Scientific Report 2008
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.
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
|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.
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
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
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
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.
Targeting of the protective antigen (PA) region of B anthracis toxin by human
Brogan, A.P., Dickerson, T.J., Janda,
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,
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,
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.