Assays, high-throughput substrate specificity

Scheme 7. High-throughput substrate specificity assays for sialidases...

The next section describes the utilization of //PLC for different applications of interest in the pharmaceutical industry. The part discusses the instrumentation employed for these applications, followed by the results of detailed characterization studies. The next part focuses on particular applications, highlighting results from the high-throughput characterization of ADMET and physicochemical properties (e.g., solubility, purity, log P, drug release, etc.), separation-based assays (assay development and optimization, real-time enzyme kinetics, evaluation of substrate specificity, etc.), and sample preparation (e.g., high-throughput clean-up of complex samples prior to MS (FIA) analysis). 

The DDI fluorescent inhibition screen is a high-throughput screen, primarily as a result of reduced analysis time, and can range from a few hundred compounds per week to a thousand per week. The assay uses recombinant microsomes prepared from insect cells infected with recombinant baculovirus containing a human CYP enzyme and individual fluorogenic substrates. In this instance the substrates are not specific and hence cannot be used in a mixed enzyme system such as HLM. In addition, this assay is more prone than the conventional inhibition screen (Section 8.3.2) to NCE interferences within the assay (i.e., inherently fluorescent NCEs, fluorescent quenching by the NCE). 

Site-specific saturation at key residues, followed by a high-throughput activity assay, produced the triple mutant F78V/L188Q/A74G, which shows a greatly increased 700-fold efficiency for the hydroxylation of w-octane and a 200-fold increase in the hydroxylation of [i-ionone at the 3-position [114], Stereoselectivity is poor, however, and all diastereomers are present in the products in comparable amounts. A five-fold mutant, the so-called (F87V)LARV, was developed in a related fashion [115] and shown to hydroxylate capric acid, a substrate not attacked by the wild-type enzyme [116]. 

The use of specific quenched fluorescent substrates (QFS) provides a rapid and sensitive method to measure peptidase activity, and is readily adaptable to high-throughput screening of potential peptidase inhibitors. In this chapter, we discuss general considerations for the development of QFS assays, and describe in detail an assay protocol for the mammalian metallopeptidase, endothelin-converting enzyme. 

The resistance of a surface to the irreversible non-specific adsorption of proteins is an essential requirement for its application as a substrate in a high-throughput device for carrying out diagnostic assays, in which cell-based assays for screening libraries of drug candidates are performed in a chip format. The protein resistance of the surface allows a cell population... 

Gel-purified dsRNA is labeled with 33P for use as a substrate in the high-throughput assay. 33P-labeled substrate has a lower background and a longer half life than substrate labeled with 32P. For secondary assays confirmation of hits in the high throughput, 96-well plate primary assay, gel-purified dsRNA substrate is labeled to a high specific activity with 32P for use in a gel-based assay. 

Many of these techniques have also been successfully utilized for the identification of inhibitors of protein-carbohydrate interactions. ELIS As are often employed, and their utility is illustrated in the efforts to identify selectin inhibitors [51-53]. However, other assays such as fluorescence polarization [33, 54] and carbohydrate affinity matrices [55] have also been utilized. These methods have provided valuable information about the substrate specificity of a number of carbohydrate-binding proteins and led to the discovery of many useful inhibitors. Still, there remains an acute need to develop effective high-throughput assays. 

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