Search PubMed⌕ Search

Biomedical subjects

Kim D Janda

Publications and source records attributed to Kim D Janda.

At least 37 records · Page 2Linked to original sources
PubMed2005

A credit-card library approach for disrupting protein-protein interactions.

Protein-protein interfaces are prominent in many therapeutically important targets. Using small organic molecules to disrupt protein-protein interactions is a current challenge in chemical biology. An important example of protein-protein interactions is provided by the Myc protein, which is frequently deregulated in human cancers. Myc belongs to the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors. It is biologically active only as heterodimer with the bHLH-ZIP protein Max. Herein, we report a new strategy for the disruption of protein-protein interactions that has been corroborated through the design and synthesis of a small parallel library composed of 'credit-card' compounds. These compounds are derived from a planar, aromatic scaffold and functionalized with four points of diversity. From a 285 membered library, several hits were obtained that disrupted the c-Myc-Max interaction and cellular functions of c-Myc. The IC50 values determined for this small focused library for the disruption of Myc-Max dimerization are quite potent, especially since small molecule antagonists of protein-protein interactions are notoriously difficult to find. Furthermore, several of the compounds were active at the cellular level as shown by their biological effects on Myc action in chicken embryo fibroblast assays. In light of our findings, this approach is considered a valuable addition to the armamentarium of new molecules being developed to interact with protein-protein interfaces. Finally, this strategy for disrupting protein-protein interactions should prove applicable to other families of proteins.

Animals↗
PubMed2005

Alpha- and beta-stilbenosides as base-pair surrogates in DNA hairpins.

The synthesis, structure, and optical spectroscopy of hairpin oligonucleotide conjugates possessing synthetic stilbene C-nucleosides (stilbenosides) are reported. Synthetic methods for selective preparation of both the alpha- and beta-stilbenosides have been developed. Both anomers are effective in stabilizing hairpin structures when used as capping groups at the open end of the hairpin base-pair domain. However, only the beta-anomer effectively stabilizes the hairpin structure when located in the interior of the base-pair domain opposite an abasic site. Similar results are obtained for hairpins possessing two stilbenosides, either adjacent to each other or with one intervening base-pair. Molecular dynamics simulations are employed to obtain averaged structures for these conjugates. The calculated structures for the capped hairpins formed with either anomer show effective pi-stacking with the adjacent base-pair. The calculated structures for the internal stilbenosides show that the alpha- and beta-anomers form extrahelical and intrahelical structures, respectively. The relative orientations of the two stilbenes in the bis-stilbenosides have been studied using a combination of exciton-coupled circular dichroism spectroscopy and molecular modeling.

Base Pairing↗
PubMed2005

Delta9-tetrahydrocannabinol immunochemical studies: haptens, monoclonal antibodies, and a convenient synthesis of radiolabeled delta9-tetrahydrocannabinol.

Immunopharmacotherapy as an approach to combat drugs of abuse has become an active area of investigation. Marijuana is the most commonly used illicit drug in the U.S. The main active chemical in marijuana is delta9-tetrahydrocannabinol (delta9-THC); hence, monoclonal antibodies with high affinity and specificity for delta9-tetrahydrocannabinol could be valuable immunopharmacotherapeutic intervention and diagnostic tools. We have synthesized immunoconjugates that induce an effective immune response to delta9-THC and describe a convenient synthesis of radiolabeled delta9-THC. We demonstrate the value and use of this probe to select anti-delta9-THC antibodies that bind delta9-THC with good affinity. The synthetic route to radiolabeled delta9-THC has enabled the correct assessment of the affinity of these antibodies to their ligand and may facilitate future binding studies between delta9-THC and its analogues and the cannabinoid receptors.

Animals↗
PubMed2005

Discovery of acetylcholinesterase peripheral anionic site ligands through computational refinement of a directed library.

The formation of beta-amyloid plaques in the brain is a key neurodegenerative event in Alzheimer's disease. Small molecules capable of binding to the peripheral anionic site of acetylcholinesterase (AChE) have been shown to inhibit the AChE-induced aggregation of the beta-amyloid peptide. Using the combination of a computational docking model and experimental screening, five compounds that completely blocked the amyloidogenic effect of AChE were rapidly identified from an approximately 200-member library of compounds designed to disrupt protein-protein interactions. Critical to this docking model was the inclusion of two explicit water molecules that are tightly bound to the enzyme. Interestingly, none of the tested compounds inhibited the related enzyme butyrylcholinesterase (BuChE) up to their aqueous solubility limits. These compounds are among the most potent inhibitors of amyloid beta-peptide aggregation and are equivalent only to propidium, a well-characterized AChE peripheral anionic site binder and aggregation inhibitor.

Acetylcholinesterase↗
PubMed2005

A blue fluorescent antibody-cofactor sensor for mercury.

[reaction: see text] A chemically programmed antibody sensor, consisting of a stilbenyl boronic acid cofactor and monoclonal antibody EP2-19G2, provides a new method of mercury detection. The fluorescent antibody sensor generates an intense powder blue fluorescence when bound to the stilbenyl boronic acid cofactor; however, it is quenched in the presence of Hg(2+) ions. The EP2-19G2-cofactor biosensor provides micromolar sensitivity and selectivity toward Hg(2+) ions over a wide range of metal ions in aqueous solution.

Antibodies, Monoclonal↗
PubMed2005

Recent advances for the treatment of cocaine abuse: central nervous system immunopharmacotherapy.

Cocaine addiction continues to be a major health and societal problem in spite of governmental efforts devoted toward educating the public of the dangers of illicit drug use. A variety of pharmacotherapies and psychosocial programs have been proposed in an effort to provide a method for alleviation of the physical and psychological symptoms of cocaine abuse. Unfortunately, these methods have been met with limited success, illustrating a critical need for new effective approaches for the treatment of cocaine addiction. Recently an alternative cocaine abuse treatment strategy was proposed using intranasal administration of an engineered filamentous bacteriophage displaying cocaine-sequestering antibodies on its surface. These phage particles are an effective vector for CNS penetration and are capable of binding cocaine, thereby blocking its behavioral effects in a rodent model. The convergence of phage display and immunopharmacotherapy has allowed for an investigation of the efficacy of protein-based therapeutics acting within the CNS on the effects of cocaine in animal models and has uncovered a new tool in the battle against cocaine addiction.

Animals↗
PubMed2005

Vitamin B2-mediated cellular photoinhibition of botulinum neurotoxin A.

Botulinum neurotoxin (BoNT) is the most toxic species known to humans and has been identified as a potential bioterrorist threat. Unfortunately, the only existing countermeasures for BoNT intoxication involve vaccinations that are only effective prior to entry of the toxin into neuronal cells. Herein, we disclose the ability of the micronutrient riboflavin (vitamin B(2)) to photooxidatively inactivate BoNT in cell-based assays without the need for toxin and riboflavin pre-exposure. In total, this study suggests that botulism neurotoxicity may be blunted with photodynamic therapy technology.

Animals↗
PubMed2005

Squaric monoamide monoester as a new class of reactive immunization hapten for catalytic antibodies.

A squaric monoester monoamide motif was employed as an effective reactive immunogen for the discovery of monoclonal antibodies with reactive residue(s) in their combining sites. Two antibodies, 2D4 and 3C8, were uncovered that enhance paraoxon hydrolysis over background. Kinetic analysis of these antibodies was performed and interestingly both undergo a single turnover event due to covalent modification within the antibody combining site. Because antibodies 2D4 and 3C8 result in covalent attachment and thus inactivation of paraoxon, they could be useful probes for investigating paraoxon intoxication.

Amides↗
PubMed2005

Exploring the scope of the 29G12 antibody catalyzed 1,3-dipolar cycloaddition reaction.

[Chemical reaction: See text] 29G12 is a murine monoclonal antibody programmed to catalyze the regio- and enantioselective 1,3-dipolar cycloaddition reaction between 4-acetamidobenzonitrile N-oxide 1a and N,N-dimethylacrylamide 2a (Toker, J. D.; Wentworth, P., Jr.; Hu, Y.; Houk, K. N.; Janda, K. D. J. Am. Chem. Soc. 2000, 122, 3244). Given the unique nature of 29G12 as a protein biocatalyst for this chemical reaction, we have investigated both the substrate specificity and mechanistic parameters of the 29G12-catalyzed process. These studies have shown that while 29G12 is specific for its dipole substrate 1a, the antibody is highly promiscuous with respect to the dipolarophiles it can process. 29G12 accepts a bulky hydrophobic dipolarophile cosubstrate, with rates of product formation up to 70-fold faster than with the original substrate 2a. In all cases, the respective isoxazoline products are produced with exquisite regio- and stereochemical control (78-98% ee). Comparison between the steady-state kinetic parameters from the 29G12-catalyzed reaction of 1a with the most efficient versus the original dipolarophile cosubstrate (2m and 2a, respectively), reveals that while the effective molarities (EM)s are almost identical (EM(2m)) 26 M; EM((2a)) 23 M), the affinity of 29G12 for the larger dipolarophile 2m is more than 1 order of magnitude higher than for 2a [Km(2m) 0.44 +/- 0.04 mM; Km(2a) 5.8 +/- 0.4 mM]. Furthermore, when 2m is the cosubstrate, the affinity of 29G12 for its dipole 1a is also greatly improved [Km(1a) 0.82 +/- 0.1 mM compared to Km(1a) 3.4 +/- 0.4 mM when 2a is the cosubstrate]. An analysis of the temperature dependence of the 29G12-catalyzed reaction between 1a and 2m reveals that catalysis is achieved via a decrease in enthalpy of activation (DeltaDeltaH 4.4 kcal mol(-1)) and involves a large increase in the entropy of activation (DeltaDeltaS 10.4 eu). The improved affinity of 29G12 for the nitrile oxide 1a in the presence of 2m, coupled with the increase in DeltaDeltaS during the 29G12-catalyzed reaction between 1a and 2m supports the notion of a structural reorganization of the active site to facilitate this antibody-catalyzed reaction.

Acrylamides↗
PubMed2005

Toward cocaine esterase therapeutics.

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).

Bacterial Proteins↗
PubMed2005

Altered retinoid homeostasis catalyzed by a nicotine metabolite: implications in macular degeneration and normal development.

Retinoids (vitamin A) serve two distinct functions in higher animals: light absorption for vision and gene regulation for growth and development. Cigarette smoking is a contributing factor for diseases that affect vision such as age-related macular degeneration and increases the risk of birth defects; however, altered retinoid homeostasis has received little attention as a potential mechanism for smoking-associated toxicities. Herein, we demonstrate that nornicotine, a nicotine metabolite and component of cigarette smoke, catalyzes the Z-to-E alkene isomerization of unsaturated aldehydes and ketones, including retinals. Despite the recent explosion in the use of organic compounds as chemical catalysts, minimal effort has been devoted to biologically relevant organocatalysis. Our study demonstrates a system in which a lowest unoccupied molecular orbital-lowering intermediate similar to the endogenous protein rhodopsin effectively catalyzes isomerization under biologically relevant conditions. The product of retinal isomerization is all-E-retinal, which in the eye is a biosynthetic precursor to N-retinylidene-N-retinylethanolamine, a hallmark of age-related macular degeneration. Furthermore, 9-Z- and all-E-retinal isomers are biosynthetic precursors to 9-Z- and all-E-retinoic acids, ligands that mediate specific cellular responses by binding to transcriptional regulatory proteins critical in growth and development. Strict maintenance of retinal isomer composition is essential for proper transcriptional regulation. Nornicotine-catalyzed retinal isomerization implies an underlying molecular mechanism for age-related macular degeneration, the birth defects associated with smoking, and other smoking-associated abnormalities that stem from disruption of retinoid metabolism.

Catalysis↗
PubMed2005

Quorum sensing in Vibrio harveyi: probing the specificity of the LuxP binding site.

Quorum sensing activity was investigated in the bacterium Vibrio harveyi using a series of both natural and nonnatural analogs of DPD, the penultimate precursor to autoinducer AI-2. The progression of molecules that were both synthesized and investigated includes enantiomeric variants, carbon-chain extension, and hydroxyl-functional group addition/deletions of DPD. The compilation of these studies reveals a binding cleft that can accommodate a number of different structural variants of DPD, albeit with invariably lower activities.

Bacterial Proteins↗
PubMed2005

Hammett correlation of nornicotine analogues in the aqueous aldol reaction: implications for green organocatalysis.

[reaction: see text] A series of meta- and para-substituted 2-arylpyrrolidines were synthesized and examined for their ability to catalyze an aqueous aldol reaction under buffered conditions. Kinetic analysis of arylpyrrolidine-catalyzed reactions displayed a linear Hammett correlation with rho = 1.14 (R(2) = 0.996), indicating that the reaction is accelerated by electron-withdrawing aryl rings. These results show promise for the development of a synthetically viable aqueous organo-catalyst.

Catalysis↗
PubMed2005

Enantioreversal in the sharpless asymmetric epoxidation reaction controlled by the molecular weight of a covalently appended achiral polymer.

Polymers such as poly(ethylene glycol) (PEG) have proven use in a variety of applications including organic synthesis. We now disclose our investigations into the recently disputed report that PEG tartrate esters can reverse the enantioselectivity of the Sharpless asymmetric epoxidation reaction. The results presented herein have clarified that the enantioselectivity of this reaction can be reproducibly reversed solely as a function of the molecular weight of the appended PEG. By preparing a range of tartrate ligands with varying PEG chains lengths, the reversal was found to occur within a molecular weight change of only 800. As the PEG chain did not affect the inherent chirality of the ligand, the enantioreversal was proposed to occur as a result of two Ti-ligand complexes which differ in their molecularity of ligand, one monomeric in ligand and the other dimeric. Support for this hypothesis was given through equilibrium measurements which revealed that the predominant species in Ti/PEG tartrate ester mixtures is a distinct 2:1 Ti-ligand complex, as opposed to the 2:2 Ti-ligand complex of traditional Sharpless asymmetric epoxidations. In total, these data represent an unrecognized property of PEG-supported catalysts that could open up new venues in the control of asymmetric reactions by means of achiral appended polymers.

Epoxy Compounds↗
PubMed2005

Fluorescent cocaine probes: a tool for the selection and engineering of therapeutic antibodies.

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.

Animals↗