uHTS Luminescent assay for identification of inhibitors of Sentrin-specific protease 7 (SENP7)
Modification of proteins by SUMO is a dynamic and reversible process. SUMOylation/deSUMOylation cycle regulates SUMOs function. Sentrin-specific proteases (SENPs) are involved in both the maturation of SUMO precursors (endopeptidase cleavage) and deconjugation of the targets (isopeptidase cleavage) [1-3]. There are seven SENPs (1, 2, 3, 5, 6, 7, 8) in humans, and several of these have been characterized as SUMO (or Nedd8) specific enzymes. ..more
BioActive Compounds: 4902
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 Proposal Number: 1R21 NS061758-01 fast track
Assay Provider: Dr. Guy Salvesen, Sanford-Burnham Medical Research Institute, San Diego, CA.
Modification of proteins by SUMO is a dynamic and reversible process. SUMOylation/deSUMOylation cycle regulates SUMOs function. Sentrin-specific proteases (SENPs) are involved in both the maturation of SUMO precursors (endopeptidase cleavage) and deconjugation of the targets (isopeptidase cleavage) [1-3]. There are seven SENPs (1, 2, 3, 5, 6, 7, 8) in humans, and several of these have been characterized as SUMO (or Nedd8) specific enzymes.
The objective of this project is to generate small molecule inhibitors specific for SENP7 (the deSUMOylating enzyme). 1536-well chemiluminescent screening assay utilizes RLRGG-aminoluciferin peptide substrate and is based on SENP7-dependent deconjugation of the aminoluciferin, which serves as a substrate for the coupled luciferase.
1. Mikolajczyk, J., Drag, M., Bekes, M., Cao, J. T., Ronai, Z. and Salvesen, G. S. (2007) Small Ubiquitin-related Modifier (SUMO)-specific Proteases: Profiling The Specificities And Activities Of Human SENPs. J Biol Chem 282, 26217-26224
2. Drag, M., Mikolajczyk, J., Krishnakumar, I. M., Huang, Z. and Salvesen, G. S. (2008) Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family. Biochem J 409, 461-469
3. Lima, C. D. and Reverter, D. (2008) Structure of the human SENP7 catalytic domain and poly-SUMO deconjugation activities for SENP6 and SENP7. J Biol Chem 283, 32045-32055
1) Z-RLRGG-aminoluciferin (Promega #X554X)
2) Luciferin Detection Reagent (LDR) Promega #V859B dissolved in 75mM Hepes, pH 7.8, 10 mM MgSO4.
3) Catalytic domain of human SENP7 (aa 640-984) - provided by Dr. Salvesen's laboratory.
4) Assay Buffer (75 mM Hepes, pH 7.8, 2 mM DTT, 1 mM EDTA, 0.1% BSA, 0.01% Tween 20)
5) Corning 1536-well white microtiter plates (Cat #3725)
SENP7 primary HTS protocol:
1) Using Labcyte Echo555, dispense 5 nl of 2 mM compound into columns 5 through 48, while 5nl of 100% DMSO into columns 1 through 4.
2) Using Thermo Scientific MultiDrop Combi nL, dispense 1 ul of 120 nM SENP7 in Assay Buffer into columns 3 through 48, while 1 ul of just Assay Buffer into columns 1 and 2.
3) Using Thermo Scientific MultiDrop Combi nL, dispense 1 ul of 160 uM Z-RLRGG-aminoluciferine in LDR to all wells of the plate.
4) Centrifuge plates briefly
5) Incubate at room temperature for 40 min.
6) Read plates on a Perkin Elmer Envision 2104 plate reader using the ultra-sensitive luminescence mode.
Compounds that demonstrated an inhibition of >= 50% at 5 uM concentration 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. Its utilization for the assay is described below.
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 demonstrated by a compound at 10 uM concentration:
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)