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BioAssay: AID 652115

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
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 Tested Compounds
 Tested Compounds
All(323506)
 
 
Active(3752)
 
 
Inactive(319778)
 
 
Inconclusive(78)
 
 
 Tested Substances
 Tested Substances
All(326702)
 
 
Active(3756)
 
 
Inactive(322868)
 
 
Inconclusive(78)
 
 
AID: 652115
Data Source: Broad Institute (7044-01_Inhibitor_SinglePoint_HTS_Activity_Set5)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-03-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 3752
Depositor Specified Assays
AIDNameTypeComment
652129Broad Institute MLPCN SirT5 Inhibitor Probe Projectsummary
Description:
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.
Protocol
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
Comment
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.

NORMALIZATION:
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.

PUBCHEM_ACTIVITY_SCORE:
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.

PUBCHEM_ACTIVITY_OUTCOME:
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
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_14.98uM_(%) (14.98μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: R21NS073049

Data Table (Concise)
Classification
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