Enzymatic inhibition assays

Figure 5.12 Diagramatic illustration of the possible correlation between compound potency in cellular and enzymatic activity assays when the cellular phenotype is a direct result of inhibition of the target enzyme. Compounds that fall into the lower left and upper right quadrants demonstrate a correlation of rank-order potency between the cellular and cell-free assays. Compounds in the upper left quadrant may represent potent enzyme inhibitors that for some reason do not achieve adequate intracellular concentrations, as described in the text. Note the absence of any compound points in the lower right quadrant. Population of this quadrant would usually be inconsistent with enzyme inhibition being the direct cause of the observed cellular phenotype.

In another report, several acetylcholinesterase (AChE) inhibitors, including tacrine, edrophonium, tetramethyl- and tetraethyl-ammonium chloride, carbofu-ran, and eserine were assayed on a chip [1045]. AChE converted the substrate, acetylthiocholine, to thiocholine. This product reacted with coumarinylphenyl-maleimide (CPM) to form thiocholine-CPM (a thioether) for LIF detection. Since the acetonitrile solvent used to dissolve CPM inhibited AChE activity, the CPM solution was added after the enzymatic reaction [1045]. Another enzyme inhibition assay using a peptide substrate was performed on a PMMA chip [1046]. 

Low-molecular-weight heparins ( 4500 Da, or 15 monosaccharide units) are isolated from standard heparin by gel filtration chromatography, precipitation with ethanol, or partial depolymerization with nitrous acid and other chemical or enzymatic reagents. Low-molecular-weight heparins differ from standard heparin and from each other in their pharmacokinetic properties and mechanism of action ("see below). The biological activity of low-molecular-weight heparin is generally measured with a factor Xa inhibition assay, which is mediated by antithrombin. 

Bacterial or enzymatic toxicity tests are used to assay the activity of organic compounds including solvents. A survey of environmental bacterial or enzymatic test systems is given by Bitton and Koopman. The principles of these test systems are based on bacterial properties (growth, viability, bioluminescence, etc.) or enzymatic activities and biosynthesis. The toxicity of several solvents were tested in bacterial or enzymatic systems, e.g., pure solvents such as phenol in growth inhibition assays (Aeromonas sp.), solvents in complex compounds such as oil derivates, solvents in environmental samples such as sediments or solvents used in the test systems.The efficiency of several test systems, e.g., Microtox tests or ATP assays, vary, e.g., looking at the effects of solvents. ... 

A flow injection immunoanalysis system connected via a sterile sampling unit to a continuous bioreactor has been used for online monitoring of monoclonal antibodies in the course of a hybridoma cell fermentation. Mouse IgG and rabbit antimouse IgG immobilized in a macroporous network have been used for an antigen inhibition assay and sandwich assay, respectively. The product of the enzymatic indicator was measured fluorimetrically. 

Inhibition Study. A proteinaceous inhibition study was conducted to study the role of the enzymatic active site in the hydrolysis and condensation of trimethylethoxysilane. Prior to reaction, trypsin was independently inhibited with an excess amount of the Bowman-Birk inhibitor (34) (4 1 BBI to trypsin mole ratio) and die Popcorn inhibitor (35) (2 1 PCI to trypsin mole ratio) in stirred neutral media for two hours. Based on standard enzymatic activity assays (36), trypsin was fully inhibited by the BBI (98%) and PCI (91%). The reactions were formulated with an 1000 1 trimethylethoxysilane to trypsin mole ratio and conducted at 25°C for three hours. The reaction products were isolated and quantitatively analyzed by GC (Table II). Although the treated enzymes were observed to catalyze the hydrolysis of trimethylethoxysilane, the condensation of trimethylsilanol was conqiletely inhibited in conq>arison to the control reactions. Notably, the rate of hydrolysis decreased in the presence of the BBI- and PCI-inhibited trypsin. Following thermal denaturation, tiie activity of trypsin was comparable to the proteinaceous inhibition experiments. Based on a standard enzymatic activity assay (36), the relative decrease in the rate of silanol condensation correlated with the enhanced stability of trypsin at higher protein concentrations (25). Consequently, it appears that non-specific interactions with trypsin including the active site promoted the hydrolysis of trimethylethoxysilane. Therefore, the active site of trypsin was determined to selectively catalyze the in vitro condensation of trimethylsilanol imder mild conditions. 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

The goal of most HTS assays for enzyme targets is to identify library components that act as inhibitors of enzymatic activity. To identify and compare inhibitory compounds, we must first define a metric that reflects the ability of a fixed concentration of compound to reduce the activity of the target enzyme. The most commonly used metric for this purpose is the inhibition percentage, which can be defined as follows ... 

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