Dose response confirmation of small molecule inhibitors of the thioesterase domain of fatty acid synthase via a kinetic, 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 ..
BioActive Compounds: 967
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 assay is to confirm hits identified in the FAS-TE primary assay (AID602261) in dose response in kinetic mode. This is accomplished via the kinetic monitoring of an enzymatic reaction utilizing a fluorogenic substrate, O-methyl fluorescein heptanoate (OMFH). By monitoring the change in fluorescence as opposed to an endpoint measurement, compounds that are interfering with the measurement of the assay via fluorescent interference can be easily identified.
A. Brief Description of the Assay:
The purpose of this assay is to confirm, in dose response and in kinetic mode, inhibitors of the FAS-TE enzyme identified in the primary screen. The readout is an endpoint fluorescence measurement of the product of the enzymatic assay.
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
DMSO, 5.50 % (0.8% from test compound)
Reaction volume, 4 uL/well
Test compound concentration,
Final DMSO concentration, 5.5%
1: Using LabCyte Echo, transfer varying volumes of test compounds in DMSO compound source plate into the test compound wells of an assay plate to achieve the appropriate test compound concentration and range. Backfill test compound wells and positive and negative control wells with DMSO to equilibrate DMSO across assay plate.
2: Using Kalypsys, dispense 2 uL of 2x reaction buffer (no enzyme) to the psotive control wells, dispense 2 uL of 2x FASTE solution to test compound wells, and then dispense 2 uL of 2x substrate solution to all wells of the assay plate.
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 at Ex.480 and Em.540 kinetically with timepoints read every 10 minutes. The slope of each reaction is calculated based on the change in fluorescence between the initial read (T0) and the read at the 50th minute.
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.
2X reaction buffer
Reagent, Working Conc.
NaCl, 100 mM
Tris-HCl, 200 mM
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.
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.
Compounds that tested with an IC50 of <=20 uM are defined as actives in this assay.
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 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 and is not applicable in this assay
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
* Activity Concentration. ** Test Concentration.
Data Table (Concise)