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Future Perspectives on Nucleic Acid Testing
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Larry J. Kricka, Paolo Fortina
Another elegant nonseparation assay strategy employs an intrasterically inactivated inhibitor DNA enzyme construct comprising a DNA capture probe linked on one end to cereus neutral protease and at the other to a phosphoramidite inhibitor of the enzyme. The enzyme activity of this conjugate is inhibited due to binding of the inhibitor to the active site of the enzyme. Binding of target DNA to the probe triggers activation of an enzyme by removing the inhibition and the resulting enzyme activity was detected using an EDANS (5-[2-aminoethyl] amino-1-naphthalene-sulfonic acid)-peptide-DABCYL (4-dimethylaminophenylazobenzoic acid) substrate. A detection limit of 10 fmol (100 pM) of a 24-mer was achieved in a PCR-independent assay.57
Identification of sulphonamide-tethered N-((triazol-4-yl)methyl)isatin derivatives as inhibitors of SARS-CoV-2 main protease
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Mai H. ElNaggar, Abdullah A. Elgazar, Ghada Gamal, Shimaa M. Hamed, Zainab M. Elsayed, Mohamed K. El-Ashrey, Amira Abood, Mahmoud A. El Hassab, Ahmed M. Soliman, Ramadan A. El-Domany, Farid A. Badria, Claudiu T. Supuran, Wagdy M. Eldehna
The enzyme inhibition experiment was conducted in 96-well, black microtiter plates with a total volume of 200 µl. A final concentration of 20 nM of the SARS-CoV-2 Mpro enzyme was used. The compounds being assessed, along with GC376 as a standard inhibitor, were pre-incubated with the enzyme at different concentrations in an assay buffer consisting of 20 mM TRIS, 1 mM EDTA, 150 mM NaCl, 1 mM DTT and the pH was adjusted to 7.3. A FRET substrate, Dabcyl-KTSAVLQSGFRKME-EDANS, was added to the mixture at final concentration of 10 µM and incubated in the dark for 3 h at room temperature. Fluorescence signals of released EDANS were estimated using a Spectrofluorometer with microplate reader accessory (Cary Eclipse, Agilent Technologies) at (excitation/emission, 355 nm/460 nm), and the blank was determined by measuring the entire reaction mixture without the enzyme. The obtained data was plotted and analysed to determine the IC50 values of the tested compounds using nonlinear regression with a variable slope.
Repurposing existing drugs: identification of SARS-CoV-2 3C-like protease inhibitors
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Wei-Chung Chiou, Meng-Shiuan Hsu, Yun-Ti Chen, Jinn-Moon Yang, Yeou-Guang Tsay, Hsiu-Chen Huang, Cheng Huang
An Edans-Dabcyl FRET platform was established, following a published protocol13. Briefly, a consensus cleavage sequence recognised by SARS-CoV-2 3CLpro was synthesised by Genomics, Taiwan, with Dabcyl at the N-terminus and Edans at the C-terminus, Dabcyl-TSAVLQ↓SGFRKME-Edans. In protease activity assays, 0.25 µM protease was incubated with 1.25 µM peptide substrate for three hours. Assays were conducted in triplicate in Eppendorf® black 96-well microplates (MA, USA) using an assay buffer containing 12 mM Tris-HCl (pH 7.5), 120 mM NaCl, 0.1 mM EDTA and 1 mM dithiothreitol (DTT), in a final volume of 100 µL. The fluorescence signal at 538 nm, at a bandwidth of 15 nm, emitted from the cleaved IQF peptide substrate after excitation at 355 nm, at a bandwidth of 10 nm, was recorded by a SPARK® multimode microplate reader (TECAN, Switzerland). The relative fluorescence units (RFU) at a gain of 131 were calculated using Spark® Control Magellan™ v2.2 software.
Inhibition of SARS-CoV 3CL protease by flavonoids
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Seri Jo, Suwon Kim, Dong Hae Shin, Mi-Sun Kim
The custom-synthesised fluorogenic substrate, DABCYL-KTSAVLQSGFRKME-EDANS (ANYGEN, Gwangju, Korea), was used as a substrate for the proteolytic assay using the SARS-CoV 3CLpro18. This substrate contains the nsp4/nsp5 cleavage sequence, GVLQ↓SG19, and works as a generic peptide substrate for many coronavirus including the SARS-CoV 3CLpro. The peptide was dissolved in distilled water and incubated with each protease. A SpectraMax i3x Multi-mode microplate reader (Molecular Devices) was used to measure spectral-based fluorescence. The proteolytic activity was determined at 310 K by following the increase in fluorescence (λexcitation = 340 nm, λemission = 490 nm, bandwidths = 9, 15 nm, respectively) of EDANS upon peptide hydrolysis as a function of time. Assays were conducted in black, 96-well plates (Nunc) in 300 μl assay buffers containing protease and substrate as follow; For the SARS-CoV 3CLpro assay, 4.05 μl of 0.074 mM protease containing 50 mM Tris pH 6.5 was incubated with 7.5 μl of 0.1 mM substrate at 310 K for 2 h before measuring Relative Fluorescence Unit (RFU). Before the assay, the emission spectra of 64 flavonoids were surveyed after illuminating at 340 nm to avoid the overlapping with the emission spectrum of EDANS. Every compound was suitable to be tested. The final concentration of the protease, peptide and chemical used at the assay was 1, 2.5 and 20 μM each. At first, the SARS-CoV 3CLpro and chemical were mixed and pre-incubated at room temperature for 1 h. The reaction was initiated by the addition of the substrate and each well was incubated at 310 K for 16 h. After that, we measured the fluorescence of the mixture on the black 96-well plate using the endpoint mode of SpectraMax i3x where the excitation wavelength was fixed to 340 nm and the emission wavelength was set to 490 nm using 9, 15 nm bandwidth, respectively. All reactions were carried out in triplicate. Among the first 64 flavonoids (Supplementary Table 1), three of them were picked up to further assay at a concentration range of 2–320 μM. The IC50 value which is the value causing 50% inhibition of the catalytic activity of the SARS-CoV 3CLpro was calculated by nonlinear regression analysis using GraphPad Prism 7.03 (GraphPad Software, San Diego, CA, USA).