uHTS identification of small molecule inhibitors of the catalytic domain of the SUMO protease, SENP1 in a FRET assay
A growing body of evidence indicates that, SUMOylation plays key homeostatic roles in the androgen mediated development and function of the prostate both by restraining the transcriptional activity of the androgen receptor (AR) and by inducing the normal process of senescence, which is a growth-arrest program that limits the lifespan of mammalian cells and prevents tumor progression. Notably, more ..
BioActive Compounds: 774
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1R03MH095586-01
Assay Provider: Jorge A. Iniguez-Lluhi, Ph.D., University of Michigan Medical School, Ann Arbor, MI
A growing body of evidence indicates that, SUMOylation plays key homeostatic roles in the androgen mediated development and function of the prostate both by restraining the transcriptional activity of the androgen receptor (AR) and by inducing the normal process of senescence, which is a growth-arrest program that limits the lifespan of mammalian cells and prevents tumor progression. Notably, recent data indicates that advanced prostate cancer cells, which are notoriously resistant to current therapies, evade SUMO-mediated homeostatic mechanisms through the specific upregulation of the SUMO protease SENP1. SENP1 is responsible for reversing the SUMOylation of multiple key proteins including AR. Notably, the gene for SENP1 is directly activated by AR and since SENP1 enhances AR activity by reversing AR SUMOylation, this leads to further SENP1 induction, and thus continued and self-sustaining suppression of SUMOylation.
The goal of this project is to identify small molecule inhibitors of the SENP1 enzyme by using a FRET-based assay to screen for selective small molecule inhibitors of the SUMO protease SENP1. This work describes the use of a FRET-based uHTS assay that utilizes the SENP1 catalytic domain and a fluorescent protein substrate to identify small molecule inhibitors from the MLPCN library of compounds.
SENP1 Assay HTS Protocol:
A. Brief Description of the Assay:
This assay identifies potential inhibitors of SENP1 enzyme. It will be measured by FRET in 1536 well plate format.
Item, source, catalog no.
His6-SENP1 Catalytic Domain, human recombinant (SENP1), Boston Biochem, E-700
SFCypet-SUMO1-SFYpet Substrate, Assay Provider, N/A
Catalase, Sigma, C30
Tris-HCl pH 8.0, Teknova, T1080
Na-EDTA, GIBCO, 15575
Sodium Chloride (NaCl), Mediatech, Inc., 46-032-CV
Beta-Mercaptoethanol (BME), Calbiochem, 444203
Bovine Serum Albumin (BSA), Sigma, A7888
Nonidet P40 (NP40), Accurate Chemical & Scientific Corp, A56009
Mol. Grade Water, Mediatech, Inc., 46-000-CM
1536 well black solid flat bottom plate, Corning, 3724
C. Assay Procedures:
1. Prepare Reagents as described in section E. Recipe.
2. Using LabCyte Echo, transfer 30 nL from a 2 mM test compound plates into assay plate Col. 5 - 48 (final concentration of test compounds is 10 uM, 0.5% DMSO). 30nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.
3. Spin plates at 1000 rpm for 1 minute in centrifuge.
4. Using the Beckman Coulter BioRAPTR, add 3 uL/well of control buffer (no enzyme control) to columns 1 and 2.
5. Using the BioRAPTR, add 3 uL/well of enzyme solution to col. 3-48 for the negative control and test compound wells.
6. Using the BioRAPTR, add 3 uL/well of substrate solution to col. 1-48.
7. Spin plates at 1000 rpm for 1 minute in centrifuge.
8. Incubate plates in the dark at room temperature for 50 minutes.
9. Read plates on the PerkinElmer EnVision using a FRET protocol that reads the CFP channel (Channel A) and the YFP channel (Channel B).
10. Conversion of substrate is determined by measuring the change in signal in the CFP channel relative to the YFP channel and is therefore expressed as a ratio of the fluorescence of Channel A (CFP) divided by the fluorescence of Channel B (YFP) after 50 minutes.
D. Plate Map:
Positive (low) control in columns 1 and 2, DMSO with substrate, no enzyme.
Negative (high) control in columns 3 and 4, DMSO with substrate and enzyme.
Test compound in columns 5-48, with substrate and enzyme.
1X Reaction Buffer
20mM Tris-Cl, pH8
1X Reaction Buffer
40pM SENP1 Enzyme in 1X Reaction Buffer (final enzyme concentration is 20pM)
50nM SFCypet-SUMO1-SFYpet Substrate in 1X Reaction Buffer (final substrate concentration is 25nM)
All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
SENP1 Enzyme should not be freeze thawed multiple times.
Storage conditions for stock solutions:
SENP1: -80 degrees.
SFCypet-SUMO1-SFYpet Substrate: -80 degrees.
Compounds with normalized or corrected %Activity >= 30% at 10 uM concentration, that have a raw RFU of less than 3081000 in the acceptor YFP channel, and have an FRatio >=0.5 and <=1.5 are defined as actives of the primary screening.
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 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
** Test Concentration.
Data Table (Concise)