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Key Concepts in Assay Development, Screening and the Properties of Lead and Candidate Compounds
Published in Venkatesan Jayaprakash, Daniele Castagnolo, Yusuf Özkay, Medicinal Chemistry of Neglected and Tropical Diseases, 2019
The commonly used fluorogenic protease substrates (which are significantly more sensitive than chromogenic substrates) contain the 7-amino-4-methyl coumarin (AMC) group (Figure 4c). Upon catalytic cleavage of the substrate, the AMC product is detected by excitation of the system at 380 nm and detects the emission at 460 nm. Alternative fluorogenic substrates make use of quenched peptides that contain a donor and quencher chemically coupled to residues within the peptide substrate which are in close spatial proximity. An example of such a substrate is one that contains the fluorescent AMC group that is quenched by resonance energy transfer to the 2,4-dinitrophenyl (DNP) group (Figure 4d) (McCartney et al. 2018). Upon catalytic cleavage of the peptide, these two groups are spatially separated, quenching is overcome and the extent of product that is produced can be determined by excitation of the system at 320 nm and detecting the emission at 405 nm. Although the fluorogenic substrates are an improvement from chromogenic substrates, they still have the unnatural fluorophore moiety within the substrate molecule that could compromise the binding modes with the enzyme.
Purification, Assay, and Standardization of t-PA
Published in Cornelis Kluft, Tissue-Type Plasminogen Activator (t-PA): Physiological and Clinical Aspects, 1988
Alternatively, fluorogenic labels can be employed, such as the fluorescent compound (β-naphtylamide which is nonfluorescent in the amide bonded form. Assays based on fluorescence are generally more sensitive, but they require the more expensive fluorimeter instead of a photometer. The nature of the aminoacids in the substrate determine its sensitivity. Tissue plasminogen activator only hydrolyses substrates with arginine as the C terminal amino acid, while urokinase also can split lysine substrates.31,141,153 The specificity is not only determined by the C terminal amino acid, but also the other amino acids can have a significant influence.31,141
Peptidases and Peptides at the Blood-Brain Barrier
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
Janet Brownlees, Carvell Williams
Several methods commonly employed in detecting and quantifying peptidase activities are (i) the use of synthetic peptides containing a chromogenic or fluorogenic leaving group, which produces a measurable signal when the peptide bond in which it participates is cleaved by an enzyme and (ii) the use of HPLC (usually on reverse phase columns) to monitor the rate of disappearance of a natural or synthetic peptide, or the appearance of cleavage products, when incubated with tissue samples/extracts containing peptidase activities. The former is generally more sensitive and less time consuming, particularly if a fluorogenic derivative is used and it has the additional advantage that the assay is usually a continuous one, whereas the use of HPLC is necessarily discontinuous. On the other hand it is not always possible to obtain a suitable fluorogenic substrate whereas the use of HPLC is not subject to such constraints (see ref. 123 for details of assay methods for some peptidases). Class-specific inhibitors can be used to ascertain to which of the four classes (aspartyl, serine, cysteine or metallo-) a particular peptidase activity belongs and the search can be further narrowed by employing known inhibitors of particular peptidases. (iii) Immunocytochemical techniques can be employed. All of these have been used to explore cerebral microvessels for the presence of peptidase activities but to date only a small number have been detected. A summary of these is shown in Table 2. In no case has the exact sub–cellular localization of these enzyme activities been established, though in some cases cytochemical methods have shown the presence of peptidases within particular cells of the microvasculature (see below).
Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Jakub Benýšek, Michal Buša, Petra Rubešová, Jindřich Fanfrlík, Martin Lepšík, Jiří Brynda, Zuzana Matoušková, Ulrike Bartz, Martin Horn, Michael Gütschow, Michael Mareš
The purified CatK zymogen (75 µM) was activated by incubation in 0.1 M sodium acetate pH 4.0 containing 2.5 mM DTT, 1 mM EDTA, and 0.3 M NaCl under an argon atmosphere at room temperature. The zymogen-like activation intermediate iCatK was obtained after 30 min of incubation, and fully activated mature enzyme mCatK after 75 min. Activation was terminated by the addition of 6-fold molar excess of the inhibitor Gü1303 or Gü2602, followed by incubation under argon atmosphere for 3 h at room temperature. The activation and inhibition were monitored with a kinetic activity assay using the fluorogenic substrate Cbz-Gly-Pro-Arg-AMC (Cbz, benzyloxycarbonyl; AMC, 7-amino-4-methylcoumarin) and Laemmli-SDS-PAGE. The processing sites were identified by N-terminal protein sequencing after electroblotting of Laemmli-SDS-PAGE gels to a PVDF membrane using a Procise 494 cLC protein sequencer (Applied Biosystems) and by peptide mapping using mass spectrometry (LC-MS/MS) on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) coupled to a UHPLC system. The LC–MS/MS data were processed with Bioworks software (Thermo). The complexes were buffer-exchanged into 20 mM sodium acetate pH 5.5 containing 2.5 mM DTT and 0.25 M NaCl, and concentrated to 3.5 mg/ml for mCatK and 5 mg/ml for iCatK using an Amicon Ultracel-10k centrifugal filter device; the inhibitors were maintained during buffer exchange and concentration in a 6-fold molar excess to mCatK/iCatK in the mixture.
A high-throughput cell-based gaussia luciferase reporter assay for measurement of CYP1A1, CYP2B6, and CYP3A4 induction
Published in Xenobiotica, 2021
Han Li, Yu-Guang Wang, Zeng-Chun Ma, Gao Yun-Hang, Song Ling, Chen Teng-Fei, Zhang Guang-Ping, Yue Gao
Compared with the assay based on fluorogenic probe, the luminogenic assay has some advantages (Cali et al. 2006, Ortega Ugalde et al. 2019). First of all, background fluorescence is indispensable for fluorogenic assay, because the residual fluorescence of unreacted probe, the fluorescence of NADPH and light scattering will produce fluorogenic background signal, which is completely dependent on the excitation beam. At the same time, the excitation and emission wavelengths may overlap with the substrate of CYP probe, which may interfere with the analysis results. This problem is avoided by the luminogenic analysis without excitation light (DeGroot et al. 2015). Moreover, Gaussia luciferase is naturally secreted and emits a high amount of light compared with Renilla luciferase because it has a much higher enzymatic activity (Tannous et al. 2005).
Digital microfluidics comes of age: high-throughput screening to bedside diagnostic testing for genetic disorders in newborns
Published in Expert Review of Molecular Diagnostics, 2018
David Millington, Scott Norton, Raj Singh, Rama Sista, Vijay Srinivasan, Vamsee Pamula
More recently, in a further demonstration of the versatility of the DMF platform, 10 distinct enzyme assays were combined in a single run [57]. The conditions targeted were Pompe, Hurler, Gaucher, Fabry, Biotinidase, Sanfilippo, Sly, GM1 gangliosidosis, and alpha and beta mannosidoses. The concept for a 10-plex enzyme assay on the same cartridge was presented, in which a dual fluorophore detector system permits combining two reagents in each of 5 reagent input wells that release different fluorophores when exposed to their targeted enzyme. One set of fluorogenic substrates is based on 4MU (detected using excitation and emission wavelengths of 360 and 460 nm, respectively) and the other set based on resorufin (detected using excitation and emission wavelengths of 570 and 590 nm, respectively). The principles of this concept were proven by comparison with data from individual assays. For higher throughput of multiple enzymatic assays of this type, a cartridge capable of analyzing 96 samples and an analyzer with a built-in dual detector would be desirable.