Search PubMed⌕ Search

Biomedical subjects

Kim D Janda

Publications and source records attributed to Kim D Janda.

At least 19 recordsLinked to original sources
PubMed2007

Selection of human antibodies against cell surface-associated oligomeric anthrax protective antigen.

The protective antigen (PA(83)) of Bacillus anthracis is the dominant antigen in natural and vaccine-induced immunity to anthrax infection. Three human single-chain variable fragments (scFvs) against cell bound PA were isolated from an antibody phage display library. Specifically, the antibodies were evaluated for their ability to bind to cell bound heptameric PA and ultimately protect against the cytotoxicity of lethal toxin. In total, all three scFvs possessed neutralizing activity against the cytotoxic effects of lethal toxin in a macrophage lysis assay. The K(d) values of the Fabs were determined, interestingly their protective effects did not parallel their affinities; hence, a simple binding argument alone to PA(63) cannot be used as the distinguishing feature for the prediction of their neutralization abilities. Immunofluorescent microscopy experiments were conducted and provided strong evidence for Fab binding to oligomeric PA on the cell surface and thus a plausible mechanism for the toxin neutralization activity that was observed. The results of this study presented herein suggest that our antibodies compete with LF-PA cell surface interactions, and thus may provide potential application of human antibodies as passive immunization prophylactics in cases of B. anthracis exposure and infection.

Amino Acid Sequence↗
PubMed2006

Unexpected acetylcholinesterase activity of cocaine esterases.

A series of proteins with cocaine esterase ability have been shown to hydrolyze acetylcholine with similar rate enhancements. Docking studies revealed that acetylcholine binds in a similar orientation as cocaine. These results suggest that acetylcholinesterase activity may be inherent to cocaine esterases.

Acetylcholinesterase↗
PubMed2006

Identification and characterization of single chain anti-cocaine catalytic antibodies.

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.

Alanine↗
PubMed2006

Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand.

Peptidic ligands can be used for specific cell targeting and the delivery of payloads into the target cell. Here we describe the screening of a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cell line Me6652/4. This strategy resulted in the selection of the cyclic 13-mer Pep42, CTVALPGGYVRVC, which showed preferential internalization into melanoma cell line Me6652/4 versus the reference cell line Me6652/56. This translocation is a receptor-mediated process that does not require electrostatic interactions nor does it involve transfer to the lysosomal compartment. The cellular receptor for Pep42 was identified as the surface membrane form of glucose-regulated protein 78 (GRP78), a member of the heat shock protein family and a marker on malignant cancer cells. The cellular uptake and intracellular trafficking of Pep42-Quantum Dot conjugates was monitored by confocal laser microscopy, and colocalization within the endoplasmic reticulum was observed. The uptake of Pep42 could be blocked by a monoclonal antibody against the identified receptor. Furthermore, Pep42 was shown to target specifically GRP78-expressing cancer cells. The in vitro cytotoxicity of a Pep42-Taxol conjugate was evaluated by flow cytometry wherein the conjugate was shown to induce apoptosis and was more effective in promoting programmed cell death in Me6652/4 cells. In summary, the data presented suggest that cyclic peptide Pep42 might be a powerful tool in the construction of drug conjugates designed to selectively kill malignant cancer cells.

Amino Acid Sequence↗
PubMed2006

N-(3-oxo-acyl)homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways.

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.

4-Butyrolactone↗
PubMed2006

Vaccination against weight gain.

Obesity endangers the lives of millions of people worldwide, through comorbidities such as heart disease, cancers, type 2 diabetes, stroke, arthritis, and major depression. New approaches to control body weight remain a high priority. Vaccines traditionally have been used to protect against infectious diseases and, more recently, for unconventional targets such as drug addiction. Methodologies that could specifically modulate the bioavailability of an endogenous molecule that regulates energy balance might provide a new foundation for treating obesity. Here we show that active vaccination of mature rats with ghrelin immunoconjugates decreases feed efficiency, relative adiposity, and body weight gain in relation to the immune response elicited against ghrelin in its active, acylated form. Three active vaccines based on the 28-aa residue sequence of ghrelin, a gastric endocrine hormone, were used to immunize adult male Wistar rats (n = 17). Synthetic ghrelin analogs were prepared that spanned residues 1-10 [ghrelin (1-10) Ser-3(butanoyl) hapten, Ghr1], 13-28 [ghrelin (13-28) hapten, Ghr2], and 1-28 [ghrelin(1-28) Ser-3(butanoyl) hapten, Ghr3], and included n-butanoyl esters at Ser-3. Groups immunized with Ghr1 or Ghr3 showed greater and more selective plasma binding capacity for the active, Ser-3-(n-octanoyl) form of ghrelin as compared with Ghr2 or keyhole limpet hemocyanin vaccinated controls. Accordingly, they gained less body weight, with sparing of lean mass and preferential reduction of body fat, consistent with reduced circulating leptin levels. The ratio of brain/serum ghrelin levels was lower in rats with strong anti-ghrelin immune responses. Effects were not attributable to nonspecific inflammatory responses. Vaccination against the endogenous hormone ghrelin can slow weight gain in rats by decreasing feed efficiency.

Amino Acid Sequence↗
PubMed2006

Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate.

Botulinum neurotoxin serotype A (BoNT/A, 1296 residues) is a zinc metalloprotease that cleaves SNAP25 to inhibit the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNT/A is a disulfide-linked di-chain protein composed of an N-terminal, thermolysin-like metalloprotease light chain domain (LC/A, 448 residues) and a C-terminal heavy chain domain (848 residues) that can be divided into two subdomains, a translocation subdomain and a receptor binding subdomain. LC/A cleaves SNAP25 between residues Gln197-Arg198 and, unlike thermolysin, recognizes an extended region of SNAP25 for cleavage. The structure of a recombinant LC/A (1-425) treated with EDTA (No-Zn LC/A) was determined. The overall structure of No-Zn LC/A is similar to that reported for the holotoxin, except that it lacks the Zn ion, indicating that the role of Zn is catalytic not structural. In addition, structures of a noncatalytic mutant LC/A (Arg362Ala/Tyr365Phe) complexed with and without an inhibitor, ArgHX, were determined. The overall structure and the active site conformation for the mutant are the same as wild type. When the inhibitor binds to the active site, the carbonyl and N-hydroxyl groups form a bidentate ligand to the Zn ion and the arginine moiety binds to Asp369, suggesting that the inhibitor-bound structure mimics a catalytic intermediate with the Arg moiety binding at the P1' site. Consistent with this model, mutation of Asp369 to Ala decreases the catalytic activity of LC/A by approximately 600-fold, and the residual activity is not inhibited by ArgHX. These results provide new information on the reaction mechanism and insight into the development of strategies for small molecule inhibitors of BoNTs.

Amino Acid Sequence↗
PubMed2006

The use of small molecules to investigate molecular mechanisms and therapeutic targets for treatment of botulinum neurotoxin A intoxication.

Botulinum neurotoxins (BoNTs) are agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and subsequent flaccid paralysis. The potent paralytic ability of these toxins has resulted in their use as a therapeutic; however, BoNTs are also classified by the Centers for Disease Control and Prevention as one of the six highest-risk threat agents of bioterrorism. Consequently, a thorough understanding of the molecular mechanism of BoNT toxicity is crucial before effective inhibitors and, ultimately, an approved drug can be developed. In this article, we systematically detail BoNT intoxication by examining each of the discrete steps in this process. Additionally, rationally designed strategies for combating the toxicity of the most potent BoNT serotype are evaluated.

Animals↗
PubMed2006

Identification of a potent botulinum neurotoxin a protease inhibitor using in situ lead identification chemistry.

[reaction: see text] Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. By using in situ lead identification chemistry, we have uncovered the first class of inhibitors that displays nanomolar potency. From a 15 microM lead compound, structure-activity relationship studies were performed granting the most potent BoNT/A inhibitor reported to date that displays an inhibition constant of 300 nM.

Botulinum Toxins, Type A↗
PubMed2006

Superactivation of the botulinum neurotoxin serotype A light chain metalloprotease: a new wrinkle in botulinum neurotoxin.

Small molecules based upon a 2-acylguanidine-5-phenyl thiophene scaffold that can activate the light chain metalloprotease of botulinum neurotoxin serotype A (BoNT LC/A) by an apparent reduction in Km are reported. On the basis of structure-activity relationships and the activation profile, one or more molecules of activator specifically bind to a defined site on the toxin, causing the observed rate enhancement. With the ever-growing clinical uses of BoNT, compounds such as those reported here may provide a method for combating the emerging adaptive immune responses to BoNT.

Botulinum Toxins, Type A↗
PubMed2006

A cyclic peptide-polymer probe for the detection of Clostridium botulinum neurotoxin serotype A.

A Botulinum neurotoxin serotype A (BoNT/A) ELISA detection system was developed based upon an 11-mer cyclic peptide, termed C11-019, that was identified through peptide phage display technology. The assay employs a sandwich format using the C11-019 cyclic peptide attached to a PEMA (poly(ethylene maleic anhydride)) matrix as the capture phase and anti-BoNT/A polyclonal antibodies as the detection phase. Results reported demonstrate that the C11-019 peptide-polymer can specifically bind to BoNT/A with no cross-reactivity to other serotypes examined in assay buffers and a variety of body fluids and foodstuffs. When a highly sensitive chemiluminescent substrate was engaged, the detection of 1 pg/mL could be readily achieved within 3h with a linear range of 0.1-1 ng/mL. These results demonstrate that an inexpensive peptide-polymer-based capture ELISA system can be used for rapid, sensitive and highly specific BoNT detection.

Animals↗
PubMed2006

Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing.

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.

4-Butyrolactone↗
PubMed2006

Targeting cell-impermeable prodrug activation to tumor microenvironment eradicates multiple drug-resistant neoplasms.

The tumor microenvironment is notably enriched with a broad spectrum of proteases. The proteolytic specificities of peptide substrates provide modular chemical tools for the rational design of cell-impermeable prodrugs that are specifically activated by proteases extracellularly in the tumor microenvironment. Targeting cell-impermeable prodrug activation to tumor microenvironment will significantly reduce drug toxicity to normal tissues. The activated prodrug attacks both tumor and stroma cells through a "bystander effect" without selectively deleting target-producing cells, therefore further minimizing resistance and toxicity. Here, we showed that legumain, the only asparaginyl endopeptidase of the mammalian genome, is highly expressed by neoplastic, stromal, and endothelial cells in solid tumors. Legumain is present extracellularly in the tumor microenvironment, associated with matrix as well as cell surfaces and functional locally in the reduced pH of the tumor microenvironment. A novel legumain-activated, cell-impermeable doxorubicin prodrug LEG-3 was designed to be activated exclusively in the tumor microenvironment. Upon administration, there is a profound increase of the end-product doxorubicin in nuclei of cells in tumors but little in other tissues. This tumor microenvironment-activated prodrug completely arrested growth of a variety of neoplasms, including multidrug-resistant tumor in vivo and significantly extended survival without evidence of myelosuppression or cardiac toxicity. The tumor microenvironment-activated prodrug design can be extended to other proteases and chemotherapeutic compounds and provides new potentials for the rational development of more effective functionally targeted cancer therapeutics.

Animals↗
PubMed2006

Complete reaction cycle of a cocaine catalytic antibody at atomic resolution.

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.

Antibodies, Catalytic↗
PubMed2006

Emerging chemical and biological approaches for the preparation of discovery libraries.

The term of combinatorial chemistry has come to embrace all types of small-molecule library strategies. In both academic and industrial settings, the development of many combinatorial techniques has been driven by a desire to generate diverse libraries of molecules. Recently, chemical approaches have been reported that range from alternative library design concepts to new synthetic procedures that allow for selective product formation in extremely short reaction times. Concurrently, biological techniques have made great strides by adapting naturally occurring strategies for library preparation and screening. With proper understanding of library design, combinatorial endeavors and molecular libraries will continue to impact on the development of the next generation of tools for modern medicine.

Combinatorial Chemistry Techniques↗
PubMed2006

Solid-phase synthesis and kinetic characterization of fluorogenic enzyme-degradable hydrogel cross-linkers.

Of critical importance in drug delivery and tissue engineering applications is the degradability of implanted polymeric materials. The use of peptide-derived cross-linkers in hydrogel design is a valuable approach by which polymeric carriers can be endowed with enzymatic degradability in a predictable, "programmable" fashion. The solid-phase synthesis strategy described herein allows for an expeditious, flexible synthesis of bis-acrylamide-derivatized peptides with complex modifications, as exemplified by the incorporation of fluorophore and quencher moieties into a matrix metalloprotease (MMP)-degradable cross-linker. The crude synthetic product was obtained in high yield and purity and purified by standard methods; it was then used directly for polymerization without the need for tedious and often nonchemoselective solution-phase modifications. Functional appendages incorporated for detection provided a direct, quantitative link between enzymatic activity and hydrogel degradation using routine methods for identification of optimal enzyme-specific degradability.

Acrylamides↗