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).

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.

Compounds that demonstrated a normalized or corrected activity of >= 50% at 15 uM concentration are defined as inhibitors of the reaction.

The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.

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

Grant Number: 1R03MH095532-01