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Biomedical subjects

K D Janda

Publications and source records attributed to K D Janda.

At least 73 records · Page 4Linked to original sources
PubMed1995

Reactive immunization.

For almost 200 years inert antigens have been used for initiating the process of immunization. A procedure is now described in which the antigen used is so highly reactive that a chemical reaction occurs in the antibody combining site during immunization. An organophosphorus diester hapten was used to illustrate this concept coined "reactive immunization." The organophosphonate recruited chemical potential from the immune response that resembled the way these compounds recruit the catalytic power of the serine hydrolases. During this recruitment, a large proportion of the isolated antibodies catalyzed the formation and cleavage of phosphonylated intermediates and subsequent ester hydrolysis. Reactive immunization can augment traditional immunization and enhance the scope of catalytic antibody chemistry. Among the compounds anticipated to be effective are those that contain appropriate reactive functionalities or those that are latently reactive, as in the mechanism-based inhibitors of enzymes.

Animals↗
PubMed1995

Suppression of psychoactive effects of cocaine by active immunization.

Cocaine is a powerfully addictive substance and new strategies are needed to treat its abuse. Generating an active immunization to cocaine offers a means of blocking the actions of the drug by preventing it from entering the central nervous system, and should have fewer side effects than treatments based on manipulation of central neurotransmitter function. The design and preparation of a cocaine immunogen requires special regard for the stability of cocaine both free and as a haptenic determinant. Immunochemistry and a well defined behavioural model were brought together to address the problem of inactivation of the psychostimulant actions of cocaine. We report here that active immunization with a new, stable cocaine conjugate suppressed locomotor activity and stereotyped behaviour in rats induced by cocaine but not by amphetamine. Moreover, following acute injection of cocaine, levels of cocaine in the striatum and cerebellum of the immunized animals were lower than those of control animals. These results suggest that immunopharmacotherapy may be a promising means by which to explore new treatments for cocaine abuse.

Animals↗
PubMed1995

Liquid-phase combinatorial synthesis.

A concept termed liquid-phase combinatorial synthesis (LPCS) is described. The central feature of this methodology is that it combines the advantages that classic organic synthesis in solution offers with those that solid-phase synthesis can provide, through the application of a linear homogeneous polymer. To validate this concept two libraries were prepared, one of peptide and the second of nonpeptide origin. The peptide-based library was synthesized by a recursive deconvolution strategy [Erb, E., Janda, K. D. & Brenner, S. (1994) Proc. Natl. Acad. Sci. USA 91, 11422-11426] and several ligands were found within this library to bind a monoclonal antibody elicited against beta-endorphin. The non-peptide molecules synthesized were arylsulfonamides, a class of compounds of known clinical bactericidal efficacy. The results indicate that the reaction scope of LPCS should be general, and its value to multiple, high-throughput screening assays could be of particular merit, since multimilligram quantities of each library member can readily be attained.

Amino Acid Sequence↗
PubMed1995

Encoded reaction cassette for the highly sensitive detection of the making and breaking of chemical bonds.

A reaction cassette has been designed for the highly sensitive detection of the making and breaking of chemical bonds. The system is envisioned as a companion device to be used in the search for antibody and other novel catalysts. The cassette also may have important clinical applications in the design of diagnostic reagents. In its fully encoded format, this methodology is capable of both detecting and decoding chemical events.

Antibodies↗
PubMed1995

Catalytic antibodies: evolution of protein function in real time.

Natural selection of enzyme function has evolved over millions to billions of years, whereas antibody induction operates over a period of weeks. If one considers how new protein functions are generated, one sees that both the immune system and natural selection have powerful methods for the generation of diversity. In this treatise we discuss antibody catalysis from an evolutionary standpoint as we consider how the elicitation of a catalytic antibody can be a useful tool for exploring the nature of biological catalysis.

Amides↗
PubMed1994

Recursive deconvolution of combinatorial chemical libraries.

A recursive strategy that solves for the active members of a chemical library is presented. A pentapeptide library with an alphabet of Gly, Leu, Phe, and Tyr (1024 members) was constructed on a solid support by the method of split synthesis. One member of this library (NH2-Tyr-Gly-Gly-Phe-Leu) is a native binder to a beta-endorphin antibody. A variation of the split synthesis approach is used to build the combinatorial library. In four vials, a member of the library's alphabet is coupled to a solid support. After each coupling, a portion of the resin from each of the four reaction vials was set aside and catalogued. The solid support from each vial is then combined, mixed, and redivided. The steps of (i) coupling, (ii) saving and cataloging, and (iii) randomizing were repeated until a pentapeptide library was obtained. The four pentapeptide libraries where the N-terminal amino acid is defined were screened against the beta-endorphin antibody and quantitated via an ELISA. The amino acid of the four pools that demonstrated the most binding was then coupled to the four tetrapeptide partial libraries that had been set aside and catalogued during the split synthesis. This recursive deconvolution was repeated until the best binders were deduced. Besides the anticipated native binder, two other members of the library displayed significant binding. This recursive method of deconvolution does not use a molecular tag, requires only one split synthesis, and can be applied to the deconvolution of nonlinear small-molecule combinatorial libraries and linear oligomeric combinatorial libraries, since it is based only on the procedure of the synthesis.

Amino Acid Sequence↗
PubMed1994

Tagged versus untagged libraries: methods for the generation and screening of combinatorial chemical libraries.

Over the past two decades the pharmaceutical industry has been driven by the biological sciences. The discovery and description of the biological mechanisms that underlie disease states accompanied by an unraveling of these mechanisms has provided drug, and more recently biotechnological, companies with a barrage of new therapeutic targets. Paradoxically, as a result of such biological and biochemical advances, new sources of drug leads are in short supply. Considerable efforts in trying to create potential drug candidates has led to the parturition of combinatorial chemical libraries. In this review I will examine some of the main technologies for generating and deducing active components from combinatorial libraries that have been segregated into two schools of thought: (i) the creation and decoding of combinatorial libraries by so-called tagged methodologies, and (ii) the production and deconvolution of chemical libraries by untagged protocols.

Amino Acid Sequence↗
PubMed1994

Antibody catalyzed cationic cyclization.

Two major goals for the design of new catalysts are the facilitation of chemical transformations and control of product outcome. An antibody has been induced that efficiently catalyzes a cationic cyclization in which an acyclic olefinic sulfonate ester substrate is converted almost exclusively (98 percent) to a cyclic alcohol. The key to the catalysis of the reaction and the restriction of the product complexity is the use of antibody binding energy to rigidly enforce a concerted mechanism in accord with the design of the hapten. Thus, the ability to direct binding energy allows the experimenter to dictate a reaction mechanism which is an otherwise difficult task in chemistry. New catalysts for cationic cyclization may be of general use in the formation of carbon-carbon and carbon-heteroatom bonds leading to multi-ring molecules including steroids and heterocyclic compounds.

Animals↗
PubMed1994

Direct selection for a catalytic mechanism from combinatorial antibody libraries.

Semisynthetic combinatorial antibody library methodology in the phage-display format was used to select for a cysteine residue in complementarity-determining regions. Libraries were panned with an alpha-phenethyl pyridyl disulfide that undergoes disulfide interchange. Out of 10 randomly picked clones, two contained an unpaired cysteine, one of which was studied. The antibody catalyzed the hydrolysis of the corresponding thioester where the electrophilic carbonyl occupies the three-dimensional space that was defined by the reactive sulfur atom during selection. The reaction operates by covalent catalysis. Although the steady-state rate enhancement relative to the activated thiol ester substrate is modest, hydrolysis of the acylated cysteine intermediate is remarkably efficient with a catalytic advantage of about four orders of magnitude. The results suggest that iterative mechanism-based selection procedures can recapitulate the enzymatic mechanisms refined through evolution.

Amino Acid Sequence↗
PubMed1993

Control of the exo and endo pathways of the Diels-Alder reaction by antibody catalysis.

Catalytic antibodies that control the reaction pathways of the Diels-Alder cycloaddition have been generated. One antibody catalyzes the favored endo and the other the disfavored exo pathway to yield the respective cis and trans adducts in enantiomerically pure form. A comparison of the x-ray structure of the hapten with the calculated geometry of the transition structure showed that [2.2.2] bicyclic compounds are excellent mimics of the transition state of the Diels-Alder reaction. To achieve catalysis and the high degree of stereoselectivity shown here, the antibody must simultaneously control the conformation of the individual reactants and their relation to each other. In the case of the disfavored process, binding energy must be used to reroute the reaction along a higher energy pathway. The rerouting of reaction pathways has become a major focus of antibody catalysis and other disfavored reactions can be expected to be catalyzed so long as the energy barrier is not extreme. The energy requirements needed for absolute control of all of the stereoisomers of many Diels-Alder reactions fall in the energy range (approximately 20 kilocalories per mole) deliverable by antibody binding.

Acrylamide↗
PubMed1993

Antibody catalysis of a disfavored chemical transformation.

Organic reactions are often limited by stereoelectronic constrains that appear along the reaction coordinate. An antibody has been generated that overcomes these constraints and catalyzes a highly disfavored chemical transformation. The antibody facilitates the difficult 6-endo-tet ring closure of an epoxy-alcohol to form a tetrahydropyran. The catalyzed process is in formal violation of what has become known as Baldwin's rules for ring-closure reactions. In addition to controlling the regiochemistry of the disfavored cyclization reaction, these catalytic antibodies resolve enantiomeric substrates to afford a stereochemically pure product. The principles demonstrated in this study may be applicable to other disfavored chemical processes.

Antibodies↗
PubMed1991

Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces.

We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of filamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules.

Antibodies, Monoclonal↗
PubMed1991

An unexpectedly efficient catalytic antibody operating by ping-pong and induced fit mechanisms.

A transition state analogue was used to produce a mouse antibody that catalyzes transesterification in water. The antibody behaves as a highly efficient catalyst with a covalent intermediate and the characteristic of induced fit. While some features of the catalytic pathway were programmed when the hapten was designed and reflect favorable substrate-antibody interactions, other features are a manifestation of the chemical potential of antibody diversity. The fact that antibodies recapitulate mechanisms and pathways previously thought to be a characteristic of highly evolved enzymes suggests that once an appropriate binding cavity is achieved, reaction pathways commensurate with the intrinsic chemical potential of proteins ensue.

Acylation↗
PubMed1991

Screening combinatorial antibody libraries for catalytic acyl transfer reactions.

A bacteriophage lambda vector system for the expression of Fab fragments from the mouse antibody repertoire in Escherichia coli has been described. We have used this system to generate a catalytic antibody from a combinatorial antibody library. Monoclonal antibody 43C9 was raised against a transition state analogue of the hydrolysis of carboxyamide. mRNA from hybridoma cells expressing this antibody was cloned into phage lambda and clones that expressed the mRNA for either the heavy or the light chain of the antibody were isolated. These individual libraries were then crossed to generate a combinatorial library in which clones coexpressed the heavy and light chains. This library was screened for antibodies/Fab fragments that bound to the original antigen with high affinity. DNA sequencing showed that these fragments were the same as those in antibody 43C9. Three different clones were found to catalyse the hydrolysis of carboxyamide. More efficient expression vectors and improved screening techniques should lead to the isolation of many more catalytic antibodies from combinatorial antibody libraries.

Acylation↗
PubMed1991

A catalytic antibody uses a multistep kinetic sequence.

Antibody NPN43C9 catalyses the hydrolysis of a p-nitrophenyl ester and p-nitroanilide. Kinetic investigations show that hydrolysis of the ester involves at least two steps whose relative importance changes with pH. We propose that the reaction proceeds via formation of an acyl intermediate that deacylates through attack by hydroxide. The rate-limiting step changes from product release at high pH to hydroxide ion-mediated hydrolysis at low pH. This multistep pathway shares similarities with that used by serine proteases.

Antibodies↗