Biomedical subjects

Amnon Albeck

Publications and source records attributed to Amnon Albeck.

7 recordsLinked to original sources

PubMed2006

Enzyme isoselective inhibitors: a tool for binding-trend analysis.

A transition-state analogue inhibitor that covalently reversibly binds to an enzyme formally consists of two parts: the chemical site, CS and the recognition site, RS. We have experimentally and theoretically demonstrated that the trend of binding affinity in a series of isoselective inhibitors (with identical RS and different CS fragments) depends mainly on their CS fragments. Isoselective inhibitors have the same affinity trend toward different enzymes of the same family with a common catalytic mechanism. Thus, very good correlation between experimentally determined and theoretically calculated Ki values was demonstrated. A practical outcome is the application of the described method as a tool for an expert analysis in virtual screening of inhibitor libraries and in the design of new enzyme inhibitors.

Binding Sites↗

PubMed2006

Enzyme isoselective inhibitors: application to drug design.

Common methodologies of computer-assisted drug design focus on noncovalent enzyme-ligand interactions. We introduced enzyme isoselective inhibition trend analysis as a tool for the expert analysis of covalent reversible inhibitors. The methodology is applied to predict the binding affinities of a series of transition-state analogue inhibitors of medicinally important serine and cysteine hydrolases. These inhibitors are isoselective: they have identical noncovalent recognition fragments (RS) and different reactive chemical fragments (CS). Furthermore, it is possible to predict the binding affinities of a series of isoselective inhibitors toward a prototype enzyme and to extrapolate the data to a target medicinally important enzyme of the same family. Rational design of CS fragments followed by conventional RS optimization could be used as a novel approach to drug design.

Drug Design↗

PubMed2004

Identification of protons position in acid-base enzyme catalyzed reactions: the hepatitis C viral NS3 protease.

General acid-base catalysis is a key element of the catalytic activity of most enzymes. Therefore, any explicit molecular modeling of enzyme-catalyzed chemical reactions requires correct identification of protons location on the catalytic groups. In this work, we apply our quantum mechanical/self-consistent reaction field in virtual solvent [QM/SCRF(VS)] method for identification of the position of protons shared by the enzyme catalytic groups and the polar groups of the inhibitor in a covalent tetrahedral complex (TC) of the hepatitis C virus NS3 protease with a peptidyl alpha-ketoacid inhibitor. To identify the relevant protonation states, we have analyzed relative stabilities of R and S configurations of the TC that depend on the specific proton distribution over the polar groups and correlated it with experimental NMR and X-ray crystallography data, both at low and neutral pH ranges. The tentative assignment of the single resonance in the (13)C NMR spectrum of the hemiketal carbon at physiological pH to the S configuration of TC is confirmed. Both R and S configurations are equally stable at acidic pH in our modeling, in good agreement with the (13)C NMR observation.

Acids↗

PubMed2004

Is there a weak H-bond --> LBHB transition on tetrahedral complex formation in serine proteases?

The transformation of a weak hydrogen bond in the free enzyme into a low-barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H-bond in the Asp102-His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self-consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102-His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme-inhibitor TC is a strong ionic asymmetric one-well hydrogen bond, in contrast to a previous suggestion that it is a weak H-bond in the former and a double-well LBHB in the latter.

Chymotrypsin↗

PubMed2004

13-Demethyl-13-substituted-13,14-dihydroretinols as potential affinity labels of retinol-binding proteins: syntheses and stability studies.

13-Demethyl-13-substituted-13,14-dihydroretinols were synthesized and their stability under various conditions was measured in order to evaluate whether they would be useful as affinity labels of retinol binding proteins and retinol metabolizing enzymes. The 13-chloro analog could not be isolated because it eliminated HCl under the Wittig reaction conditions of its preparation. The trans- and cis-13,14-epoxy analogs are stable in non-protic organic solvents, but undergo an elimination reaction under various chromatographic conditions and in mixtures of organic solvents with water or alcohol. The 13-hydroxy and 13-methoxy analogs are stable in aqueous solutions and are therefore suitable for biological studies.

Affinity Labels↗

PubMedDate not supplied

Free nonimmobilized ligands as a tool for purification of proteins.

Purification of proteins on a large scale is a complex multistep process, and alternative economic strategies are required. This study presents a novel approach (Affinity Sinking, AS) for purification of native proteins utilizing nonimmobilized modified ligands. The nonimmobilized state of the ligand circumvents the need for immobilizing ligands to polymeric supports. Therefore, purification from large volumes can be accomplished without the use of industrial-scale affinity columns. The mechanism of product capture is formation and precipitation of a specific [target-protein/modified-ligand] complex by using a soluble interconnecting entity that generates an insoluble [target-protein/modified-ligand/interconnecting entity] sediment containing the target protein. Rabbit IgG and two glycoproteins were purified accordingly, utilizing free avidin (as the interconnecting entity) and either desthiobiotinylated-protein A (DB-ProA) or desthiobiotinylated-concanavalin A (DB-ConA) as the modified ligand. The recovery yields for the IgG and the two glycoproteins were 80-86% and 70-75%, respectively. Target proteins are eluted from the generated pellet nearly without disrupting the [modified-ligand/interconnecting entity] macro-complex, thus enabling a practical procedure of recovering target proteins. Leaching of the DB-ProA ligand under eluting conditions (pH 3) was found to be lower than 1%. The two modified ligands, DB-ProA and DB-ConA, were regenerated without any chromatographic procedure in 80% and 85%-89% yield, respectively. The advantage of excluding the polymeric component from the purification process and obtaining highly purified proteins has been demonstrated, and it implies that other contaminants (e.g. endotoxins, prions, host DNA) could be excluded as well, thereby reducing the number of purification steps in a typical downstream process.

Animals↗

PubMedDate not supplied

A general approach for antibody purification utilizing [Protein A-catechol:Fe3+] macro-complexes.

A general platform for antibody purification utilizing free nonimmobilized Protein A modified with the strong metal chelator catechol (ProA-CAT) and Fe3+ ions is presented. The mechanism of purification requires formation and precipitation of macro-complexes composed of [ProA-CAT:IgG:Fe3+]. Target IgGs are eluted directly from the precipitates (i.e. pellets) at pH 3 in high yields (71-80%) and high purity (>95%), without dissociating the [ProA-CAT:Fe3+] insoluble macro-complex.

Animals↗