uHTS identification of SUMO1-mediated protein-protein interactions
There are no published patents or studies specifically describing small molecule inhibitors of proteinprotein interactions mediated by the Small Ubiquitin-like Modifications (SUMO). Given this gap, our rationale in developing a HTS assay is based on a need for discovering small molecule probes that can specifically act as "chemical modulators" of SUMO-mediated signaling and cellular functions. An more ..
BioActive Compounds: 1202
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1R21NS066498-01
Assay Provider: Yuan Chen, Ph.D., City of Hope, Duarte, CA
There are no published patents or studies specifically describing small molecule inhibitors of proteinprotein interactions mediated by the Small Ubiquitin-like Modifications (SUMO). Given this gap, our rationale in developing a HTS assay is based on a need for discovering small molecule probes that can specifically act as "chemical modulators" of SUMO-mediated signaling and cellular functions. An important objective of this research program is to provide new insight into the regulation of cancer cell resistance to chemo and radiation therapy. A small molecule probe will be utilized to address our key hypothesis:
inhibitors of the SUMOdependent protein-protein interactions can sensitize DNA damaging chemotherapeutic drugs and radiation for cancer therapy by inhibiting the Non-Homologous-End-Joining(NHEJ) DNA repair pathway.
Primary assay is a TR-FRET based on the N-terminal-fluorescein tagged-SUMO 1 specific sequence peptide identified from NMR studies by Dr. Chen (F-S1: Fluorescein-linker- DNEIEVIIVWEKK) binding to purified GST-tagged SUMO1 protein, which is then captured by the terbiumlabeled mouse anti-GST-antibody from Cisbio (Tb-anti-GST). The binding event brings the fluorescein acceptor moiety in close proximity to the Tb-donor to allow time-resolved lanthanide fluorescence from the terbium. In an orthogonal assay, the direct fluorescence polarization change of free F-S1 upon binding to the larger SUMO1 is monitored.
Hay, R.T., Protein Modification by SUMO. Trends Biochem Sci, 2001.26 (5).
Saitoh, H. and J. Hinchey, Functional heterogeneity of small ubiquitin-related proetin modifiers SUMO-1 versus SUMO-2/3. J Biol Chem, 2000.275(9): p6252-8.
Bartek, J. and Z. Hodny, SUMO boosts the DNA damage response barrier against cancer. Cancer Cell, 2010.17(1):p. 9-11
SUMO-1 protein (Assay provider Lab)
FITC-F1 Peptide (BioPeptide)
Anti-GST Terbium antibody (CisBio 61GSTTLAL)
Assay plate: Aurora 1536 white Solid Bottom Plate
DTT (Sigma 43816)
Tween-20 (MP Biomedicals)
HEPES (Sigma H0887 1M)
I. Compound Addition:
1- Using LabCyte Echo, transfer 20 nL from 2 mM compound source plate into assay plate Columns 5-48 (final concentration of test compounds is 20 uM, 1.0% DMSO), and 20 nL of DMSO to control wells in Columns 1-4.
II. Reagent Addition
2- Make up a 2X stock solution of SUMO1 and Cisbio Tb anti-GST antibody.
3- Make up at 2X stock solution of F-S1 peptide.
4-Incubate protien/ab mixture for 1 hr.
5-Add 2ul protein/ab mixture to wells 3-48.
6-Add 2ul buffer/ab to wells 1-2.
7-Spin down plates without lids on Vspin at 2000 rpm for 2 min.
8-Add 2ul peptide to entire plate.
9-Spin down plates without lids on Vspin at 2000 rpm for 1 min.
10- Put lid on, and then incubate plates at room temp for 1hr.
IV. Reading plates:
11- Read plates on envision using a TR-FRET protocol em340/ and ex520/485.
Compounds that demonstrated a normalized or corrected % activity >= 40% are defined as active in the assay.
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)