Dose-response confirmation of uHTS inhibitor hits of Sentrin-Specific Protease 8 in a Caspase-3 Fluorescence assay
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: 28
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.
The goal of this assay is to confirm hits in "uHTS Fluorescent Assay Using Nedd8 Protein Substrate for Identification of Inhibitors of Sentrin-Specific Protease 8 (SENP8)", AID 602440. In this biochemical assay, Caspase-3 deconjugates the aminocoumarin (AMC) label from DEVD peptide. The release of AMC is monitored through detection of unquenched fluorescence at Ex/Em 350/450 nm. The purpose of this counterscreen assay is to deprioritize cysteine-reactive compounds that inhibit Caspase-3, an out-of-family protease, relatively to the SENP family enzymes. SENP inhibitors that also inhibit Caspase-3 would be considered nonselective. This high throughput selectivity assay was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening
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 Caspase-3 - provided by Dr. Salvesen's laboratory.
2) Z-DEVD-AMC (Anaspec, Cat #60303-5)
3) Assay Buffer (20 mM Pipes, pH 7.2, 10 mM DTT, 0.1 mM EDTA, 10% sucrose, 0.1% BSA, 0.1% CHAPS)
4) Corning 1536-well black flat bottom plates (Cat #3724)
SENP8/Nedd8 confirmatory HTS protocol:
1) Using Labcyte Echo555, dispense 40 nL of serially diluted compounds into assay plate columns 5 - 48, and 40 nL of 100% DMSO into assay plate columns 1 - 4.
2) Using Thermo Scientific Multidrop Combi, dispense 2 muL of 20 muM Z-DEVD-AMC in Assay Buffer to all assay wells.
3) Using Thermo Scientific Multidrop Combi, dispense 2 muL of Assay Buffer into assay plate columns 1 and 2.
4) Using Thermo Scientific Multidrop Combi, dispense 2 muL of 1 nM Caspase-3 in Assay Buffer into columns 3 - 48 of the first plate.
5) Using Eppendorf centrifuge 5810, centrifuge plate at 1500 rpm for 1 minute.
6) Read plate on BMG Labtech PHERAstar Plus plate reader at Ex/Em 350/450 nm in fluorescence intensity kinetic Plate mode for 5 cycles, 219 seconds apart, for roughly 19 minutes.
7) Repeat steps 4 through 6 for subsequent plates, initiating reaction with Caspase-3 at 20-minute intervals.
Compounds with an IC50 <= 60 uM 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.
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:
The primary scoring is not applicable in this assay
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
* Activity Concentration. ** Test Concentration.
Data Table (Concise)