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1.
J Biol Chem ; 286(19): 17351-8, 2011 May 13.
Article in English | MEDLINE | ID: mdl-21454495

ABSTRACT

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using "quorum sensing," which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key Glu(H95) provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11·AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.


Subject(s)
Ligands , Quorum Sensing/genetics , Staphylococcus aureus/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Crystallography, X-Ray/methods , Gene Expression Regulation, Bacterial , Immunoglobulin Fragments/chemistry , Immunoglobulin G/chemistry , Luminescent Proteins/metabolism , Models, Molecular , Peptides/chemistry , Protein Binding , Protein Interaction Mapping , Quorum Sensing/immunology , Signal Transduction
2.
Science ; 319(5867): 1232-5, 2008 Feb 29.
Article in English | MEDLINE | ID: mdl-18309081

ABSTRACT

The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedented ability to produce bright luminescence and has been used as a biosensor in various applications. We show that the prolonged luminescence is not stilbene fluorescence. Instead, the emissive species is a charge-transfer excited complex of an anionic stilbene and a cationic, parallel pi-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody.


Subject(s)
Antibodies, Monoclonal/chemistry , Electrons , Immunoglobulin Variable Region/chemistry , Stilbenes/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex , Binding Sites, Antibody , Crystallization , Crystallography, X-Ray , Fluorescence , Fluorescence Polarization , Haptens/chemistry , Haptens/immunology , Hydrophobic and Hydrophilic Interactions , Immunoglobulin Variable Region/immunology , Ligands , Luminescence , Mutation , Oxidation-Reduction , Protein Structure, Tertiary , Spectrometry, Fluorescence , Spectrum Analysis , Stilbenes/immunology , Tryptophan/chemistry
3.
Bioorg Med Chem Lett ; 18(5): 1632-6, 2008 Mar 01.
Article in English | MEDLINE | ID: mdl-18243696

ABSTRACT

Tumor targeting peptides are promising vehicles for site-directed cancer therapy. Pep42, a cyclic 13-mer oligopeptide that specifically binds to glucose-regulated protein 78 (GRP78) and internalized into cancer cells, represents an excellent vehicle for tumor cell-specific chemotherapy. Here, we report the synthesis and evaluation of Pep42-prodrug conjugates that contain a cathepsin B-cleavable linker, resulting in the traceless release of drug inside the cancer cells.


Subject(s)
Heat-Shock Proteins/chemistry , Molecular Chaperones/chemistry , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Endoplasmic Reticulum Chaperone BiP , Humans , Ligands , Molecular Structure
4.
Mol Immunol ; 45(9): 2710-4, 2008 May.
Article in English | MEDLINE | ID: mdl-18304641

ABSTRACT

The Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, uses acyl-homoserine lactone-based quorum sensing systems to control its pathogenicity. One of its quorum sensing factors, N-3-oxo-dodecanoyl-homoserine lactone, has been shown not only to mediate bacterial quorum sensing but also to exert cytotoxic effects on mammalian cells. The monoclonal antibody RS2-1G9 generated against a 3-oxo-dodecanoyl-homoserine lactone analogue hapten was able to protect murine bone marrow-derived macrophages from the cytotoxic effects and also prevented the activation of the mitogen-activated protein kinase p38. These data demonstrate that an immunopharmacotherapeutic approach to combat P. aeruginosa infections might be a viable therapeutic option as the monoclonal antibody RS2-1G9 can readily sequester bacterial N-3-oxo-dodecanoyl-homoserine lactone molecules, thus interfering with their biological effects in prokaryotic and eukaryotic systems.


Subject(s)
4-Butyrolactone/analogs & derivatives , Antibodies, Monoclonal/immunology , Homoserine/analogs & derivatives , Macrophages/immunology , Pseudomonas aeruginosa/physiology , Quorum Sensing/physiology , 4-Butyrolactone/immunology , 4-Butyrolactone/metabolism , Animals , Cell Line , Cells, Cultured , Enzyme Activation , Haptens/immunology , Homoserine/immunology , Homoserine/metabolism , Macrophages/cytology , Macrophages/metabolism , Mice , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Pseudomonas aeruginosa/immunology , p38 Mitogen-Activated Protein Kinases/metabolism
5.
J Mol Biol ; 368(5): 1392-402, 2007 May 18.
Article in English | MEDLINE | ID: mdl-17400249

ABSTRACT

A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing.


Subject(s)
4-Butyrolactone/analogs & derivatives , Antibodies, Monoclonal/chemistry , Protein Structure, Tertiary , Pseudomonas aeruginosa/physiology , Signal Transduction/physiology , 4-Butyrolactone/chemistry , 4-Butyrolactone/metabolism , Antibodies, Monoclonal/metabolism , Binding Sites , Crystallography, X-Ray , Ethylene Glycol/chemistry , Ethylene Glycol/metabolism , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Models, Molecular , Molecular Structure , Protein Engineering
6.
J Mol Biol ; 365(3): 722-31, 2007 Jan 19.
Article in English | MEDLINE | ID: mdl-17084858

ABSTRACT

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.


Subject(s)
Antibodies, Catalytic/immunology , Cocaine/immunology , Alanine/genetics , Amino Acid Sequence , Animals , Antibodies, Catalytic/chemistry , Cocaine/chemistry , Cocaine/metabolism , Complementarity Determining Regions/chemistry , DNA Mutational Analysis , Esters/chemistry , Esters/metabolism , Hydrolysis , Kinetics , Mice , Molecular Sequence Data , Mutant Proteins/immunology , Mutation/genetics , Sequence Alignment
7.
J Biol Chem ; 281(39): 28822-30, 2006 Sep 29.
Article in English | MEDLINE | ID: mdl-16893899

ABSTRACT

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). Here we have shown that p38 and eIF2alpha phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.


Subject(s)
4-Butyrolactone/analogs & derivatives , Bone Marrow Cells/microbiology , Gene Expression Regulation , Macrophages/microbiology , 4-Butyrolactone/chemistry , 4-Butyrolactone/physiology , Amino Acid Motifs , Animals , Bone Marrow Cells/cytology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Pseudomonas aeruginosa/metabolism , RNA, Viral/metabolism , Signal Transduction
8.
J Am Chem Soc ; 128(9): 2802-3, 2006 Mar 08.
Article in English | MEDLINE | ID: mdl-16506750

ABSTRACT

Many bacterial pathogens coordinate their virulence factor expression in a cell density-dependent manner. This population-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). N-Acyl homoserine lactones (AHLs) are used by over 70 Gram-negative bacterial species as autoinducers. Inhibition of QS signaling might represent a new target for antimicrobial therapy. Here we report the hapten design, synthesis, generation of monoclonal antibodies (mAbs) against AHLs, and the evaluation of these mAbs for their ability to blunt QS signaling and inhibit virulence factor expression in P. aeruginosa. The mAbs can be envisioned as a tool for future investigations into AHL-based QS, which may aid in gaining new insights into the pathogenesis of P. aeruginosa and may ultimately lead to the development of new strategies to combat bacterial diseases.


Subject(s)
4-Butyrolactone/analogs & derivatives , Antibodies, Monoclonal/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Haptens/pharmacology , 4-Butyrolactone/antagonists & inhibitors , 4-Butyrolactone/immunology , 4-Butyrolactone/physiology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibody Specificity , Gene Expression Regulation, Bacterial/physiology , Haptens/chemistry , Haptens/immunology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/immunology
9.
Structure ; 14(2): 205-16, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16472740

ABSTRACT

Antibody 7A1 hydrolyzes cocaine to produce nonpsychoactive metabolites ecgonine methyl ester and benzoic acid. Crystal structures of 7A1 Fab' and six complexes with substrate cocaine, the transition state analog, products ecgonine methyl ester and benzoic acid together and individually, as well as heptaethylene glycol have been analyzed at 1.5-2.3 angstroms resolution. Here, we present snapshots of the complete cycle of the cocaine hydrolytic reaction at atomic resolution. Significant structural rearrangements occur along the reaction pathway, but they are generally limited to the binding site, including the ligands themselves. Several interacting side chains either change their rotamers or alter their mobility to accommodate the different reaction steps. CDR loop movements (up to 2.3 angstroms) and substantial side chain rearrangements (up to 9 angstroms) alter the shape and size (approximately 320-500 angstroms3) of the antibody active site from "open" to "closed" to "open" for the substrate, transition state, and product states, respectively.


Subject(s)
Antibodies, Catalytic/chemistry , Cocaine/chemistry , Models, Molecular , Antibodies, Catalytic/metabolism , Benzoic Acid/chemistry , Binding Sites, Antibody , Catalysis , Cocaine/analogs & derivatives , Cocaine/immunology , Cocaine/metabolism , Crystallography , Ethylene Glycols/chemistry , Immunoglobulin Fab Fragments/chemistry
10.
J Am Chem Soc ; 127(28): 10016-7, 2005 Jul 20.
Article in English | MEDLINE | ID: mdl-16011362

ABSTRACT

Cocaine is among the most reinforcing of all drugs of abuse, yet no effective pharmacotherapy is available. Herein, we report the development and characterization of phage-displayed cocaine esterases with pharmacologically relevant kinetic parameters (kcat/Km approximately 104 M-1 s-1).


Subject(s)
Bacteriophages/chemistry , Bacteriophages/enzymology , Carboxylic Ester Hydrolases/biosynthesis , Carboxylic Ester Hydrolases/chemistry , Cocaine/chemistry , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacteriophages/genetics , Carboxylic Ester Hydrolases/genetics , Escherichia coli/genetics , Hydrolysis , Kinetics
12.
J Am Chem Soc ; 127(8): 2477-84, 2005 Mar 02.
Article in English | MEDLINE | ID: mdl-15725002

ABSTRACT

Cocaine is a highly addictive drug, and despite intensive efforts, effective therapies for cocaine craving and addiction remain elusive. In recent years, we and others have reported advances in anti-cocaine immunopharmacotherapy based on specific antibodies capable of sequestering the drug before it reaches the brain. In an effort to obtain high affinity therapeutic anti-cocaine antibodies, either whole IgGs or other antibody constructs, fluorescence spectroscopic techniques could provide a means of assisting selection and engineering strategies. We report the synthesis of a series of cocaine-fluorophore conjugates (GNC-F1, GNC-F2, GNC-I) and the functional evaluation of these compounds against single-chain Fv antibodies obtained via crystallographic analysis/engineering and against commercially available anti-cocaine monoclonal antibodies with a wide range of cocaine-binding affinities. From these studies, we determined that the GNC-F2 fluorophore reproduced affinity constants obtained using [(3)H]-labeled cocaine. We anticipate that the readily synthesized and nonradioactive GNC-F2 will find use both as a tool for bioimaging and in the high-throughput selection and engineering of potential therapeutic antibodies against cocaine.


Subject(s)
Antibodies, Monoclonal/chemistry , Cocaine/analogs & derivatives , Fluorescent Dyes/chemical synthesis , Immunoconjugates/chemistry , Immunoglobulin Fragments/chemistry , Animals , Antibodies, Monoclonal/immunology , Base Sequence , Cocaine/chemistry , Cocaine/immunology , Humans , Immunoconjugates/immunology , Immunoglobulin Fragments/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Kinetics , Mice , Models, Molecular , Molecular Sequence Data , Peptide Library , Protein Engineering , Spectrometry, Fluorescence
13.
Proc Natl Acad Sci U S A ; 101(28): 10416-21, 2004 Jul 13.
Article in English | MEDLINE | ID: mdl-15226496

ABSTRACT

Cocaine addiction continues to be a major health and social problem in the United States and other countries. Currently used pharmacological agents for treating cocaine abuse have proved inadequate, leaving few treatment options. An alternative is to use protein-based therapeutics that can eliminate the load of cocaine, thereby attenuating its effects. This approach is especially attractive because the therapeutic agents exert no pharmacodynamic action of their own and therefore have little potential for side effects. The effectiveness of these agents, however, is limited by their inability to act directly within the CNS. Bacteriophage have the capacity to penetrate the CNS when administered intranasally. Here, a method is presented for engineering filamentous bacteriophage to display cocaine-binding proteins on its surface that sequester cocaine in the brain. These antibody-displaying constructs were examined by using a locomotor activity rodent model to assess the ability of the phage-displayed proteins to block the psychoactive effects of cocaine. Results presented demonstrate a strategy in the continuing efforts to find effective treatments for cocaine addiction and suggest the application of this protein-based treatment for other drug abuse syndromes.


Subject(s)
Cocaine-Related Disorders/therapy , Inovirus , Administration, Intranasal , Animals , Brain/virology , Cocaine/metabolism , Dopamine Uptake Inhibitors/metabolism , Inovirus/metabolism , Male , Psychomotor Agitation , Rats , Rats, Wistar
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