Aldolase Catalytic Antibodies Research Articles

Computational elucidation and validation of the three-dimensional structure of humanized aldolase catalytic antibody 38C2

Catalytic antibodies are immunoglobulin proteins that are capable of catalyzing multiple reactions with diverse substrates. Aldolase catalytic antibody 38C2 catalyzes aldol and retro-aldol reactions via an enamine mechanism. Therefore, 38C2 has a high potential to be used in prodrug activation, and it is currently developed for selective chemotherapy. For medical applications, its humanization is essential, and therefore, the understanding of the three-dimensional (3D) spatial atomistic structure of 38C2 is mandatory. In this study, it was attempted to construct the 3D atomic structure of humanized abzyme 38C2 using computational methods. A homology modeled structure was simulated for 100 ns with classical molecular dynamics simulations for its dynamics stability. The accuracy of the constructed model was further evaluated with various theoretical methods. The binding of four selected natural substrates to the constructed structure was studied in detail to further validate the model. Finally, to evaluate the reaction readiness of the constructed protein, the first step of the catalytic reaction has been successfully carried out with QST3/IRC calculations using the DFT/B3LYP-6-31G level of theory in the presence of extracted catalytic residues with the preserved coordinates in implicit water. Hence, the reaction readiness of the proposed protein structure, along with all the other validation tests, strongly proves that the modeled structure has high accuracy. This study, therefore, sheds new light on the structure, mechanism of action and applications of the 38C2 abzyme by constructing and validating its full 3D atomistic model. Further, this highly reliable modeled structure will expedite and facilitate future 38C2-based drug discovery. Communicated by Ramaswamy H. Sarma

ChemInform Abstract: Broadening the Aldolase Catalytic Antibody Repertoire by Combining Reactive Immunization and Transition State Theory: New Enantio- and Diastereoselectivities.

Nine efficient aldolase antibodies were generated by using hapten 1. This hapten unites reactive immunization and the transition state analogue approach in a single molecule. Characterization of two of these antibodies reveals that they are highly proficient (up to 1000-fold better than any other antibody catalyst) and enantioselective catalysts for aldol and retro-aldol reactions and exhibit enantio- and diastereoselectivities opposite to that of antibody 38C2.

Studies toward the duocarmycin prodrugs for the antibody prodrug therapy approach

A tricyclic precursor for the synthesis of the prodrugs of pro-1,2,9,9a-tetrahydrocyclopropa[c]benz-[e]indole-4-one tetramethoxyindolecarboxamide (CBI-TMI) was prepared using the ring-closing metathesis approach. The tricyclic intermediate was converted to an advanced precursor of a CBI-TMI prodrug equipped with a linker presumably suitable for activation using the aldolase catalytic antibody 38C2. An attempted 38C2-catalyzed two-step activation of the hydroxy-pro-CBI intermediate involving retro-aldol and the β-elimination reactions was also examined.

Substrate Arrays for Fluorescence‐Based Enzyme Fingerprinting and High‐Throughput Screening

Indirect release of fluorogenic phenols such as umbelliferone was used as a chemical principle to prepare fluorogenic substrates for a variety of enzymes in which the enzyme-reactive group is separated from the fluorescent reporter group, allowing structural and functional diversification. Fluorescent probes were obtained for enzymes useful for asymmetric synthesis including aldolase catalytic antibodies, transaldolases, alcohol dehydrogenases, lipases, epoxide hydrolases, proteases, and Bayer-Villiger monoxygenases. Arrays of structurally related substrates were prepared and used to record the activity of enzymes on multiple substrates simultaneously in parallel formats such as microtiter plates, microarrays, and substrate cocktails, leading to enzyme-specific activity patterns. Arrays of nonlabeled substrates were assayed using indirect product sensors such as the adrenaline-periodate back-titration assay for 1,2-diols or the copper-calcein assay for amino acids. Many of the fluorogenic substrates and sensors described here are either commercially available or accessible in one or two simple synthetic steps and can be used for high-throughput screening of enzyme activities.

Selection of phage-displayed peptides that bind to a particular ligand-bound antibody

Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.

Breaking the one antibody–one target axiom

Studies at the interface of chemistry and biology have allowed us to develop an immunotherapeutic approach called chemically programmed antibodies (cpAbs), which combines the merits of traditional small-molecule drug design with immunotherapy. In this approach, a catalytic antibody catalyzes the covalent conjugation of a small molecule or peptide to the active site of the antibody, effectively recruiting the binding specificity of the conjugated molecule to the antibody. In essence, this technology provides the tools for breaking the "one antibody-one target axiom" of immunochemistry. Our studies in this area have focused on using the chemistry of the well studied aldolase catalytic antibodies of which mAb 38C2 is a member. Previously, we explored reversible assembly of cpAbs available through diketone chemistry. In this article, we explore a unique proadapter assembly strategy wherein an antibody 38C2-catalyzed transformation unveils a reactive tag that then reacts to form a stable covalent bond with the antibody. An integrin alpha(v)beta 3 antagonist was synthesized with the designed proadapter and studied using human breast cancer cell lines MDA-MB-231 and MDA-MB-435. We demonstrate that this approach allows for (i) the effective assembly of cpAbs in vitro and in vivo, (ii) selective retargeting of 38C2 to integrin alpha(v)beta 3 expressing breast cancer cell lines, (iii) intracellular delivery of cpAbs into cells, (iv) dramatically increased circulatory half-life, and (v) substantial enhancement of the therapeutic effect over the peptidomimetic itself in animal models of breast cancer metastasis. We believe that this technology possesses potential for the treatment and diagnosis of disease.

有機触媒を利用するエナミン機構に基づく反応：アルドラーゼ抗体から低分子のアミノ酸およびアミン触媒まで

Enamines are key intermediates in both organic chemistry and enzyme catalysis. Natural aldolase enzymes form an enamine intermediate from aldehydes and ketones in order to catalyze aldol reactions under mild ambient conditions. We have developed man-made aldolase catalytic antibodies and small peptides that catalyze aldol reactions, in which catalyst molecules form enamines from aldehydes and ketones in situ, like natural aldolase enzymes do. Through these studies, we have also developed small amine- and amino acid-catalyzed bond-forming reactions that use an enamine mechanism and that can be performed under mild ambient conditions. These low molecular weight amines form enamine intermediates in situ and the enamine reacts with electrophiles to afford products with high diastereo- and enantioselectivities. Our organocatalytic methodologies include catalytic asymmetric aldol, Mannich, Michael, and Diels-Alder reactions. Our catalysts, from aldolase antibodies to small organic amine and amino acid catalyst molecules, emulate many of the features of nature's enzymes.

Flow cytometric screening of aldolase catalytic antibodies

High-throughput screening of cells expressing active catalytic antibody clones by flow cytometry is described. A fluorogenic retro-aldol retro-Michael substrate was designed and synthesized with incorporation of a chloromethyl moiety for intracellular retention. Hybridoma or transfected mammalian cells expressing catalytic antibody molecules could be rapidly isolated.

A tunable hydrogel for encapsulation and controlled release of bioactive proteins.

A N,N-dimethylacrylamide-based hydrogel (2) with the new cross-linker (ethylenedioxy) bis[2,2'-(N-acryloylamino)ethane] (1) has been prepared, and its physicochemical properties in aqueous solution were studied. Three different native proteins (lysozyme, bovine serum albumin, and rabbit IgG) were encapsulated within the polymeric matrix 2, and the kinetics of their release from the swollen hydrogel were determined. The rate of protein release exhibits a clear dependence on both the molecular weight of the protein and the amount of cross-linker utilized to prepare the hydrogel. This is reflected by the fact that the low molecular weight proteins are released at an increased rate versus higher molecular weight proteins. In addition a greater amount of protein is released from the hydrogels with a lower percentage of cross-linker. The polymerization procedure used in this study is sufficiently mild to safeguard the functional integrity of attendant biomolecules as determined by the retention of catalytic activity of encapsulated alpha-chymotrypsin and aldolase catalytic antibody 38C2. The potential utility of these hydrogels for the controlled release of bioactive agents in vivo is strengthened by both their lack of toxicity against human dermal fibroblasts and their lack of immunogenicity in mice.

Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism.

Carbon-carbon bond formation is an essential reaction in organic chemistry and the use of aldolase enzymes for the stereochemical control of such reactions is an attractive alternative to conventional chemical methods. Here we describe the crystal structures of a novel class II enzyme, 2-dehydro-3-deoxy-galactarate (DDG) aldolase from Escherichia coli, in the presence and absence of substrate. The crystal structure was determined by locating only four Se sites to obtain phases for 506 protein residues. The protomer displays a modified (alpha/beta)(8) barrel fold, in which the eighth alpha-helix points away from the beta-barrel instead of packing against it. Analysis of the DDG aldolase crystal structures suggests a novel aldolase mechanism in which a phosphate anion accepts the proton from the methyl group of pyruvate.

Proline-Catalyzed Direct Asymmetric Aldol Reactions

Most enzymatic transformations have a synthetic counterpart. Often though, the mechanisms by which natural and synthetic catalysts operate differ markedly. The catalytic asymmetric aldol reaction as a fundamental C-C bond forming reaction in chemistry and biology is an interesting case in this respect. Chemically, this reaction is dominated by approaches that utilize preformed enolate equivalents in combination with a chiral catalyst.1 Typically, a metal is involved in the reaction mechanism.1d Most enzymes, however, use a fundamentally different strategy and catalyze the direct aldolization of two unmodified carbonyl compounds. Class I aldolases utilize an enamine based mechanism,2 while Class II aldolases mediate this process by using a zinc cofactor.3 The development of aldolase antibodies that use an enamine mechanism and accept hydrophobic organic substrates has demonstrated the potential inherent in amine-catalyzed asymmetric aldol reactions.4 Recently, the first small-molecule asymmetric class II aldolase mimics have been described in the form of zinc, lanthanum, and barium complexes.5,6 However, amine-based asymmetric class I aldolase mimics have not been described in the literature.7 Here we report our finding that the amino acid proline is an effective asymmetric catalyst for the direct aldol reaction between unmodified acetone and a variety of aldehydes. Recently we developed broad scope aldolase antibodies that show very high enantioselectivities, have enzymatic rate accelerations, and use the enamine mechanism of class I aldolases.4 During the course of these studies, we found that one of our aldolase catalytic antibodies (Aldolase Antibody 38C2, Aldrich) is an efficient catalyst for enantiogroup-differentiating aldol cyclodehydrations of 2,6-heptanediones to give cyclohexenones, including the Wieland-Miescher ketone.8,9 These intramolecular reactions are also catalyzed by proline (Hajos-Eder-Sauer-Wiechert reaction)10 and it has been postulated that they proceed via an enamine mechanism.11 However, the proline-catalyzed direct intermolecular asymmetric aldol reaction has not been described. Further, there are no asymmetric small-molecule aldol catalysts that use an enamine mechanism.7 Based on our own results and Shibasaki’s work on lanthanum-based small-molecule aldol catalysts,4,6 we realized the great potential of catalysts for the direct asymmetric aldol reaction. We initially studied the reaction of acetone with 4-nitrobenzaldehyde. Reacting proline (30 mol %) in DMSO/acetone (4:1) with 4-nitrobenzaldehyde at room temperature for 4 h furnished aldol product (R)-1 in 68% yield and 76% ee (eq 1). This result

Broadening the Aldolase Catalytic Antibody Repertoire by Combining Reactive Immunization and Transition State Theory: New Enantio- and Diastereoselectivities.

Nine efficient aldolase antibodies were generated by using hapten 1. This hapten unites reactive immunization and the transition state analogue approach in a single molecule. Characterization of two of these antibodies reveals that they are highly proficient (up to 1000-fold better than any other antibody catalyst) and enantioselective catalysts for aldol and retro-aldol reactions and exhibit enantio- and diastereoselectivities opposite to that of antibody 38C2.

A stereoselective fluorogenic assay for aldolases: Detection of an anti-selective aldolase catalytic antibody

Abstract Stereoisomeric aldols 6a-d undergo retroaldolization to give 3-oxopropyl umbelliferyl ether 5 and subsequently umbelliferone 4 by β-elimination, leading to a fluorescence increase at λ em = 460 ± 20 nm ( λ ex = 360 ± 20 nm). This fluorogenic assay for aldolases operates in cell culture media and can be used to search for new stereoselective aldolase biocatalysts. Aldolase antibody 38C2 (Aldrich no. 47,995-0) catalyzes stereoselectively the retroaldolization of (S)-anti aldol 6c .

Stereoselectivity of aldolase catalytic antibodies

Abstract Enolate- and enamine-mediated versions of the aldol condensation can be catalyzed by antibodies. Antibody 78H6, an antibody against the quaternary ammonium hapten 1, catalyzes the enolate-mediated condensation of keto-aldehyde 2 to form aldol 3a and 3b and the subsequent β-elimination to form enone 4. Catalysis of both steps is promoted by a carboxylate side-chain on the antibody acting as a general base. Antibody 78H6 catalyzes this process 104 times more efficiently than acetate. Despite of this efficiency, stereochemical control occurs only in the elimination step by kinetic resolution. The stereochemistry of the carbon–carbon bond forming step from 2 to 3, which is not rate-limiting in this system, cannot be controlled by the antibody. Antibody 72D4, another antibody raised against hapten 1, can be combined with primary amine 5 to form an artificial aldolase catalyzing the aldol condensation of aldehydes such as 6 with acetone to form aldols 7a/b. Catalysis occurs via a covalent enamine mechanism, whereby condensation of the amine cofactor 5 with acetone to form the corresponding enamine 9, is promoted within the hydrophobic binding pocket of the antibodies by exclusion of water. In contrast to the enolate mediated reaction, the carbon–carbon bond formation is rate-limiting in the enamine mechanism, and the antibody shows a high level of stereoselectivity in this step. Stereoselectivity is a consequence of conformational control by a hydrogen bonding group on the antibody that activates the aldehyde carbonyl. This activation furthermore allows chemoselective catalysis of aldolization over β-elimination to occur. These experiments demonstrate that reaction design has a critical influence on the stereoselectivity and outcome of antibody-catalyzed aldol condensations.

Aldolase Antibodies of Remarkable Scope

This paper describes the substrate specificity, synthetic scope, and efficiency of aldolase catalytic antibodies 38C2 and 33F12. These antibodies use the enamine mechanism common to the natural Class I aldolase enzymes. Substrates for these catalysts, 23 donors and 16 acceptors, have been identified. The aldol acceptor specificity is expected to be much broader than that defined here since all aldehydes tested, with the exception polyhydroxylated aldehydes, were substrates for the antibodies. 38C2 and 33F12 have been shown to catalyze intermolecular ketone−ketone, ketone−aldehyde, aldehyde−ketone, and aldehyde−aldehyde aldol addition reactions and in some cases to catalyze their subsequent dehydration to yield aldol condensation products. Substrates for intramolecular aldol reactions have also been defined. With acetone as the aldol donor substrate a new stereogenic center is formed by attack on the si-face of the aldehyde with ee's in most cases exceeding 95%. With hydroxyacetone as the donor substrate, ...

Therapeutic Antibody Technology 97

Almost 200 antibody aficionados attended the Therapeutic Antibody Technology 97 meeting, held September 21-24, 1997 at the Holiday Inn, Union Square in the heart of San Francisco, CA. The meeting was sponsored by the Palo Alto Institute of Molecular Medicine and organized by James W. Larrick (PAIMM) and Dennis R. Burton (Scripps Research Institute). The meeting featured excellent discussions on many interesting talks and a number of poster presentations. It is likely that another meeting will be organized in 2 years, however in the meantime, an effort is underway to organize a 'Virtual Antibody Society' to be set up on the web server at Scripps Research Institute in La Jolla, CA (Questions and comments on this project can be sent to: Jwlarrick@aol.com or Burton@scripps.edu). Richard Lerner (Scripps) gave the keynote address on 'Catalytic Antibodies', describing recent work with Carlos Barbas on so-called reactive immunization to generate a high activity aldolase catalytic antibody. This antibody, soon to be described in an article in Science, is the first commercially available catalytic antibody.

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.

Design and synthesis of chiral and racemic phosphonate-based haptens for the induction of aldolase catalytic antibodies

A novel strategy for the generation of aldolase catalytic antibodies, based on the use of antibody-catalyzed enol ester hydrolysis as a 'trigger' to generate a reactive enolate intermediate, is described. A model system to test this strategy was developed and substrate 8 was synthesized. However, the targeted bifunctional haptens 11 and 33 were synthetically inaccessible, and therefore the alternative phosphonate hapten 39 was prepared. The key step in the synthesis of 39 was the direct generation of an unprotected phosphonate precursor via coupling of the secondary alcohol 37 with CH3P(O)Cl2. The chiral counterpart of hapten 39 was also synthesized from alcohol 46, prepared by Corey's asymmetric reduction method. One polyclonal antibody preparation generated from 39 appeared to catalyze the hydrolysis of the secondary acetate 49, but not the desired aldol cyclization of 8. Possible rationales for this finding are discussed.

ChemInform Abstract: (S)‐Selective Retroaldol Reaction and (R)‐Selective β‐Elimination with an Aldolase Catalytic Antibody.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

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.