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BioAssay: AID 624325

Single concentration confirmation of uHTS inhibitor hits of the thioesterase domain of fatty acid synthase via a fluorescence intensity assay

This study will focus on developing drug-like inhibitors/probes against fatty acid synthase, an enzyme that is essential for growth of solid tumors. Notably, FAS has only marginal importance in adults because dietary fat provides for normal physiology. The link between FAS and cancer was uncovered in 1994 when Frank Kuhajda found that the OA-519 antigen, a marker for poor prognosis in breast more ..
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 Tested Compounds
 Tested Compounds
All(2252)
 
 
Active(1009)
 
 
Inactive(1243)
 
 
 Tested Substances
 Tested Substances
All(2255)
 
 
Active(1009)
 
 
Inactive(1246)
 
 
AID: 624325
Data Source: Burnham Center for Chemical Genomics (SBCCG-A865-FAS-TE-CP-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-06-22

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1009
Related Experiments
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AIDNameTypeComment
602261uHTS identification of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a fluorescence intensity assayScreeningdepositor-specified cross reference
602265Summary assay for small molecule inhibitors of the thioesterase domain of fatty acid synthaseSummarydepositor-specified cross reference
624326Dose response confirmation of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a kinetic, fluorescence intensity assayConfirmatorysame project related to Summary assay
624327Dose response confirmation of uHTS inhibitor hits of the thioesterase domain of fatty acid synthase via a fluorescence intensity assayConfirmatorysame project related to Summary assay
651670SAR analysis of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a fluorescence intensity assayConfirmatorysame project related to Summary assay
652164SAR confirmation of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a fluorescence intensity assayConfirmatorysame project related to Summary assay
652165SAR confirmation of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a kinetic, fluorescence intensity assayConfirmatorysame project related to Summary assay
652286SAR confirmation of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a kinetic, fluorescence intensity assay, round 2Confirmatorysame project related to Summary assay
652287SAR confirmation of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a fluorescence intensity assay, round 2Confirmatorysame project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1R03MH095532-01
Assay Provider: Dr. Jeffrey Smith, Sanford-Burnham Medical Research Institute, San Diego CA

This study will focus on developing drug-like inhibitors/probes against fatty acid synthase, an enzyme that is essential for growth of solid tumors. Notably, FAS has only marginal importance in adults because dietary fat provides for normal physiology. The link between FAS and cancer was uncovered in 1994 when Frank Kuhajda found that the OA-519 antigen, a marker for poor prognosis in breast and prostate cancer, is actually fatty acid synthase. A number of subsequent immunohistochemical analyses showed that increased expression of FAS is a hallmark of all major cancers. The correlation between expression of FAS and poor prognosis strongly suggests that this enzyme is mechanistically linked to disease progression, providing a strong rationale for pursuing the development of FAS inhibitors.

The FAS protein contains six enzymatic domains and an acyl-carrier protein (ACP). The final enzymatic pocket is a thioesterase, which liberates the final product (palmitate) from its link to the ACP. It is the thioesterase domain of FAS which we plan to target here. To our knowledge, no thioesterase (TE) has ever been targeted for drug development. The goal of this high-throughput assay is to identify hit compounds for the FAS-TE domain. This is accomplished via an enzymatic reaction utilizing a fluorogenic substrate, O-methyl fluorescein heptanoate (OMFH).

The goal of this assay is to confirm hits in "uHTS identification of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a fluorescence intensity assay", AID 602261.
Protocol
A. Brief Description of the Assay:
The purpose of this assay is to find inhibitors of the FAS-TE enzyme. The readout is endpoint fluorescence from the product of the enzymatic assay.
B. Materials:
Item, Source, Cat #
NaCl, Fisher, BP358-212
Trizma base, Sigma, T1503
HCl, Fisher, A466
TCEP, Sigma, 646547
Brij35, Sigma, B4184
Sarcosine, Sigma, 131776
FAS-TE, SBMRI protein production facility, N/A
OMFH, CPCCG chemistry, N/A
OMF, Research Organics, 0143M
DMSO, Sigma, D2650
Molecular Grade Water, Cellgro, 46-000-CM
Aurora low-base plates, Aurora Biotech, 00029844
C. Final Assay Conditions:
Reagent, Final Concentration
NaCl, 50 mM
Tris-HCl, 100 mM
TCEP, 1 mM
Brij35, 0.005 %
Sarcosine, 500 mM
FAS-TE enzyme, 0.9 uM
OMFH, 0.03
DMSO, 5.75 % (0.75% from test compound)
Reaction volume, 4 uL/well
Test compound concentration,
Final DMSO concentration, 5.75% at 15 uM test compound
D. Procedures:
Step:
1: Using LabCyte Echo, transfer 30 nL of test compounds from a 2 mM compound source plate into assay plate Cols. 5-48 (final concentration of test compounds is 15 uM). Also transfer 30 nL of 100% DMSO into assay plate Cols. 1-4.
2: Using Kalypsys, dispense 2 uL of 2x reaction buffer (no enzyme) to each well on columns 1 and 2, dispense 2 uL of 2x FASTE solution to each well on columns 3-48, and then dispense 2 uL of 2x substrate solution to each well on columns 1-48.
3: Spin plates at 1000 rpm for 1 minute on Vspin.
4: Incubate for 60 min at room temp.
5: Read plates on PerkinElmer Envision.
E. Plate Map:
Positive (Low) control (P) in columns 1 and 2, DMSO and O-methyl fluorescein hexanoate substrate but no enzyme.
Negative (High) control (N) in columns 3 and 4, DMSO, O-methyl fluorescein hexanoate and enzyme
Test compound in columns 5 - 48, Test compound and O-methyl fluorescein hexanoate and enzyme.
F. Recipe:
2X reaction buffer
Reagent, Working Conc.
NaCl, 100 mM
Tris-HCl, 200 mM
TCEP,
Brij35, 0.01 %
Sarcosine, 1000 mM
in molecular grade water
2x FAS-TE enzyme solution
Reagent, Working Conc.
FAS-TE enzyme, 1.8 uM
in 2x reaction buffer
2x substrate solution
Reagent, Working Conc.
OMFH, 60 uM
DMSO, 10% in molecular grade water
5 M NaCl, 14.6 g of NaCl + water to 50 mL.
1 M Tris-HCl pH 7.5, 60.6 g of Trizma base + water + HCl to pH 7.5 and total volume 500 mL.
5 M Sarcosine, 222.8 g of sarcosine + water to 500 mL, filter sterilize and store at 4 degrees C.
G. Note:
1. Use fresh molecular grade water to make all reagents.
2. All reagents can be kept on ice to up to 4 hours during the assay.
Comment
Compounds that demonstrated a % activity >= 50% at 15 uM concentration in at least one replicate are defined as inhibitors of the reaction.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Activity at 15 uM_Mean (15μM**)Mean % inhibition in primary screeningFloat%
2%Activity at 15 uM_1 (15μM**)% inhibition for the first replicateFloat%
3%Activity at 15 uM_2 (15μM**)% inhibition for the second replicateFloat%
4%Activity at 15 uM_3 (15μM**)% inhibition for the third replicateFloat%
5Value_1 (15μM**)Measured value of the sample FloatRFU
6Value_2 (15μM**)Measured value of the sample FloatRFU
7Value_3 (15μM**)Measured value of the sample FloatRFU
8Mean High_1Mean fluorescent signal of negative controls in the corresponding plate FloatRFU
9Mean High_2Mean fluorescent signal of negative controls in the corresponding plate FloatRFU
10Mean High_3Mean fluorescent signal of negative controls in the corresponding plate FloatRFU
11STD Deviation High_1Standard deviation (n=64) of negative controls in the corresponding plateFloatRFU
12STD Deviation High_2Standard deviation (n=64) of negative controls in the corresponding plateFloatRFU
13STD Deviation High_3Standard deviation (n=64) of negative controls in the corresponding plateFloatRFU
14Mean Low_1Mean fluorescent signal of positive controls in the corresponding plateFloatRFU
15Mean Low_2Mean fluorescent signal of positive controls in the corresponding plateFloatRFU
16Mean Low_3Mean fluorescent signal of positive controls in the corresponding plateFloatRFU
17STD Deviation Low_1Standard deviation (n=64) of positive controls in the corresponding plate FloatRFU
18STD Deviation Low_2Standard deviation (n=64) of positive controls in the corresponding plate FloatRFU
19STD Deviation Low_3Standard deviation (n=64) of positive controls in the corresponding plate FloatRFU

** Test Concentration.
Additional Information
Grant Number: 1R03MH095532-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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