Dose Response confirmation of small molecule inhibitors originally identified via uHTS of Artemis endonuclease activity via a fluorescence intensity assay
Nonhomologous DNA end joining (NHEJ) is the primary DNA repair pathway in human cells for the repair of double-strand DNA breaks. Like most DNA repair pathways, NHEJ relies on three enzyme activities: a nuclease to remove damaged DNA, polymerases to fill-in new DNA, and a ligase to restore DNA strand integrity. The primary nuclease for NHEJ is Artemis, which is activated when a protein kinase more ..
BioActive Compounds: 69
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 Number: 1 R03 MH095489-01A1
Assay Provider: Michael Lieber, M.D., Ph.D., University of Southern California, Los Angeles, CA
Nonhomologous DNA end joining (NHEJ) is the primary DNA repair pathway in human cells for the repair of double-strand DNA breaks. Like most DNA repair pathways, NHEJ relies on three enzyme activities: a nuclease to remove damaged DNA, polymerases to fill-in new DNA, and a ligase to restore DNA strand integrity. The primary nuclease for NHEJ is Artemis, which is activated when a protein kinase called DNA-PKcs makes contact with a double-stranded DNA end at a chromosomal break site. Artemis is an endonuclease which nicks the DNA at transitions between single- and double-stranded DNA. It is very important for repairing a critical subset of double-strand breaks caused by X-rays, as found in therapeutic radiation. Artemis is even more critical for the repair of DNA damage caused by Type II topoisomerase inhibitors, such as the etoposide, VP-16, which is used in many human cancer chemotherapy regimens. It has been shown that human pre-B acute lymphoblastic lymphoma (ALL) cells that lack Artemis are much more susceptible to the cytotoxic effects of VP-16 at concentrations well below those used clinically. Therefore, inhibitors of Artemis might be synergistic with topoisomerase II inhibitors. Hence, complete inhibition of Artemis activity by a small molecule would be expected to recapitulate this topoisomerase II inhibitor hypersensitivity.
In addition to being critical in repairing etoposide and a subset of X-ray induced DNA damage, the most essential role of Artemis is one that is specific to B and T cell precursors, and the ALL malignancies derived from those precursors. The endonuclease activity of Artemis includes not only its ability to cut 5' and 3' overhangs (which are its role in etoposide and X-ray repair), but also the opening of DNA hairpins which are involved in the immunoglobin and T-cell receptor gene rearrangement process called V(D)J recombination. Without Artemis, the hairpins remain unopened, the DNA ends are not joined efficiently or at all, and the cell cycle is markedly delayed while the cells attempt a variety of less efficient pathways for their repair.
For the reasons stated above, it has been hypothesized that a small molecule that strongly inhibits Artemis might have strong potential therapeutic use for human ALL and other human cancers. The purpose of the assay described herein, is to confirm hits in dose response derived from the HTS campaign (AID 686954) used to identify potential inhibitors of Artemis endonuclease activity. This was accomplished via the development and performance of an assay that utilizes a quenched, labeled substrate that fluoresces upon endonucleolytic cleavage.
Sequence: DCLRE1C DNA cross-link repair 1C [ Homo sapiens (human) ]
Artemis HTS Protocol
A. Brief Description of the Assay:
This assay was used to confirm hits derived from the HTS campaign to identifiy compounds that inhibit Artemis endonuclease activity. The assay uses a haipin oligomer as a substrate that has been labeled with 6-FAM at the 5' end and the quencher, BHQ-1, at the 3' end (5' - /6-FAM/-T*T*T*T*TT TTG CCA GCT GAC GCG CGT CAG CTG GC-/BHQ-1/-3'). Artemis activity is determined by measuring the change in fluorescent intensity in the assay as the BHQ quencher and 6-FAM are separated as a result of overhang cleavage near the 5' end of the DNA oligo.
Item, Source, Cat #
Glycerol, MP Biomedicals, 194680
NaCl powder, Molecular Biology Tested, Sigma-Aldrich, S3014-5KG
1 M Trizma hydrochloride solution pH 7.4, molecular biology grade, Sigma-Aldrich, T2663-1L
1 M KCl solution, molecular biology grade, Teknova, P0320
1 M MnCl2 solution, molecular biology grade, Fisher Scientific, BP541-100
1 M DL-Dithiothreitol solution (DTT), molecular biology grade, Sigma-Aldrich, 43816
Molecular Biology Grade H2O, Mediatech, 46-000-CM
Artemis enzyme, Assay Provider, n/a
DNA oligo [Custom Mid-Scale Molecular Beacon (5' - /6-FAM/-T*T*T*T*TT TTG CCA GCT GAC GCG CGT CAG CTG GC-/BHQ-1/-3'), 32 bases, *thioester bonds], Integrated DNA Technologies, 83099375
1536 well black solid flat bottom Medium-Binding plate, Corning, 3724
1. Using LabCyte Echo, transfer varying volumes from a Echo qualified plate containing test compounds in DMSO into assay plate to achieve desired dose concentrations and range. Transfer DMSO to the positive and negative control wells and backfill dose response wells to equilibrate DMSO volumes and final assay concentration across the assay plate.
Note: Artemis enzyme should be thawed on ice, and DNA at room temperature in dark just prior to use in assay. Prepare Reagents as described in sections D. Recipe. Avoid exposure to light as much as possible. Add the Artemis Enzyme 30 min prior to use in assay. Keep the enzyme solution on ice.
2. Set up Combi and clean tubings as described in section Instrument setting.
3. Using Combi, dispense 2 ul/well of 20mM MnCl2 Buffer Solution into columns 1-48.
4. Using Combi, dispense 2 ul/well of Storage Buffer Solution into columns 1-2.
5. Using Combi, dispense 2 ul/well of Artemis Enzyme Solution into columns 3-48.
6. Spin the plate at 1000 rpm for 1 min using a centrifuge.
7. Cover the plate with Kalypsys lid (seal the holes of the lid if it hasn't been already) and incubate plates at room temperature for 30 min.
8. Using Combi, dispense 2 ul/well of DNA Substrate Solution into columns 1-48.
9. Spin the plate at 1000 rpm for 1 min using a centrifuge.
10. Cover the plate with Kalypsys lid and incubate at 37degrees C for 60 min.
11. Read the plate on a plate reader capable of reading fluorescence at Ex. 485 nm, Em. 520 nm with the temperature set at 37 degrees C.
Storage Buffer Preparation:
25 mM Tris-HCl pH 7.4, 10% Glycerol, 100 mM NaCl (Powder, Molecular Biology Tested)
Note: Make single batch large enough for the entire screen and store the aliquots in -80 degrees C.
20 mM MnCl2 Buffer Solution:
25 mM Tris-HCl pH 7.4, 10 mM KCl, 20 mM MnCl2, 1mM DTT, Molecular Biology Grade Water
Artemis Enzyme Solution (in 0 mM MnCl2 Buffer):
25mM Tris-HCl pH 7.4, 10mM KCl, 1mM DTT, 0.09mg/mL Artemis Enzyme (0.03 mg/ml FAC), Molecular Biology Grade Water
Note: Make the buffer component first and add Artemis Enzyme 30 min before dispense. Keep solution in ice.
Storage Buffer Solution
25mM Tris-HCl pH 7.4, 10 mM KCl, 1mM DTT, 0.09 mg/mL Storage Buffer (0.03 mg/ml FAC), Molecular Biology Grade Water
DNA Substrate Solution:
25 mM Tris-HCl pH 7.4, 10 mM KCl, 10 mM MnCl2, 1mM DTT, 0.36uM DNA Substrate (0.12 uM FAC), Molecular Biology Grade Water
Note: Add DNA substrate last. Keep the solution away from light.
E. Special Notes:
1. All reagents should be made up according to manufacturer's recommendations or otherwise in Mol. Grade Water.
2. Storage conditions after reagents are made up:
Artemis enzyme, -80 degrees, one time use only (do not use previously freeze-thawed samples)
DNA, -20 degrees, light sensitive
DTT, -20 degrees
Tris-HCl, KCl, MnCl2, 4 degrees or room temperature
3. Preparation of DNA oligo substrate requires heating to 80-85 degrees C, followed by slow cooling to yield the hairpin formation.
Compounds that have an IC50 of less than 20 uM are defined as actives in this confirmatory 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 and is not applicable to 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)