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Biochemical Assays for the Discovery of TDP1 Inhibitors

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Biochemical Assays for the Discovery of TDP1 Inhibitors Published OnlineFirst July 14, 2014; DOI: 10.1158/1535-7163.MCT-13-0952 Molecular Cancer Models and Technologies Therapeutics Biochemical Assays for the Discovery of TDP1 Inhibitors Christophe Marchand1, Shar-yin N. Huang1, Thomas S. Dexheimer2, Wendy A. Lea2, Bryan T. Mott2, Adel Chergui1, Alena Naumova1, Andrew G. Stephen3, Andrew S. Rosenthal2, Ganesha Rai2, Junko Murai1, Rui Gao1, David J. Maloney2, Ajit Jadhav2, William L. Jorgensen4, Anton Simeonov2, and Yves Pommier1 Abstract Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I–, alkylation-, and chain terminator–induced DNA damage, whereas TDP2 repairs topoisom- erase II–induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors. Mol Cancer Ther; 13(8); 2116–26. Ó2014 AACR. Introduction consideration for combination therapies with existing Topoisomerase I (Top1)–mediated cleavage complexes anticancer treatments. There is currently no reported resulting from the trapping of Top1 by DNA lesions TDP1 inhibitor exhibiting a synergistic effect when used including abasic sites, oxidized bases, carcinogenic in combination with a Top1 inhibitor. Yet, the usefulness adducts (1–3), and anticancer Top1 inhibitors (topotecan, of a combination therapy with a TDP1 and a Top1 inhib- irinotecan, and non-camptothecin Top1 inhibitors; refs. 4, itor in the clinic is supported by genetic evidence. Genetic 5) are removed by TDP1 (for review, see refs. 6, 7). TDP1 inactivation of TDP1 confers hypersensitivity to camp- acts by cleaving the covalent bond between a 30-DNA tothecin (CPT) in human cells (18–20), murine cells (21, phosphate group and the catalytic tyrosine residue of the 22), chicken cells (17, 23), and in yeast (24). In addition, trapped Top1 (8–10). TDP1 can also remove a broad range mutation of the catalytic histidine to an arginine residue at of 30-blocking DNA lesions, including 30-phoshoglyco- position 493 (H493R) results in the accumulation of cova- lates (11, 12), 30-nucleosides (13, 14), and chain-terminat- lent TDP1-DNA intermediates (13), ultimately leading to ing anticancer and antiviral nucleotide analogs (15). TDP1 the rare autosomal recessive neurodegenerative disease has also been shown to act as a backup repair pathway for called spinocerebellar ataxia with axonal neuropathy topoisomerase II (Top2) cleavage complexes (16, 17). Both (SCAN1; ref. 25); SCAN1 cells are hypersensitive to CPT Top1 and Top2 are pharmacologic targets for widely used (18–21). Because there is yet no available TDP1 inhibitor anticancer drugs. Therefore, TDP1 inhibitors are under active in cells, an indirect way to inhibit the TDP1 pathway is actually to block PARP activity. Indeed, we recently showed that PARP1 is a critical cofactor of TDP1 in cells, Authors' Affiliations: 1Developmental Therapeutics Branch and Labora- acting by stabilizing TDP1 and facilitating its recruitment tory of Molecular Pharmacology, Center for Cancer Research, National to Top1cc damage sites (26). This mechanism is one of the Cancer Institute; 2National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda; 3Cancer Research Tech- underlying molecular mechanisms by which PARP inhi- nology Program, Leidos Biomedical Research, Inc., Frederick National bitors synergize with Top1 inhibitors (27–29). Laboratory for Cancer Research, Frederick, Maryland; and 4Department of Chemistry, Yale University, New Haven, Connecticut The discovery of TDP1 inhibitors has been challenging because previously known inhibitors either lack selectiv- Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). ity or cellular efficiency suitable for drug development (30). We previously reported the development and opti- Corresponding Authors: Christophe Marchand, NIH, 37 Convent Dr, Bethesda, MD 20892. Phone: 301-435-2463; Fax: 301-402-0752; E-mail: mization of a quantitative high-throughput screening [email protected]; and Yves Pommier, NIH, 37 Convent Dr, (qHTS) assay based on the AlphaScreen technology for Bethesda, MD 20892. Phone: 301-496-5944; E-mail: [email protected] the discovery of TDP1 inhibitors (31). In this study, we doi: 10.1158/1535-7163.MCT-13-0952 report the development of novel biochemical assays with Ó2014 American Association for Cancer Research. increased stringency for the confirmation of chemical hits 2116 Mol Cancer Ther; 13(8) August 2014 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst July 14, 2014; DOI: 10.1158/1535-7163.MCT-13-0952 TDP1 and TDP2 Inhibitor Screening À À À À obtained from our qHTS campaign using libraries at the 37C. TDP1-deficient (Tdp1 / ) cells, and TDP1 / cells National Center for Advancing Translational Sciences complemented with human TDP1 (hTDP1) in chicken (http://www.ncats.nih.gov/research/reengineering/ncgc/ DT40 B cell line, have previously been reported and des- ncgc.html), and the use of TDP2 for counterscreening. We cribed here (17). also discuss the importance of reaction conditions and counter screening for the characterization of TDP1-selective qHTS assay inhibitors. TDP1 enzyme in the HTS buffer containing 1Â PBS, pH 7.4, 80 mmol/L KCl, and 0.01% Tween-20 (31) was dis- pensed at 3 mL into 1536-well Kalypsys black solid bottom Materials and Methods plates (Kalypsys). Compounds and controls (23 nL) were Chemicals transferred via a Pin Tool station (Kalypsys) at 23 nL. The JLT048 (CAS# 664357; 4-(5-[{[1-(2-fluorobenzyl)- plates were incubated for 15 minutes at room tempera- 2,5-dioxo-4-imidazolidinylidene] methyl]}-2furyl)benzoic ture, and then 1 mL of DNA substrate (Supplementary Fig. acid) was purchased from ChemBridge Corporation. CPT S1) was added to start the reaction. After 5 minutes and veliparib were obtained from the Drug Synthesis and incubation at room temperature, 1 mL of AlphaScreen Chemistry Branch, Developmental Therapeutics Program, donor/acceptor bead mix (PerkinElmer Lifesciences) was DCTD, NCI. added, and the plates were further incubated for 10 All reactions were performed under argon in oven- minutes at room temperature. The detection was then dried or flame-dried glassware. All commercially avail- performed on a PerkinElmer Envision reader (PerkinEl- able reagents were purchased from Sigma Aldrich and mer Lifesciences). Compounds were first classified as used as received. All experiments were monitored by having full titration curves, partial modulation, partial analytical thin layer chromatography performed on curve (weaker actives), single-point activity (at highest Silicycle silica gel 60 A glass-supported plates with 0.25- concentration only), or inactive (32). For all active com- mm thickness. Yields are not optimized. Low-resolution pounds, a score range was given for each curve class type mass spectra (electrospray ionization) were acquired on given above. Active compounds received a Pubchem an Agilent Technologies 6130 quadrupole spectrometer Activity_Score between 40 and 100. Inconclusive com- coupled to an Agilent Technologies 1200 series HPLC. pounds received Pubchem Activity_Score between 1 High-resolution mass spectrum-electron ionization spray and 39. All inactive compounds received a Pubchem (HRMS-ESI) were obtained on an Agilent Technologies Activity_Score of 0. 1200 series Dual Absorbance Detector HPLC system equipped with a Phenomenex Luna 75 Â 3 mm, C18, 3- Whole cell extract TDP1 assay mm column at 45C [UV detection at 220 nm and 254 nm, DT40 hTDP1 were collected, washed, and centrifuged. band width (BW) 8 nm; flow rate: 0.8 mL/min (increas- Cell pellets were then resuspended in 100 mL of CelLytic M ing); injection volume: 1.0 mL; sample solvent: 100% meth- cell lysis reagent (Sigma-Aldrich C2978). After 15 minutes anol; sample conc.: 0.01 mg/mL; mobile phase A: water on ice, lysates were centrifuged at 12,000 g for 10 minutes, with 0.1% acetic acid; mobile phase B: acetonitrile with and supernatants were transferred to a new tube. Protein 0.1% acetic acid] coupled to an Agilent 6210 time-of-flight concentrations were determined using a Nanodrop spec- mass spectrometer [ion source: Duel ESI, minimum range: trophotometer (Invitrogen), and whole cell extracts 115 m/z, maximum range: 1,400 m/z, scan rate: 0.9 sec- (WCE) were stored at À80 C. This method of protein onds, gas temperature: 340C, gas flow: 10 L/min, neb- concentration determination is acceptable in this case ulizer: 50 PSI, ion polarity: positive, VCap: 3,500 V, frag- because cellular nucleic acids have been precipitated and mentor: 175 V, skimmer1: 65 V, OctopoleRFPeak: 250 V, discarded during the precipitation step described above reference mass: enabled (Agilent P/N G1969–85001)]. and because the amount of WCE to be used is determined Data were analyzed using Agilent Masshunter Worksta- by the dilution of WCE required to achieve 30% to 40% 0 tion Data Acquisition (v B.02.00, Patch 1,2,3) and Agilent of TDP1 cleavage in the assay. A 5 -[32P]–labeled single- 0 Masshunter Qualitative Analysis (v B.02.00, Build stranded DNA oligonucleotide containing a 3 -phospho- 2.0.197.7, Patch 3). Preparation steps for NCGC00183974 tyrosine (N14Y; ref.
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