Epi Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2)
Name: Epi Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2) ..more
BioActive Compounds: 1241
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Sanjay Adhikari, Georgetown University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA035193-01A1
Grant Proposal PI: Sanjay Adhikari
External Assay ID: TPD2_INH_EPIABS_1536_1X%INH PRUN
Name: Epi Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2)
The TopoisomeraseII (TopII) family is an important class of topoisomerases that produces transient 5? DNA-protein crosslinks [1, 2]. Drugs targeting these enzymes act by stabilizing the crosslinks, producing a covalent DNA-protein complex [3, 4]. Despite their clinical efficacy, drugs that target TopII (TopII poisons) may lead to negative side-effects, including secondary malignancies. The repair of TopII-DNA complexes is poorly understood. Recently, a human 5?- tyrosine phosphodiesterase (hTDP2) has been identified for the excision of TopII-DNA adducts [5, 6]. As hTDP2 repairs the cytotoxic adducts produced by TopII poisons, it is likely that effective inhibition of hTDP2 can be targeted to improve the efficacy of TopII poisons and reduce the side-effects. It has been shown that the knockdown/knockout of TDP2 activity in A549 (non-small cell lung cancer cell line, NSCLC) and DT40 cells increased sensitivity to the TopII targeting agent etoposide, demonstrating that hTDP2 can be a target for adjuvant chemotherapy . Our hypothesis is effective inhibition of hTDP2 will increase the cancer cell killing efficacy of TopII poisons like etoposide . Therefore, hTDP2 inhibitor screening, especially in a high-throughput manner, will expedite drug discovery.
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2. Wang, J.C., Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Q Rev Biophys, 1998. 31(2): p. 107-44. PMID 9794033.
3. Cutts, S.M., et al., The power and potential of doxorubicin-DNA adducts. IUBMB Life, 2005. 57(2): p. 73-81. PMID 16036566
4. Habermeyer, M., et al., Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity. Chem Res Toxicol, 2005. 18(9): p. 1395-404. PMID 16167831
5. Cortes Ledesma, F., et al., A human 5'-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage. Nature, 2009. 461(7264): p. 674-8. PMID 19794497.
6. Bahmed, K., K.C. Nitiss, and J.L. Nitiss, UnTTrapping the ends: a new player in overcoming protein linked DNA damage. Cell Res, 2010. 20(2): p. 122-3. PMID 20118967.
7. Zeng, Z., et al., TDP2/TTRAP is the major 5'-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular resistance to topoisomerase II-induced DNA damage. J Biol Chem, 2011. 286(1): p. 403-9. PMID 21030584.
8. Bandele, O.J., S.J. Clawson, and N. Osheroff, Dietary polyphenols as topoisomerase II poisons: B ring and C ring substituents determine the mechanism of enzyme-mediated DNA cleavage enhancement. Chem Res Toxicol, 2008. 21(6): p. 1253-60. PMID 18461976.
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TDP2, hTDP2, tyrosyl-DNA phosphodiesterase 2, PRUN, primary, singlicate, biochemical, inhibit, inhibitor, inhibition, inh, p-nitrophenol, nitrophenol, hydrolysis, T5PNP, PNP, p-nitrophenyl-thymidine-5?-phosphate, cancer, DNA repair, excise, topoisomerase, phosphodiesterase, substrate, absorbance, colorimetric, chromogenic, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute
The purpose of this assay is to identify compounds that act as inhibitors of the activity of tyrosyl DNA phosphodiesterase 2 (TDP2). TDP2 is a divalent cation-dependent enzyme that repairs TopII-associated DNA strand breaks. It is hypothesized that inhibitors of TDP2 may serve as useful adjuvants in combination with cancer drugs such as etoposide.
In this biochemical assay, recombinant human TDP2 protein is incubated at 37 degrees Celsius with the T5PNP substrate in the presence of Mg2+-containing assay buffer.T5PNP is a substrate for snake venom phosphodiesterase, as well as a substrate for TDP2. As a substrate for TDP2, T5PNP is used to mimic the TopII-DNA complex. TDP2 cleaves the phosphodiester bond in T5PNP, and the chromogenic p-nitrophenol group is released. As time increases the TDP2 enzyme will increasingly catalyze hydrolysis of the T5PNP substrate, resulting in increased release of p-nitrophenol and detection at 415nM wavelength. Compounds are tested in singlicate at a final nominal concentration of 12.8microM.
Prior to the start of the assay, 2 ul of a solution containing T5PNP subtrate (final concentration 5mM) in assay buffer (50mM Tris-HCl pH7.5, 1mM DTT, 1mM MgCl2, 50mM KCl and 100ug/ml BSA) was dispended into a all wells of a 1536 well plate. Next, 39nL of test compound in DMSO or DMSO alone (1% final concentration) was added to the appropriate wells. The assay was started by dispensing 1 ul of a solution contanting TDP2 enzyme (120nM final concentration) in assay buffer to wells in columns 4-48 and 1ul of assay buffer alone to wells in columnes 1-3. Plates were centrifuged and incubated for 2hrs at 37 degrees Celsius at which time fluorescence intensity was measured (Ex. 405nm and Em. 405nm) using a EnVision microplate reader (Perkin Elmer).
Prior to further calculations, the following formula was used to calculate Epi Absorbance (EPIABS)
EPIABS = -log10( sample / background)
Sample is defined as the fluorescent intensity of wells containing test compounds or DMSO.
Background is defined as the fluorescent intensity of wells containing buffer and T5PNP only.
The % inhibition for each well was then calculated as follows:
%_Inhibition = ( EPIABS_Test_Compound - MedianEPIABS_Low_Control ) / ( MedianEPIABS_High_Control - MedianEPIABS_Low_Control ) * 100
Test_Compound is defined as wells containing test compound, TDP2 and T5PNP.
High_Control is defined as wells containing only Buffer and T5PNP.
Low_Control is defined as wells containing DMSO, TDP2 and T5PNP.
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-22, and for inactive compounds 22-0.
List of Reagents:
TDP2 (supplied by Assay Provider)
Tris Base(Sigma, 93349)
DTT (Sigma, 43815)
BSA (Sigma, A2153)
KCl (Sigma, P9333)
T5PNP (Sigma, T4510)
1536 well plates (Corning 7254)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)