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Scientific Report 2008


Chemistry




A Merging of Chemistry and Biology


K.D. Janda, J. Ashley, K. Capková, S. De Lamo Marin, J. Denery, T. Dickerson, A. Di Mola, B. Ellis, L. Eubanks, K. Fukuchi, C. Hernandez, G. Kaufmann, C. Lowery, S. Mahajan, A. Mayorov, G. McElhaney, J. Mee, A. Moreno, Y. Nakai, A. Nguyen, A. Nunes, J. Park, A. Rohrbach, C. Saccavini, 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. Representative examples of our results are given for 3 research programs: inhibition of bacterial virulence via the disruption of bacterial communication, discovery of a link between drug abuse and cardiovascular disease, and selection and characterization of human neutralizing antibodies against Bacillus anthracis toxin.

Infection Control By Antibody-Mediated Interference With Bacterial Communication

The ability of microorganisms to coordinate their gene expression according to population density has been termed quorum sensing. This chemical exchange of information among single-cell organisms is mediated by secreted signaling molecules termed autoinducers. Important biological and clinical aspects of quorum sensing include the regulation of bacterial virulence factors and the formation of biofilms; hence, inhibition of signaling associated with quorum sensing could provide a promising new strategy for the attenuation of bacterial infections. Indeed, analogs of autoinducers have been used as small-molecule antagonists in several quorum-sensing circuits as a means of signaling interference. Alternatively, we have pioneered an antibody-based strategy to combat quorum sensing through disruption of signal transmission.

Recently, we applied our antibody-based technology to the interference of the quorum-sensing circuits of Staphylococcus aureus. This microorganism is the most common cause of hospital-acquired infections, including diseases ranging from skin infections and food poisoning to life-threatening nosocomial infections. The increasing resistance of S aureus isolates to glycopeptide antibiotics, most prominently vancomycin, is a major concern in intensive care units, and an alternative strategy to combat this pathogen is urgently required.

Staphylococcus aureus uses a set of 4 cyclic autoinducing peptides (AIP-1—AIP-4) to regulate its quorum-sensing machinery, which is responsible for orchestrating the expression of virulence genes. Thus, inhibition of the S aureus system would result in decreased pathogenicity. We generated a monoclonal antibody, AP4-24H11, to sequester AIP-4 (Fig. 1). This antibody was elicited against a rationally designed hapten (AP4, Fig. 1) and efficiently interfered with the quorum sensing of S aureus in vitro, as determined by real-time polymerase chain reaction analysis and inactivation of AP4-24H11 by synthetic AIP-4. Importantly, AP4-24H11 suppressed both S aureus—induced dermal injury in a mouse model of abscess formation in vivo and provided complete protection against a lethal S aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy as treatment of bacterial infections in which quorum sensing controls the expression of virulence factors.
Fig. 1. Structure of the S aureus autoinducer AIP-4 and AP4 hapten used to generate the quorum-quenching antibody AP4-24H11.


A Link Between Chronic Methamphetamine Use and Cardiovascular Disease

The rapid spread of methamphetamine abuse across the United States is as alarming as the propensity of the drug to induce severe addiction and the health-related consequences of addiction. Whereas before 2001 methamphetamine use occurred predominantly in the western United States, its use now is extending rapidly throughout the United States and across different ethnic groups. The threat that methamphetamine now poses to society underscores the need to more thoroughly examine the ramifications of chronic methamphetamine abuse.

In addition to causing severe dopaminergic neurotoxic effects, chronic methamphetamine self-administration induces increasing drug tolerance that correlates with escalating intake. Although the molecular mechanism behind pharmacologic tolerance is not fully elucidated, we hypothesized that methamphetamine covalently modifies endogenous proteins in a process known as glycation (Fig. 2) before reaching the brain and mediating its well-characterized stimulant effects. Glycation reactions, collectively termed the Maillard reaction, have been studied for decades in the food industry in the development of flavor and color; however, Maillard products can also assume a biologically hazardous role when synthesized in vivo. Acquiring the ability to cross-link proteins, these irreversible reaction products, termed advanced glycation end products (AGEs), have gained notoriety for their participation in a range of pathologic changes.
Fig. 2. Reaction scheme of methamphetamine protein glycation as initiated by glucose and methamphetamine.


Protein glycation by methamphetamine induces an immune response against these modified proteins, which could lead to sequestration of drug and, ultimately, the development of tolerance. We have shown that this drug-dependent glycation mechanism is operative in vivo. We detected antibodies against methamphetamine-derived AGEs in rats that chronically self-administered the drug, and we noted a direct relationship between the level of methamphetamine intake and the respective antibody titers against methamphetamine-glycated proteins.

Additionally, we detected increased levels of proinflammatory and other cytokine molecules, particularly vascular endothelial growth factor. AGE-associated upregulation of this growth factor has been associated with the onset of heart disease, but these effects had not been previously associated with methamphetamine-derived AGEs. Because AGEs can alter protein function in vivo and participate in various diseases, methamphetamine-derived AGEs provide an unrecognized molecular mechanism for the development of vasculitis and other cardiovascular maladies with high incidence in chronic methamphetamine users.

Human Neutralizing Antibodies Against Anthrax Toxin

A less-than-adequate therapeutic plan for the treatment of anthrax in the 2001 bioterrorism attacks has highlighted the importance of developing alternative or complementary therapeutic approaches for biothreat agents. Vaccination against B anthracis for protection against anthrax has been known for more than a century. However, the prolonged vaccination schedules and induction times required for an immune response are serious drawbacks, because the therapeutic window for treatment of anyone exposed to a deliberate release of
B anthracis is limited. Alternatively, recently developed antibiotic prophylaxis for the treatment of persons exposed to anthrax, although important, would also be of lesser value if the infection were caused by an antibiotic-resistant strain.

Passive immunization has provided an attractive avenue as a treatment both before and after exposure to B anthracis. Indeed, in many animal studies, passive transfer of antiserum successfully provided protection against anthrax. Furthermore, passive immunization could have advantages over active vaccination and antibiotic treatments via few toxic effects, high specificity, the capability for stockpiling large quantities of the antiserum, and immediate protection against a biological attack.

Using a phage-displayed human single-chain variable fragment (scFv) antibody library, we selected and characterized several human monoclonal neutralizing antibodies against the toxin of B anthracis. In total, 15 clones with distinct sequences and high specificity for the protective antigen region of the anthrax toxin (Fig. 3) were analyzed by using biophysical and cell-based cytotoxicity assays. From this panel of antibodies, a set of neutralizing antibodies was identified, and the potency of protection was established by using a macrophage cytotoxicity assay. Among the neutralizing antibodies identified, 1 clone had excellent affinity for the protective antigen region of the anthrax toxin and provided superior protection from lethal toxin in the cell cytotoxicity assay. Our results add to the ever-growing arsenal of immunologic and functional analysis of monoclonal antibodies to the exotoxins of anthrax. In addition, the antibodies may be new candidates for prophylactic and therapeutic agents.
Fig. 3. Targeting of the protective antigen (PA) region of B anthracis toxin by human monoclonal antibodies.


Publications

Brogan, A.P., Dickerson, T.J., Janda, K.D. Nornicotine-organocatalyzed aqueous reduction of α ,β -unsaturated aldehydes. Chem. Commun. (Camb.) Issue 46:4952, 2007.

Capková, K., Yoneda, Y., Dickerson, T.J., Janda, K.D. Synthesis and structure-activity relationships of second-generation hydroxamate botulinum neurotoxin A protease inhibitors. Bioorg. Med. Chem. Lett. 17:6463, 2007.

Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcic, G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B., Lerner, R.A. Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex. Science 319:1232, 2008.

Dickerson, T.J., McKenzie, K.M., Hoyt, A.S., Wood, M.R., Janda, K.D., Brenner, S., Lerner, R.A. Phage escape libraries for checkmate analysis. Proc. Natl. Acad. Sci. U. S. A. 104:12703, 2007.

Ino, A., Dickerson, T.J., Janda, K.D. Positional linker effects in haptens for cocaine immunopharmacotherapy. Bioorg. Med. Chem. Lett. 17:4280, 2007.

Kaufmann, G.F., Park, J., Janda, K.D. Bacterial quorum sensing: a new target for anti-infective immunotherapy. Expert Opin. Biol. Ther. 8:719, 2008.

Kaufmann, G.F., Park, J., Mee, J.M., Ulevitch, R.J., Janda, K.D. The quorum quenching antibody RS2-1G9 protects macrophages from the cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone. Mol. Immunol. 45:2710, 2008.

Lowery, C.A., Dickerson, T.J., Janda, K.D. Interspecies and interkingdom communication mediated by bacterial quorum sensing. Chem. Soc. Rev. 37:1337, 2008.

Park, J., Jagasia, R., Kaufmann, G.F., Mathison, J.C., Ruiz, D.I., Moss, J.A., Meijler, M.M., Ulevitch, R.J., Janda, K.D. Infection control by antibody disruption of bacterial quorum sensing signaling. Chem. Biol. 14:1119, 2007.

Park, J., Kaufmann, G.F., Bowen, J.P., Arbiser, J.L., Janda, K.D. Solenopsin A, a venom alkaloid from the fire ant Solenopsis invicta, inhibits quorum-sensing signaling in Pseudomonas aeruginosa. J. Infect. Dis. 198:1198, 2008.

Richardson, H.N., Zhao, Y., Fekete, E.M., Funk, C.K., Wirsching, P., Janda, K.D., Zorrilla, E.P., Koob, G.F. MPZP: a novel small molecule corticotropin-releasing factor type 1 receptor (CRF1) antagonist. Pharmacol. Biochem. Behav. 88:497, 2008.

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.

Willis, B., Eubanks, L.M., Dickerson, T.J., Janda, K.D. The strange case of the botulinum neurotoxin: using chemistry and biology to modulate the most deadly poison. Angew. Chem. Int. Ed. 47:8360, 2008.

Willis, B., Eubanks, L.M., Wood, M.R., Janda, K.D., Dickerson, T.J., Lerner, R.A. Biologically templated organic polymers with nanoscale order. Proc. Natl. Acad. Sci. U. S. A. 105:1416, 2008.

Xu, Y., Hixon, M.S., Dawson, P.E., Janda, K.D. Development of a FRET assay for monitoring of HIV gp41 core disruption. J. Org. Chem. 72:6700, 2007.

Yoneda, Y., Steiniger, S.C., Capkova, K., Mee, J.M., Liu, Y., Kaufmann, G.F., Janda, K.D. A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy. Bioorg. Med. Chem. Lett. 18:1632, 2008.

Zarebski, L.M., Vaughan, K., Sidney, J., Peters, B., Grey, H., Janda, K.D., Casadevall, A., Sette, A. Analysis of epitope information related to Bacillus anthracis and Clostridium botulinum. Expert Rev. Vaccines 7:55, 2008.

Zhou, B., Carney, C., Janda, K.D. Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin. Bioorg. Med. Chem. 16:1903, 2008.

Zhou, B., Pellett, S., Tepp, W.H., Zhou, H., Johnson, E.A., Janda, K.D. Delineating the susceptibility of botulinum neurotoxins to denaturation through thermal effects. FEBS Lett. 582:1526, 2008.

Zhou, H., Zhou, B., Ma, H., Carney, C., Janda, K.D. Selection and characterization of human monoclonal antibodies against Abrin by phage display. Bioorg. Med. Chem. Lett. 17:5690, 2007.

 

Kim D. Janda, Ph.D.
Professor

Andrew Bin Zhou, Ph.D.
Assistant Professor



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