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Biomedical subjects

R A Lerner

Publications and source records attributed to R A Lerner.

At least 19 recordsLinked to original sources
PubMed1998

Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide.

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and block gap junction communication in a structurally specific manner. Herein, the structural features of oleamide required for inhibition of the gap junction-mediated chemical and electrical transmission in rat glial cells are defined. The effective inhibitors fall into two classes of fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the most effective, and its structural requirements for inhibition of the gap junction are well defined. It requires a chain length of 16-24 carbons of which 16-18 carbons appears optimal, a polarized terminal carbonyl group capable of accepting but not necessarily donating a hydrogen bond, a Delta9 cis double bond, and a hydrophobic methyl terminus. Within these constraints, a range of modifications are possible, many of which may be expected to improve in vivo properties. A select set of agents has been identified that serves both as oleamide agonists and as inhibitors of fatty acid amide hydrolase, which is responsible for the rapid inactivation of oleamide.

Amidohydrolases↗
PubMed1997

The sleep-inducing lipid oleamide deconvolutes gap junction communication and calcium wave transmission in glial cells.

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction-mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18beta-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.

Animals↗
PubMed1997

Immune versus natural selection: antibody aldolases with enzymic rates but broader scope.

Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.

Animals↗
PubMed1997

On roads not taken in the evolution of protein catalysts: antibody steroid isomerases that use an enamine mechanism.

Reactive immunization has emerged as a new tool for the study of biological catalysis. A powerful application resulted in catalytic antibodies that use an enamine mechanism akin to that used by the class I aldolases. With regard to the evolution of enzyme mechanisms, we investigated the utility of an enamine pathway for the allylic rearrangement exemplified by Delta5-3-ketosteroid isomerase (KSI; EC 5.3.3.1). Our aldolase antibodies were found to catalyze the isomerization of both steroid model compounds and steroids. The kinetic and chemical studies showed that the antibodies afforded rate accelerations up to a factor of 10(4) by means of an enamine mechanism in which imine formation was the rate-determining step. In light of our observations and the enzyme studies by other workers, we suggest that an enamine pathway could have been an early, viable KSI mechanism. Although this pathway is amenable to optimization for increased catalytic power, it appears that certain factors precluded its evolution in known KSI enzymes.

Antibodies, Catalytic↗
PubMed1997

Making chemistry selectable by linking it to infectivity.

The link between recognition and replication is fundamental to the operation of the immune system. In recent years, modeling this process in a format of phage-display combinatorial libraries has afforded a powerful tool for obtaining valuable antibodies. However, the ability to readily select and isolate rare catalysts would expand the scope of library technology. A technique in which phage infection controlled the link between recognition and replication was applied to show that chemistry is a selectable process. An antibody that operated by covalent catalysis to form an acyl intermediate restored phage infectivity and allowed selection from a library in which the catalyst constituted 1 in 10(5) members. Three different selection approaches were examined for their convenience and generality. Incorporating these protocols together with well known affinity labels and mechanism-based inactivators should allow the procurement of a wide range of novel catalytic antibodies.

Antibodies, Catalytic↗
PubMed1997

Chemical selection for catalysis in combinatorial antibody libraries.

For the past decade the immune system has been exploited as a rich source of de novo catalysts. Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chemical reactions. In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes. Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function. A method that allows for the direct chemical selection for catalysis from antibody libraries was so devised, whereby the positive aspects of hybridoma technology were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis. This methodology is based on a purely chemical selection process, making it more general than biologically based selection systems because it is not limited to reaction products that perturb cellular machinery.

Animals↗
PubMed1996

Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.

Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.

Amides↗
PubMed1996

On signal sequence polymorphisms and diseases of distribution.

We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.

Base Sequence↗
PubMed1996

Cationic cyclopropanation by antibody catalysis.

Reactions involving highly reactive carbocations play a central role in many important chemical processes, such as cyclization reactions. However, the potential for controlling the pathways of such reactions to obtain energetically disfavoured (but desirable) products has been hard to realize because of the difficulties inherent in controlling the conformation and chemical environment of the carbocation intermediates. Antibody catalysts, with their high specificity and binding energies, can provide the degree of conformational and chemical control necessary for directing such reactions. Here we show how antibody catalysis can guide cationic cyclization reactions selectively to form products (in high yield) that would otherwise be highly disfavoured. Most notable is the formation of a strained bicyclic compound containing a rare cyclopropane group. To explain our results, we propose a common reaction scheme in which the key step is the formation of a highly reactive protonated cyclopropane intermediate; subtle structural modifications to the substrate (the compound on which the catalytic antibody acts) lead to dramatic differences in the structure of the final product.

Antibodies, Catalytic↗
PubMed1996

Synthesis of five enantiomerically pure haptens designed for in vitro evolution of antibodies with peptidase activity.

A series of five haptens have been synthesized for use in vitro selection experiments from combinatorial antibody libraries. Haptens were designed for the recruitment of serine and cysteine protease reaction mechanisms for the cleavage of Phe-Ala and Phe-Phe (L,L) dipeptide analogues. For the selection of transition state stabilization, PheP(O)Ala (7) and PheP(O)Phe (10) derivatives were synthesized using the Mitsunobu approach where Phe(P) represents the phosphonic acid analogue of phenylalanine and (O)Phe and (O)Ala represent (L.)-beta-phenyllactic and (L)-lactic acid, respectively. Optically pure peptidyl diazomethyl ketones 16 and 22 were synthesized for selection of the catalytic ensemble of cysteine proteases. An optically pure dipeptidyl boronic acid 26 was synthesized for the selection of the catalytic ensemble of serine proteases. A strategy for the evolution of catalytic antibodies using these haptens was developed which includes mechanism-based selections. Since mechanism based selections result in covalent trapping of species from libraries, diol and disulfide containing haptenic linkers were developed for the oxidative or reductive release of selected catalysts.

Antibodies, Catalytic↗
PubMed1996

Using the process of reactive immunization to induce catalytic antibodies with complex mechanisms: aldolases.

The process of reactive immunization has been used to induce efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. Reactive immunogens are those that react chemically during induction of the immune response. This same reaction is used later in catalysis. In essence one immunizes with the equivalent of a mechanism-based inhibitor. The difference is that instead of inhibiting a mechanism, a mechanism is induced. This advance allows the experimenter to dictate the exact mechanism by which catalytic antibodies proceed. The hapten used in the present study is a 1,3-diketone that both traps the requisite lysine residue to initiate formation of the enamine and induces a binding pocket that overcomes the entropic barrier of this bimolecular reaction.

Acetone↗
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

Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes.

Antibodies that catalyze the aldol reaction, a basic carbon-carbon bond-forming reaction, have been generated. The mechanism for antibody catalysis of this reaction mimics that used by natural class I aldolase enzymes. Immunization with a reactive compound covalently trapped a Lys residue in the binding pocket of the antibody by formation of a stable vinylogous amide. The reaction mechanism for the formation of the covalent antibody-hapten complex was recruited to catalyze the aldol reaction. The antibodies use the epsilon-amino group of Lys to form an enamine with ketone substrates and use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond. The antibodies control the diastereofacial selectivity of the reaction in both Cram-Felkin and anti-Cram-Felkin directions.

Acetone↗
PubMed1995

From molecular diversity to catalysis: lessons from the immune system.

By combining the enormous molecular diversity of the immune system with basic mechanistic principles of chemistry, one can produce catalytic antibodies that allow control of reactions in ways heretofore not possible. Mechanistic and structural studies of these antibodies are also providing insights into important aspects of enzymatic catalysis and the evolution of catalytic function. Moreover, the ability to rationally direct the immune response to generate selective catalysts for reactions ranging from pericyclic and redox reactions to cationic rearrangement reactions underscores the chemical potential of this and other large combinatorial libraries.

Amino Acid Sequence↗
PubMed1995

Chemical characterization of a family of brain lipids that induce sleep.

A molecule isolated from the cerebrospinal fluid of sleep-deprived cats has been chemically characterized and identified as cis-9,10-octadecenoamide. Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat, and human, indicating that these compounds compose a distinct family of brain lipids. Synthetic cis-9,10-octadecenoamide induced physiological sleep when injected into rats. Together, these results suggest that fatty acid primary amides may represent a previously unrecognized class of biological signaling molecules.

Animals↗
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↗