qHTS for Inhibitors of human tyrosyl-DNA phosphodiesterase 1 (TDP1): qHTS in cells in presence of CPT
Human tyrosyl-DNA phosphodiesterase 1 (TDP1) is a novel repair gene, and we propose to use it as a new target for anticancer drug development. TDP1 is not an essential protein, but under treatment with topoisomerase I poison (camptothecin: CPT), TDP1 works as a critical factor for cell survival. To directly identify novel TDP1 inhibitors active in a cellular environment, we have knocked-out the more ..
BioActive Compounds: 49243
Human tyrosyl-DNA phosphodiesterase 1 (TDP1) is a novel repair gene, and we propose to use it as a new target for anticancer drug development. TDP1 is not an essential protein, but under treatment with topoisomerase I poison (camptothecin: CPT), TDP1 works as a critical factor for cell survival. To directly identify novel TDP1 inhibitors active in a cellular environment, we have knocked-out the Tdp1 gene in chicken DT40 cells (Tdp1-/-) and generated a complemented counterpart cells that contains a stable transfection of the human TDP1 gene (Tdp1-/-;hTDP1 cells). For the primary screen, Tdp1-/-;hTDP1 cells will be exposed to small molecules in the presence or absence of CPT, and their growth kinetics will be evaluated after 48 hours by measuring ATP activity. If a given compound shows a synergistic effect with CPT, this compound could inhibit the repair pathway of CPT-induced lesions including the TDP1-mediated repair pathway. The hit compounds will then be evaluated in the presence or absence of CPT using Tdp1-/- cells. If a compound shows synergistic effect with CPT in Tdp1-/-;hTDP1 cells, but not with Tdp1-/- cells, such compound could be involved in the TDP1-mediated repair pathway inhibition. In tertiary assays, biochemical gel-based assays will be used to assess whether the hit compounds specifically target TDP1.
To this end, we have screened this cell line against the MLSMR in two primary screens. In one screen we add CPT and in the other we screen in the absence of CPT; this AID represents the screen where CPT was present.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH095538
Assay Submitter (PI): Yves Pommier, NCI
DT40-hTDP1 cells with 20 nM Camptothecin (add 20 microL of 1 mM CPT stock in 1 L of cell culture medium) were dispensed at 400 cells/5microL/well in tissue culture treated 1536-well white wall/solid bottom assay plates (Greiner Bio-One North America, NC, U.S.A.) using a Multidrop Combi 8 channel dispenser (Thermo Fisher, Waltham, MA, USA). 23 nL compounds and controls were transferred using the pin tool (Kalypsys, San Diego, CA, USA) to the assay plates. The assay plates were then incubated at 37 masculineC for a minimum 48 hr. Three microL of Cell Titer Glo solution was added to the plates and incubated at RT in dark for 30 min. Luminescence was read using ViewLux (Perkin Elmer) with 1 sec exposure slow speed, high gain and 2x binning.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)