TFMU-ADPr

TFMU-ADPr (0-500 μM, 0-120 μM; 5 minutes) serves as a continuous fluorescent substrate for hPARG (full-length PARG from Homo sapiens), ttPARG (PARG from Tetrahymena thermophila) and hARH3 (full-length ARH3 from H. sapiens), enabling robust enzyme kinetic assays. The corresponding K_M values for the three enzymes are 66.2 μM, 210 μM and 6.3 μM, respectively^[1].
TFMU-ADPr (200 μM) can report the combined hydrolase activity of PARG and ARH3 in U2OS cell lysates, and this activity is detected in both wild-type cells and ARH3 knockout cells (the latter represents PARG-only activity)^[1].
TFMU-ADPr (0-50 μM) serves as an effective reporter substrate for detecting the inhibitory activity of hARH3, which is confirmed by the potent inhibitory effect of the endogenous metabolite ADPr-Arg (K_i=18 nM)^[1].
TFMU-ADPr (in a dose-response manner; incubation duration 30 min) potently binds SARS-CoV-2 Macro1 (IC₅₀=0.59 μM), human MacroD1, human MacroD2, VEEV Macro, and human PARP9 Macro2; compared with ADPr, it shows significantly improved binding potency for all tested macrodomains except CHIKV Macro^[2].
TFMU-ADPr (2 mM; 5 d) binds to the conserved ADPr-binding pocket of SARS-CoV-2 Macro1, and its TFMU group forms favorable interactions with hydrophobic residues in the adjacent hydrophobic groove, thereby contributing to high binding affinity^[2].
TFMU-ADPr acts as an inhibitor of the ADPr hydrolase activity of SARS-CoV-2 Macro1 and human MacroD1 in cell lysates^[2].
Experimental procedure for in vitro detection using TFMU-ADPr mainly focuses on enzyme kinetics and inhibition assays, as specified below^[3]:
1. Experimental Preparation:
Establish a reaction system in a 384-well plate. The system contains 50 mM Na₂HPO₄ (pH 7.4), 10 mM MgCl₂, 5 mM DTT, TFMU-ADPr at a specified concentration as the substrate, and the enzyme to be tested (such as hARH1, hARH3, etc.). For inhibitor studies, pre-incubate the enzyme with the inhibitor at room temperature for 5 minutes first.
2. Reaction Initiation and Signal Detection:
After shaking the reaction system for 5 seconds, record the fluorescence signal using a Molecular Devices SpectraMax M3 microplate reader. Set the excitation wavelength (λ_Ex) to 385 nm and the emission wavelength (λ_Em) to 502 nm (or 495 nm). Read each well 6 times in low-gain mode, and record the fluorescence value every 5 seconds for a continuous detection duration of 15 min.
3. Data Processing:
Determine the initial reaction rate by fitting the linear segment of the reaction progress curve. Fit the initial rate to the substrate concentration, use the nonlinear curve fitting algorithm in GraphPad Prism 6, and analyze enzyme kinetic parameters based on the Michaelis-Menton equation. For inhibition assays, use enzyme-free reactions and inhibitor-free reactions as positive and negative controls, respectively, calculate the inhibition percentage, and obtain relevant inhibition parameters through dose-response curve fitting.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.