MLPCN SirT-5 Measured in Biochemical System Using Imaging - 7044-01_Inhibitor_SinglePoint_HTS_Activity_Set5
Keywords: SirT-5, NAD-dependent desuccinylase and demalonylase, succinyl lysine modification, malonyl lysine modification, transcriptional and metabolic regulation. ..more
BioActive Compounds: 3752
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 expereiments 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) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
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.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 15.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
** Test Concentration.
Data Table (Concise)