uHTS Fluorescent Assay Using Nedd8 Protein Substrate for Identification of Inhibitors of Sentrin-Specific Protease 8 (SENP8)
The human peptidase clan CE is defined by seven proteases with a common fold and catalytic mechanism, commonly known as SENPs [1-3]. SENPs 1, 2, 3, 5, 6, and 7 are specific for processing SUMO precursors and removing SUMO conjugated to protein substrates, while the most divergent member of the family, SENP8, also called NEDP1  or DEN1 , is essential for the similar metabolism of the small more ..
BioActive Compounds: 2340
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, La Jolla, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH094200-01
Assay Provider: Dr. Guy Salvesen, Sanford-Burnham Medical Research Institute, La Jolla, CA.
The human peptidase clan CE is defined by seven proteases with a common fold and catalytic mechanism, commonly known as SENPs [1-3]. SENPs 1, 2, 3, 5, 6, and 7 are specific for processing SUMO precursors and removing SUMO conjugated to protein substrates, while the most divergent member of the family, SENP8, also called NEDP1  or DEN1 , is essential for the similar metabolism of the small ubiquitin-related modifier Nedd8 [4, 6]. The major targets for neddylation are cullin proteins that function as part of the catalytic core of cullin-RING ubiquitin ligases (CRLs) . The substrates of CRLs crucially regulate cell cycle progression, DNA replication, and DNA damage response mechanisms, and thus the Neddylation machinery constitutes a set of valuable drug targets for cancer therapy .
Prior SENP uHTS campaigns using penta-peptide based assays (AIDs 2575, 2582, 434986, 489001), did not identify selective inhibitors of SENP8, likely due to targeting only the catalytic center. The aim of this lead discovery campaign is to identify novel SENP8 selective probes that target the physiologically relevant substrate-bound conformation of SENP8 induced by Nedd8 protein binding. Potent and selective SENP8 chemical probes would provide invaluable tools to help elucidate the function of this therapeutically important enzyme, and may ultimately lead to the development of new anticancer therapies.
In order to find selective SENP8 ligands and to explore possible allosteric binding sites, we developed a novel Fluorescent Intensity uHTS assay that utilizes physiological protein substrates and is based on SENP8-dependent deconjugation of the aminocoumarin from the Nedd8-AMC substrate. This screening assay was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN).
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
4. Mendoza, H. M., Shen, L. N., Botting, C., Lewis, A., Chen, J., Ink, B., and Hay, R. T. (2003)
NEDP1, a highly conserved cysteine protease that deNEDDylates Cullins. J Biol Chem 278,
5. Gan-Erdene, T., Nagamalleswari, K., Yin, L., Wu, K., Pan, Z. Q., and Wilkinson, K. D. (2003)
Identification and characterization of DEN1, a deneddylase of the ULP family. J Biol Chem 278,
6. Wu, K., Yamoah, K., Dolios, G., Gan-Erdene, T., Tan, P., Chen, A., Lee, C. G., Wei, N.,
Wilkinson, K. D., Wang, R., and Pan, Z. Q. (2003) DEN1 is a dual function protease capable of
processing the C terminus of Nedd8 and deconjugating hyper-neddylated CUL1. J Biol Chem
7. Petroski, M. D., and Deshaies, R. J. (2005) Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6, 9-20.
8. Petroski, M. D. (2010) Mechanism-based neddylation inhibitor. Chem Biol 17, 6-8.
1) Full-length SENP8 - provided by Dr. Salvesen's laboratory.
2)Nedd8 AMC (R&D Systems, Boston Biochem, Cat #UL-552)
3) Pro-Nedd8 protein substrate - purified by Sanford-Burnham Protein Facility
4) Assay Buffer (75 mM Hepes, pH 7.8, 2 mM DTT, 0.5 mM EDTA, BSA 0.1%, 0.005% Tween 20)
5) Corning 1536-well black flat bottom plates (Cat #3726)
SENP8/Nedd8 primary HTS protocol:
1) Using Labcyte Echo555, dispense 20 nl of 2 mM compound into columns 5 - 48, while 20nl of 100% DMSO into columns 1 - 4.
2) Using Beckman BioRAPTR, dispense 1 ul of 12 pM SENP8 in Assay Buffer into columns 3 - 48, while 1 ul of just Assay Buffer into columns 1 and 2.
3) Using Beckman BioRAPTR, dispense 1 ul of 1.6 uM Pro-Nedd8 and 300 nM Nedd8 AMC in Assay Buffer to all wells of the plate.
4) Using Eppendorf centrifuge 5810, centrifuge plates at 1500 rpm for 1 minute
5) Incubate at room temperature for 30 min.
6) Read plates on Perkin Elmer Envision 2104 plate reader at Ex/Em 355/460 nm in fluorescence intensity mode.
Compounds that demonstrated a normalized inhibition of >= 30% or corrected inhibition of >= 40% at 20 uM concentration are defined as actives in this 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)