MLPCN SirT-5 Measured in Biochemical System Using Imaging - 7044-01_Inhibitor_Dose_CherryPick_Activity_Set5
Keywords: SirT-5, NAD-dependent desuccinylase and demalonylase, succinyl lysine modification, malonyl lysine modification, transcriptional and metabolic regulation. ..more
BioActive Compounds: 58
Keywords: SirT-5, NAD-dependent desuccinylase and demalonylase, succinyl lysine modification, malonyl lysine modification, transcriptional and metabolic regulation.
Assay Overview: This work aims to develop a high-throughput assay to find/screen for specific inhibitors of SirT-5 which can be used to study the biological function of SirT-5. This includes the function of two novel SirT-5 protein post-translational modifications, lysine succinylation and malonylation.
Expected Outcome: This assay utilizes a succinyl peptide conjugated to 7-amino-4-methylcoumarin (AMC), ISGASE(SuK)-AMC, where SuK represents succinyl lysine. This substrate is not fluorescent by itself but is a substrate for SirT5 which catalyzes the removal of the succinyl group. Loss of the succinyl group makes the substrate molecule suseptable to the trypsin cleavage, producing the fluorescent product 7-amino-4-methylcoumarin (AMC) which results in an increase in fluorescenct signal. Inhibitors of either SirT5 or trypsin will result in a decreased fluorescence emission. Future experiments will determine whether the active compounds inhibit the SirT5 reaction or whether they interfere with the subsequent tryspsin cleavage reaction.
1.) A 10 mM solution of ISGASE(SuK)-AMC is prepared as a DMSO stock solution. This solution is diluted to a 20 uM working solution I with reaction buffer containing 20 mM Tris-HCl, 1 mM dithiothreitol, 100 uM nicotinamide adenine dinucleotide, pH 7.4.
2.) A second working solution is prepared using the 90 uM stock SirT-5 solution by diluting the stock solution using PBS, pH 7.4 to a final concentration of 0.6 uM.
3.) The enzyme reaction is initiated by the simultaneous 2.5 uL additions of working solutions from #1 and #2 above into a 1536-well, black Aurora plate. This results in a reaction with a final substrate ISGASE(SuK)-AMC concentration of 10 uM and a final SirT-5 concentration of 0.3 uM.
4.) The reaction is allowed to proceed for 1 hr. at room temperature.
5.) 10X Trypsin (0.5% Trypsin-EDTA) is diluted to 1X (0.05%) by the addition of H20. 2.5 uL of 1X trypsin is then added to the reaction and incubated at room temperature for 1 hr (final trypsin concentration 0.0167% = 0.33X).
6.) After the 1 hr. incubation, the reaction progress is evaluated by monitoring the fluorescence emission at 460 nm. This can be done using an Envision with 50/50 splitter, Ex. 380 nm and Em 460 nm. Or using the View Lux equipped with the same filter set.
* Human SirT 5 was expressed and purified by the Lin laboratory at Cornell.
* ISGASE(SuK) was synthesized and purified by the Lin laboratory at Cornell.
* ISGASE(K) was synthesized and purified by the Lin laboratory at Cornell
* Suramin*hexasodium was purchased from Enzo, cat. # ALX-430-022
* 0.5% Trypsin-EDTA (10X) was purchased from Gibco (Life Technologies), cat. # 15400-054
PRESENCE OF CONTROLS: Neutral control wells (NC; n=144) and positive control wells (PC; n=144) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)