The fluorogenic peptide substrate Mca-YVADAPK(Dnp)-OH (R&D Systems, Bio-Techne, Minneapolis, MN), was diluted from stock (4 mM) to a final concentration of 1 mM in dimethyl sulfoxide (DMSO). All monitored reactions with ACE2 were conducted in black, 96-well, flat-bottom, tissue culture-treated microplates (Falcon, Corning, NY) in 100 μL of PBS (pH 7.4) at room temperature with substrate (1–5 μL, 10–50 μM final concentration, DMSO concentration maintained at ≤5% [v/v]) added immediately before measurement. An equal volume of ACE2 was added to solutions of the supramolecular structure and incubated for 1 h for all experiments unless stated otherwise. ACE2 activity was monitored continuously (every 5 min for 2 h total) by measuring fluorescence intensity (λex = 320 nm, λem = 405 nm) upon substrate hydrolysis using a SpectraMax M3 microplate reader (Molecular Devices, San Jose, CA). The initial velocity for each reaction was determined from the rate of fluorescence evolved over the 5- to 20-min time course (the slope from linear regression analysis of this region). The extent of enzymatic inhibition (used as a reflection of the docking efficiency of ACE2 to the filament surface) was determined as the measured initial velocity of a tested condition relative to free ACE2 at the same ACE2 and substrate concentration.
Mca yvadapk dnp oh
Manufactured by R&D Systems
Sourced in United States
Mca-YVADAPK(Dnp)-OH is a synthetic peptide that can be used as a substrate for caspase-3 and related proteases. It contains a fluorometric reporter group (Mca) and a quencher (Dnp) that allow for the detection of protease activity.
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Lab products found in correlation
Lisinopril, by Merck Group (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Fl600 microplate fluorescence reader, by Agilent Technologies (2 mentions)
Dx600, by Phoenix Pharmaceuticals (2 mentions)
Six well collagen 1 coated plates, by BD (2 mentions)
Mln4760, by AnaSpec (2 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Biotek synergy 2, by Agilent Technologies (1 mentions)
Sinergy ht, by Agilent Technologies (1 mentions)
8 protocols using mca yvadapk dnp oh
1
Fluorogenic Peptide Substrate-Based ACE2 Assay
2
Fluorogenic Assay for PreP Activity
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The fluorogenic peptide substrate Mca-Y-V-A-D-A-P-K(Dnp)-OH (R&D Systems, Catalog # ES007) was used to measure the activity of PreP. The reaction was monitored on a Synergy Neo microplate reader using an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Reactions were carried out at 37 °C, using 5 nM PreP with various concentrations of substrate V (5, 10, 20, or 40 μM) in 200 μL of buffer containing 20 mM Tris, pH7.7, 150 mM NaCl, 1 mM β-mercaptoethanol. Degradation of substrate V was assessed by monitoring the fluorescence increase for 10 min at 30-s intervals. To calculate enzymatic activity, background subtraction and linear regression fitting were used to find the initial velocity, whereas specific activity was determined by comparing the maximal fluorescence converted from the known quantity of substrate V by PreP.
3
ACE-2 Enzymatic Activity Measurement
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IMR-90s raised and treated in six-well collagen I-coated plates (BD Biosciences, Bedford, MA) were harvested in ice-cold complete Tris-HCl buffer (Tris-HCl (pH 6.5), 1× Complete Protease Inhibitor Cocktail EDTA-free (Roche, Indianapolis, IN), and lisinopril (50 μg/l; Sigma-Aldrich). lisinopril was added to the buffer as a means to block the activity of ACE. In a half-area black 96-well microtiter plate, the fluorogenic peptide substrate for ACE-2, MCA-YVADAPK(Dnp)-OH (R&D Systems, Minneapolis, MN) was added at a final concentration of 10 μmol/l to 30 μl cell lysate (in a total volume of 50 μl using complete Tris-HCl buffer) on ice. DX600 (at a final concentration of 10 µmol/l; Phoenix Pharmaceuticals, Burlingame, CA), a competitive inhibitor of ACE-2, was also added to half of the wells to compare enzymatic ACE-2 activity inhibition. The plate was warmed to room temperature and the fluorescence was read on a plate reader (310/20 nm excitation and 420/50 nm emission) in a BioTek FL600 Microplate Fluorescence Reader (BioTek, Burlington VT) for 30 min. Kinetic readings were normalized to their respective DNA concentrations.
4
Screening FDA-Approved Drugs for ACE2 Modulators
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Calu-3 cells were treated serially with individual compounds contained within the Johns Hopkins Drug Library (JHDL), which includes a series of FDA-approved drugs [46 (link)] (kindly provided by Dr. Jun O. Liu, Johns Hopkins University) at 10μM for 18h, and the ACE2 activity in cell culture medium was determined by measuring the fluorescence intensity of ACE2 substrate Mca-YVADAPK(Dnp)-OH (Catalog no. ES007; R&D Systems) [15 (link)]. In brief, samples were diluted in assay buffer (50 mM MES, 300 mM NaCl, 10 nM ZnCl2, and 0.01% Brij-35 [pH 6.5]) and incubated with ACE2 substrate, with or without the ACE2 inhibitor MLN4760 (1 nM, catalog no. 62337; AnaSpec), at 37 °C for 45 min. The fluorescence intensity was determined using a multiplate fluorescence reader at excitation 330 nm and emission 405 nm. Hits that enhance ACE2 activity in the culture medium >4 folds than that of the control vehicle are selected to determine the dose and time-dependent responses further. Our lead compound from these studies is herein labeled “H4”.
5
Measuring Renal ACE2 Enzymatic Activity
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Renal ACE2 enzymatic activity was determined following incubation with a fluorogenic peptide substrate [ACE2 substrate:fluorogenic peptide VI (FPS VI), Mca-YVADAPK(Dnp)-OH (catalogue no. ES007; R&D Systems, Minneapolis, MN, USA)] as previously described [24 (link)]. In brief, tissue samples were homogenized in ACE buffer (75 mmol l−1 Tris–HCl (pH 7.5), 1 mol l−1 NaCl and 0.5 mmol l−1 ZnCl2). Homogenates were centrifuged, and the resulting supernatants were used for analysis. All assays were performed in duplicate. A reaction mixture containing 0.1 M NaCl and 10 mM captopril in ACE2 buffer was added to each supernatant. Fluorescence was emitted due to the breakdown of the FPS VI peptide and was measured using a microplate reader (BioTekSynergy™ 2; BioTek, Winooski, VT, USA) every minute for 120 min immediately after the addition of the fluorogenic peptide substrate at 37°C (320 nM excitation and 405 nM emission). The total fluorescence was corrected according to the protein content, as determined by the Bradford assay [23 (link)]. Data were presented in fluorescence units per minute normalized to the total protein concentration.
6
ACE2 Activity Assay in Muscle Tissue
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The ACE2 activity assay was performed as previously described with minor modifications [38] (link). Tissue was homogenized in ACE2 buffer (75 mM Tris pH 7.5, 0.5 μM ZnCl2) in ice with an Ultraturrax (Kinematica, Littau, Switzerland). Tissue extracts were centrifuged at 4°C for 10 minutes at 10,000 g and supernatant was retained. The fluorescence emitted by breakdown of the fluorogenic peptide Mca-Y-V-A-D-A-P-K(Dnp)-OH (10 uM; R&D Systems, USA) was measured in a reaction mix containing 50 ug of total protein from tissue extracts, 0.1 M NaCl, and 10 μM captopril in ACE2 buffer to a final volume of 100 μl. Fluorescence emitted was measured at 2-minute intervals over 46 minutes at 37°C at 320 nM excitation and 405 nM emission (Biotek instruments, Sinergy HT, USA). The slope of the linear range of data represents ACE2 activity (fluorescence units/min) and was corrected for total micrograms of protein. The activity of 0.5 nM recombinant mouse ACE2 (R&D Systems, USA) was used as the positive control. Data were expressed as percentage of mdx ACE2 activity relative to wt DIA muscle or as ACE2 specific activity.
7
ACE-2 Activity Measurement in IMR-90 Cells
8
Quantifying ACE2 Activity in BALF
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