| Assay Method Information | |
| | Test on the inhibitory activity of the compounds of the invention against PRMT5 enzyme by radioisotopic method |
| Description: | Test method: A 1× enzyme reaction buffer (10 mM Tris 8.0 (Sigma, Cat. No. T2694-1L), 0.01% Tween-20 (Sigma, Cat. No. P2287-100 ML), 1 mM DTT (Sigma, Cat. No. D0632-10G)) was prepared. PRMT5 (Active Motif, Cat. No. 31921) and [3H]SAM (PerkinElmer, Cat. No. NET155V001MC) were added to the 1×enzyme reaction buffer, to prepare a 25/15× mixed solution (PRMT5 final concentration: 5 nM, [3H]SAM final concentration: 0.3 μM). 15 μL of this solution was transferred into a 384-well microplate (Corning 384-well Polypropylene Storage Microplates, Cat. No. 3657) with various concentrations of the compounds (DMSO final concentration 1%), and incubated at room temperature for 60 minutes. A polypeptide substrate, GL-27 (Ac-SGRGKGGKGLGKGGAKRHRKVGG-K (Biotin) (GL Biochem, Cat. No. 342095)), was added into the 1×enzyme reaction buffer to prepare a 25/10× substrate solution. Then 10 μL of the polypeptide substrate solution (final concentration of the polypeptide substrate: 100 nM) was added, a reaction was stirred at room temperature for 120 minutes, and then 5 μL 6× ice cold SAM (Sigma, Cat. No. A7007-100 MG) solution was added to stop the reaction (SAM final concentration: 0.125 mM). 25 L of the reaction mixture was transferred into a FlashPlate (Streptavidin FlashPlate HTS PLUS, High Capacity, 384-well, Perkin Elmer, Cat. No. SMP410A001PK), and incubated at room temperature for 1 h. After washed three times with distilled water containing 0.1% Tween-20, the microplate was read on a MicroBeta reader for CPM data (Counts Per Minute). After the CPM raw data of the compounds at various concentrations were obtained, the data were normalized according to Inh %=(Max-Sample)/(Max-Min)*100%, and the enzyme activity inhibition rate Inh % at each concentration point was obtained (wherein Max is the CPM value of a positive well with the enzyme, Min is the CPM value of a negative well without the enzyme, and Sample is the CPM value of the sample well treated with the compounds). Then the inhibition rate Inh % (Y) corresponding to each concentration (X) was input in EXCEL, and the IC50 value (the half maximal inhibitory concentration) of each compound was calculated with the XLfit plug-in according to the built-in four-parameter fitting equation Y=Bottom+(Top-Bottom)/(1+(IC50/X)*HillSlope). |
| Affinity data for this assay | |
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