The inhibitor compounds were prepared as stock solutions in DMSO and diluted hundredfold with HEPES buffer (50 mm HEPES, pH 7.5, 0.1 mg mL−1 bovine serum albumin, 0.1% Triton-X100) to micromolar concentrations. Volumes of 50 μL of 200 nM SARS-CoV-2 PLpro (Elabscience) in HEPES buffer were added to the wells of a black 96-well microtiter plate (Greiner Bio One). Volumes of 50 μL of the inhibitor solutions or 1% DMSO in HEPES buffer (positive control) were added. The resulting solutions (100 nm SARS-CoV-2 PLpro 0.5% DMSO, 0.1–0.75 μm test compound or blank HEPES buffer) were mixed and incubated at 37 °C for 10 min. A volume of 100 μL of 20–8000 μm of substrate (Z-Arg-Leu-Arg-Gly-Gly-AMC, Bachem) was added to all wells. The resulting solutions were mixed and the fluorescence emission was measured immediately every minute for 1 h (λexc = 355 nm; λem = 460 nm) at 37 °C using a Victor™ X4 Perkin Elmer 2030 multilabel reader. The enzyme activity of PLpro was represented by the Michaelis Menten equation (eqn (1)) and the Km and Vmax values were calculated with Lineweaver–Burk equation (eqn (2)) where the slope is Km/Vmax, the intersection with the Y-axis corresponds to 1/Vmax, and intersection with the X-axis corresponds to −1/Km.
Z arg leu arg gly gly amc
Manufactured by Bachem
Sourced in Switzerland
Z-Arg-Leu-Arg-Gly-Gly-AMC is a synthetic peptide substrate used in biochemical assays. It consists of the amino acid sequence Z-Arg-Leu-Arg-Gly-Gly linked to the fluorescent reporter 7-amino-4-methylcoumarin (AMC). This substrate can be used to measure the activity of proteases that cleave at the arginine residues.
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Lab products found in correlation
5 protocols using z arg leu arg gly gly amc
1
Inhibition Kinetics of SARS-CoV-2 PLpro Protease
2
SARS-CoV-2 PL^pro Enzyme Inhibition Assay
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A slightly modified
protocol from a previous publication40 (link) was
used. The PLpro enzyme (Elabscience) was thawed on ice
and activated by dilution to 10.0 ng/μL with HEPES buffer. The
enzyme solution was further diluted with HEPES buffer to 0.5 ng/μL.
Twenty microliters of the enzyme solution was mixed with 5 μL
of increasing concentrations of the complex [2% (v/v) DMSO] diluted
in assay buffer in the dark. The substrate (Z-Arg-Leu-Arg-Gly-Gly-AMC,
Bachem Bioscience) was diluted to 10 μM, and 25 μL was
added to the enzyme mixture. The mixture was incubated for 60 min
at 37 °C with slow shaking. The generated fluorescence signal
(λex = 355 nm; λem = 460 nm) was
recorded with a Synergy H4 (BioTek) microplate reader. As a positive
control, the known inhibitor GRL-0617 (IC50 = 5 ±
2 μM) was used.
protocol from a previous publication40 (link) was
used. The PLpro enzyme (Elabscience) was thawed on ice
and activated by dilution to 10.0 ng/μL with HEPES buffer. The
enzyme solution was further diluted with HEPES buffer to 0.5 ng/μL.
Twenty microliters of the enzyme solution was mixed with 5 μL
of increasing concentrations of the complex [2% (v/v) DMSO] diluted
in assay buffer in the dark. The substrate (Z-Arg-Leu-Arg-Gly-Gly-AMC,
Bachem Bioscience) was diluted to 10 μM, and 25 μL was
added to the enzyme mixture. The mixture was incubated for 60 min
at 37 °C with slow shaking. The generated fluorescence signal
(λex = 355 nm; λem = 460 nm) was
recorded with a Synergy H4 (BioTek) microplate reader. As a positive
control, the known inhibitor GRL-0617 (IC50 = 5 ±
2 μM) was used.
3
High-Throughput Screening for SARS-CoV and MERS-CoV PLpro Inhibitors
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The 25 000-compound
Life Chemicals library was screened against the two PLpro cysteine
proteases from SARS-CoV and MERS-CoV. All assays against SARS-PLpro
were done in duplicate, and those against MERS-PLpro were done in
a single pass in black 384-well plates (Matrix Technologies). The
SARS-PLpro enzyme (20 nM final concentration) was prepared in an assay
buffer (50 mM HEPES, pH 7.5, 0.01% Triton X-100 (v/v), 0.1 mg mL–1 BSA, and 2 mM GSH). The MERS-PLpro enzyme (400 nM
final concentration) was prepared in the same assay buffer with 5
mM DTT in place of 2 mM GSH. A total of 30 μL of enzyme solution
was dispensed into wells, and then 200 nL of 10 mM compounds (50 μM
final concentrations) was added and incubated for 5 min. Enzyme reactions
were initiated with 10 μL of substrate Z-Arg-Leu-Arg-Gly-Gly-AMC
(Bachem Bioscience; 50 μM and 75 μM for SARS- and MERS-PLpro,
respectively) dissolved in the assay buffer and incubated for 6 min,
followed by adding 10 μL of 10% SDS (w/v) as a stop solution.
Fluorescence intensity was monitored at 360 nm (excitation) and 450
nm (emission).
Life Chemicals library was screened against the two PLpro cysteine
proteases from SARS-CoV and MERS-CoV. All assays against SARS-PLpro
were done in duplicate, and those against MERS-PLpro were done in
a single pass in black 384-well plates (Matrix Technologies). The
SARS-PLpro enzyme (20 nM final concentration) was prepared in an assay
buffer (50 mM HEPES, pH 7.5, 0.01% Triton X-100 (v/v), 0.1 mg mL–1 BSA, and 2 mM GSH). The MERS-PLpro enzyme (400 nM
final concentration) was prepared in the same assay buffer with 5
mM DTT in place of 2 mM GSH. A total of 30 μL of enzyme solution
was dispensed into wells, and then 200 nL of 10 mM compounds (50 μM
final concentrations) was added and incubated for 5 min. Enzyme reactions
were initiated with 10 μL of substrate Z-Arg-Leu-Arg-Gly-Gly-AMC
(Bachem Bioscience; 50 μM and 75 μM for SARS- and MERS-PLpro,
respectively) dissolved in the assay buffer and incubated for 6 min,
followed by adding 10 μL of 10% SDS (w/v) as a stop solution.
Fluorescence intensity was monitored at 360 nm (excitation) and 450
nm (emission).
4
High-Throughput Screening for SARS and MERS PLpro Inhibitors
5
Screening of FDA-Approved Drugs Against SARS-CoV-2
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Fluorogenic peptide substrate Dabcyl-KTSAVLQSGFRKME -Edans of 3CLpro was synthesized by Yuan Yu Ltd. (Taiwan). The fluorogenic substrate peptide (Z-Arg-Leu-Arg-Gly-Gly-AMC) for PLpro was purchased from Bachem (Bubendorf, Switzerland). The collections of 1,068 and 2,701 FDA-approved drugs were purchased from Target Molecule Corp. (Boston, MA, USA) and Selleck Chemicals (Houston, TX, USA), respectively. After screening, the active compounds in bulk size (e.g., 25 mg) were purchased from the companies for IC50, EC50, and CC50 measurements. The plasmid miniprep kit, DNA gel extraction kit, and Ni-NTA resin were purchased from Qiagen (Hilden, Germany). FXa and the protein expression kit, including pET32a and pET16b vectors as well as competent Escherichia coli JM109 and BL21(DE3) cells were obtained from Novagen (Merck KGaA, Darmstadt, Germany). The SARS-CoV-2 virus used in this study was SARS-CoV-2/NTU13/TWN/human/2020 (accession identifier [ID] EPI_ISL_422415) as previously described (50 (link)). All commercial buffers and reagents were of the highest grade.
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