Analysis of Non-Competitive Enzyme Inhibitors by Thermometric Titrimetry

Abstract

A new thermometric method for the analysis of non-competitive enzyme inhibitors has been developed. Advantages and basic requirements of the method are discussed. In the case studied, 30 nanograms of silver ions were detected.     

Mixed S/P Ligands from Carbohydrates: Synthesis and Utilization in Asymmetric Catalysis

Abstract

New chiral mixed sulfur/phosphorus ligands derived from carbohydrates are reported. These ligands were found to be efficient catalyst precursors for palladium-catalyzed asymmetric substitution of 1,3-diphenylpropenyl acetate with dimethyl malonate or benzylamine (up to 96% ee), and for rhodium-catalyzed methyl acetamidocinnamate hydrogenation (up to 92% ee).     

Synthesis of 1,6-Anhydro-2,3-di-O-farnesyl-5-O-([{phosphonomethyl}phosphinyl]methyl)-α-d-galactofuranose: A Zaragozic Acid-Presqualene Pyrophosphate Hybrid

Abstract

A multi-step synthesis of a new potential inhibitor (i.e., compound 2) of squalene synthase is described, starting from D-galactose. The title compound is a hybrid of presqualene pyrophopshate and the recently discovered zaragozic acids, which are picomolar competitive inhibitors of the enzyme squalene synthase.     

An Enzyme Immunoassay of Catalytically Active Proteases

Abstract

A method is proposed to assay catalytically active protease. The method is based on the subsequent inter action of the enzyme with a protein inhibitor immobi lized on polysterene and with antibodies labelled with a high-activity enzyme. Optimal conditions have been chosen for assaying alkaline protease from Bacillus subtilis (subtilisin) by ELISA with peroxidase as label, employing two protein serine protease inhibitors isolated from duck egg white and maize seeds. The proposed method enables the detection of the catalyti cally active enzyme at concentrations as low as 1 ng/ml for 1.5 hr. The c. v. of the assay is less than 5%. The method can be used to determine the equilibrium con stant of the enzyme-inhibitor reaction in solution.     

Synthesis and NMR-Spectroscopical Studies of Fluorinated Arylmethyl-Phosphinic Acid Derivatives

Abstract

Since Horiguchi in 1959 isolated CILIATIN from rumen protozoa the class of amino phosphonic and phosphinic acids has generated widespread interests directed towards synthetical problems and biological applications such as insecticides, hgicides, virostatica and enzyme inhibitors. Recent efforts have been devoted to more convenient preparations and stereospecific aspects. Here we wish to report on a facile method for the synthesis of C-fluorinated aminoalkane-mono- and bis-phosphinic acids and corresponding derivatives.     

Pyrolysis of Amino Acid Derived Stabilised Ylides as a Route to Chiral Acetylenic Amines and Amino Acids and Gaba Analogues

Abstract

We recently reported the pyrolysis of stabilised ylides as a method for overall conversion of carboxylic acids to homologous acetylenic esters and terminal alkynes.^1,2 This has now been applied successfully to amino acids. A wide range of alkoxycarbonyl protected amino acids have been converted to the stable crystalline ylides 1. These have been fully characterised, and upon FVP, eliminate Ph₃PO to afford the protected acetylenic amino acids 2 in good yield and without significant racemisation. Subsequent reactions of these extremely versatile intermediates have been used to gain access to a wide variety of chiral amine and amino acids of great interest as potential selective enzyme inhibitors and components for modified peptide structures.     

Determination of Oxalate with Immobilized Oxalate Oxidase in an Enzyme Thermistor

Abstract

This paper describes a rapid enzymic procedure, based on calorimetry, for specific determination of oxalic acid. Oxalate is oxidized by immobilized oxalate oxidase to hydrogen peroxide and carbon dioxide. The heat generated by this reaction is measured in a calorimetric device, the enzyme thermistor. Oxalate concentrations as low as 0.02 mM can be determined. Also described is purification and immobilization of the enzyme, as well as the effect of some of its inhibitors. The urinary oxalate content of 16 persons was determined using the enzyme thermistor. For samples containing a very low concentration of oxalate (less than 0.2 mM) an extraction step with tributylphosphate can be introduced to purify and concentrate the oxalate. The oxalate content in different kinds of food was also determined.     

Green synthesis of diethyl((2-iodo-4-(trifluoromethyl)phenyl)amino)(aryl)methyl)phosphonates as potent α-glucosidase inhibitors

Abstract

Novel diethyl((2-iodo-4-(trifluoromethyl)phenyl)amino)(aryl)methyl)phosphonates (4a-j) were synthesized via a simple and efficient one pot by three-component condensation reaction (Kabachnik-Fields reaction) of 2-iodo-4-trifluoromethyl aniline, aromatic aldehydes and diethyl phosphite in presence of anatase TiO₂ nanoparticles as catalyst under solvent free conditions. The molecular docking studies of synthesized compounds with α-glucosidase enzyme revealed that these compounds have strong α-glucosidase inhibitory activity. The synthesized compounds (4a-j) are also screened for in vitro α-glucosidase inhibitory activity and the results showed compound 4i as the strongest inhibitor and compounds 4a, 4b, 4f and 4g as stronger inhibitors even better than the reference standard acarbose.     

L-proline derivatives based on a calix[4]arene scaffold as chiral organocatalysts for the direct asymmetric aldol reaction in water

Abstract

A new series of water-compatible proline catalysts (4–6) derived from calixarene bearing a hydrophobic nature have been synthesised. It was found that the compound 4 was a highly efficient organocatalyst for aldol reactions occurred in the water. Under optimised reaction conditions, high yields (up to 82%), good enantioselectivities (ee up to 81%) and high diastereoselectivities (dr up to 91:9) were obtained.     

Isosteres of Natural Phosphates. Methylene and Hydroxyrnethylene Analogues of Tyrosine O-Phosphate

Abstract

The ability of phosphonic acid analogues isosteric with natural phosphate esters to Serve as inhibitors of enzymatic phosphate hydrolysis has been documented in a wide variety of systems.¹ The use of such an analogue in place of the natural phosphate ester provides a functionality which the enzyme may not be able to distinguish from the natural ester, but which is incapable of being hydrolyzed. In some instances the use of hydroxymethylene analogues has resulted in a greater degree of recognition, and resultant inhibition of hydrolytic activity, than the simple methylene analogues.² On this basis, the methylene and hydroxymethylene analogues of tyrosine O-phosphate appear to be reasonable candidates to Serve as inhibitors for phosphoprotein phosphatases and alkaline phosphatase, and as probes for biological mechanisms.     

Exploring the structural aspects of ureido-amino acid-based APN inhibitors: a validated comparative multi-QSAR modelling study

ABSTRACT

The zinc-dependent enzyme aminopeptidase N (APN) is a member of the M1 metalloenzyme family. The multi-functionality of APN as a peptidase, a receptor and a signalling molecule has provided it the access to influence a number of disease conditions namely viral diseases, angiogenesis, cellular metastasis and invasion including different cancer conditions. Hence, the development of potent APN inhibitors is a possible route for the treatment of diseases related to the activity of APN. In this study, different QSAR approaches have been adopted to identify the structural features of a group of hydroxamate-based ureido-amino acid derivative APN inhibitors. This study was able to identify different constitutional aspects of these APN inhibitors which are important for their inhibitory potency. Additionally, some of these observations were also aligned with the observations of previously performed QSAR studies conducted on different APN inhibitors. Therefore, the results of this study may help to design potent and effective APN inhibitors in the future.     

The Preparation,Characterization and Properties of Catalase Immobilized on Crosslinked Gellan

In the present paper, the reaction of chemical immobilization of catalase on a crosslinked macromolecular carrier of a polysaccharide structure (gellan) is studied. The influence of some reaction parameters (enzyme/carrier, activator/carrier ratios, duration) on the activity of enzymatic products is analyzed. The kinetics of the biocatalytic process, stability under different pH and temperature conditions, and the inhibitors effect were studied for the immobilized enzymes.     

Investigating interactions between chloroquine/hydroxychloroquine and their single enantiomers and angiotensin-converting enzyme 2 by a cell membrane chromatography method

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS-CoV-2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high-expression angiotensin-converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin-converting enzyme 2 receptor. Molecular docking assay and detection of SARS-CoV-2 spike pseudotyped virus entry into angiotensin-converting enzyme 2-HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin-converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R-Chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S-chloroquine or the racemate, R-chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R-hydroxychloroquine showed better inhibitory effects than S-hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R-chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability and inhibitory effects compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS-CoV-2 pseudovirus from entering cells was weaker than R-hydroxychloroquine/hydroxychloroquine (racemate).     

DER OXIDATIVE ABBAU VON THIOPHOSPHIN- THIOPHOSPHON- UND THIOPHOSPHORSÄUREESTERN MIT HYPOCHLORIT

Abstract

The thioesters R¹R²P(X)SR (X=O,S) 1 to 4 are degradated oxidatively forming R¹R²P(O)OH and R-SO³H. The influence of the following parameters on the oxidation course is investigated using standard conditions: pH, concentration of the hypochlorite, quality of the organic phase and cooperation of phase transfer catalysts and hypochlorite cations.

The thioesters 1 to 4 are degradated by hypochlorite with different rates depending on the type of the ligands R¹ and R² and the employment of an optimal pH. An analytical evaluation is possible.

Die Thioester R¹R²P(X)SR (X=O,S) 1 bis 4 werden durch Hypochlorit oxidativ zu R¹R²P(O)OH und RSO₃H abgebaut. Der Einfluß folgender Parameter auf den Oxidationsverlauf unter Standardbedingungen wird untersucht: pH, Hypochloritkonzentration, Art der organischen Phase, Mitwirkung von Phasentransfer-Katalysatoren und der Hypochlorit Kationen.

Die Thioester 1 bis 4 werden mit Hypochlorit in Abhängigkeit vom Typ der Liganden R¹ und R² und nach Einstellen eines optimalen pH-Wertes unterschiedlich schnell abgebaut. Dieser Unterschied ist analytisch auswertbar.     

A theoretical study of angiotensin-converting enzyme inhibitors

Summary INDO molecular orbital calculations are reported for 35 selected angiotensin-converting enzyme inhibitors. QSARs are developed between pI₅₀ data and molecular electronic indices. The QSARs obtained reflect the importance of both charge-charge interactions between inhibitor and receptor and of specific interactions between groups on the inhibitor with points around the molecule which are postulated to correspond to binding sites at the receptor.     

A Prospect for the Analysis of Species Acting as Enzyme Inhibitors as Illustrated by Heavy Metal Inhibition

Abstract

A flow system incorporating an amperometric glucose oxidase enzyme electrode has been used to study the inhibitory effects of 16 metal cations on glucose oxidase. Only copper(II), mercury(II) and silver(I) caused any significant inhibition. the enzyme electrode could be reactivated by EDTA, the reactivation being most effective for copper(II) and least so for silver(I). Other complexing agents were tried for reactivation but proved to be unsatisfactory.

The ability to reactivate the enzyme on the electrode following copper(II) inhibition, and the linear response of the system to the level of this inhibitor according to I/A = -9.49 × 10^?7 log([Cu]/M) + 4.84 × 10^?8; r = 0.994 between 2.5 × 10^?4M and 5 × 10^?3M [Cu]²⁺ indicates a prospect for the use of a flow system for determining enzyme inhibitors in samples.     

An Approach for Visualization of the Active Site of Enzymes with Unknown Three-Dimensional Structures

Abstract

A new approach for virtual characterization of the active site structure of enzymes with unknown three-dimensional (3D) structure has been proposed. It includes analysis of data on enzyme interaction with reversible competitive inhibitors, their 3D structures and moulding of the substrate-binding region. The superposition of ligands in biologically active conformations allows to determine the shape and dimension of the active site cavity accommodating these compounds. Monoamine oxidase A (MAO-A), a “typical” enzyme with unknown spatial organisation, was used to test this method. The correctness of such approach was validated by the analysis of HIV protease interaction with its inhibitors using 3D structures of their complexes. Mould of the substrate/inhibitor binding site can be used for the visualization of this binding site and for searching new ligands in molecular databases.     

Synthesis of Methyl 6′-Deoxy- and 6′-Thiolactosaminides and Their Inhibitory Activity Toward CMP-NeuNAc:D-galactoside-(2→6)-α-D-sialyltransferase

Abstract

Specific inhibitors of glycosyltransferases have become of interest1 not only for investigation of carbohydrate-participating cell-surface phenomena but also for practical use such as chemotherapeutic reagents. Glycosyltransferases catalyze the transfer of glycosyl moieties from nucleotide donors to oligosaccharide acceptors. Therefore, two kinds of substrate-analog inhibitors are possible. The donor analogs have been rather well studied, but are not specific. On the other hand, glycosyltransferases have in general smct acceptor specifkity. Recently, acceptor analogs which inhibit the corresponding glycosyltransferases were reported^2-5 and as expected were acceptor-specific inhibitors.     

In silico study directed towards identification of the key structural features of GyrB inhibitors targeting MTB DNA gyrase: HQSAR,CoMSIA and molecular dynamics simulations

ABSTRACT

Mycobacterium tuberculosis DNA gyrase subunit B (GyrB) has been identified as a promising target for rational drug design against fluoroquinolone drug-resistant tuberculosis. In this study, we attempted to identify the key structural feature for highly potent GyrB inhibitors through 2D-QSAR using HQSAR, 3D-QSAR using CoMSIA and molecular dynamics (MD) simulations approaches on a series of thiazole urea core derivatives. The best HQSAR and CoMSIA models based on IC₅₀ and MIC displayed the structural basis required for good activity against both GyrB enzyme and mycobacterial cell. MD simulations and binding free energy analysis using MM-GBSA and waterswap calculations revealed that the urea core of inhibitors has the strongest interaction with Asp79 via hydrogen bond interactions. In addition, cation-pi interaction and hydrophobic interactions of the R₂ substituent with Arg82 and Arg141 help to enhance the binding affinity in the GyrB ATPase binding site. Thus, the present study provides crucial structural features and a structural concept for rational design of novel DNA gyrase inhibitors with improved biological activities against both enzyme and mycobacterial cell, and with good pharmacokinetic properties and drug safety profiles.