-tetrahydrocannabinol (Δ9-THC), the active component of marijuana, was an ideal candidate for binding the allosteric PAS of acetylcholinesterase because of the lipophilic nature and tricyclic structure of the active component. This theory arose from our previous studies of compounds whose rigid, aromatic scaffolds enabled them to disrupt protein-protein interactions. By modeling Δ9-THC binding to acetylcholinesterase in silico in collaboration with A.J. Olson, Scripps Research, we determined that the ABC-fused ring of the Δ9-THC scaffold could interact with the tryptophan 86 indole side chain and backbone carbonyls phenylalanine 123 and serine 125 of acetylcholinesterase. Although Δ9-THC competitively inhibited the catalytic activity of acetylcholinesterase only moderately (Ki = 10.2 μM), the potential for Δ9-THC to block substrate entry into the active site of the catalytic triad and thereby inhibit acetylcholinesterase activity is enhanced by its superior binding affinity to PAS in comparison with known PAS binders. In addition, we found that Δ9-THC not only completely blocked the effect of acetylcholinesterase on Aβ aggregation but also had significantly greater potency than propidium, one of the most effective aggregation inhibitors reported to date (Fig. 2).
In total, we have shown that Δ9-THC competitively inhibits acetylcholinesterase and prevents acetylcholinesterase-induced aggregation of Aβ. Although the psychoactive properties of Δ9-THC preclude its application as drug for treatment of Alzheimer's disease, its superior efficacy in inhibiting aggregation of Aβ, as compared with medications approved by the Food and Drug Administration, support study of Δ9-THC analogs as drug targets for the treatment of the signs, symptoms, and progression of Alzheimer's disease.
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., 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.
Dickerson, T.J., McKenzie, K.M, Hoyt, A.S., Wood, M.R., Janda, K.D., Brenner, S.B., Lerner, R.A. Phage escape libraries for checkmate analysis. Proc. Natl. Acad. Sci. U. S. A. 104:12703, 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.
Ino, A., Dickerson, T.J., Janda, K.D. Positional linker effects in haptens for cocaine immunopharmacotherapy. Bioorg. Med. Chem. Lett. 17:4280, 2007.
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, 2006.
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.
Treweek, J., Wee, S., Koob, G.F., Dickerson, T.J., Janda, K.D. Self-vaccination by methamphetamine glycation products chemically links chronic drug abuse and cardiovascular disease. Proc. Natl. Acad. Sci. U. S. A. 104:11580, 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.