title: Biochemical assays for the discovery of TDP1 inhibitors.
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 topoisomerase 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.
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute; marchanc@mail.nih.gov pommier@nih.gov.
authors.forename: Christophe
authors.initials: C
authors.lastname: Marchand
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute;
authors.forename: Shar-yin N
authors.initials: SY
authors.lastname: Huang
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda;
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Dexheimer
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda;
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Lea
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda;
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Mott
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute;
authors.forename: Adel
authors.initials: A
authors.lastname: Chergui
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute;
authors.forename: Alena
authors.initials: A
authors.lastname: Naumova
authors.affiliation: Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland; and.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Stephen
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda;
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Rosenthal
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda;
authors.forename: Ganesha
authors.initials: G
authors.lastname: Rai
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute;
authors.forename: Junko
authors.initials: J
authors.lastname: Murai
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute;
authors.forename: Rui
authors.initials: R
authors.lastname: Gao
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda;
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda;
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.affiliation: Department of Chemistry, Yale University, New Haven, Connecticut.
authors.forename: William L
authors.initials: WL
authors.lastname: Jorgensen
authors.affiliation: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda;
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute; marchanc@mail.nih.gov pommier@nih.gov.
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002645:Chickens; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004357:Drug Synergism; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*
chemical_list: D000970:Antineoplastic Agents; D010446:Peptide Fragments; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; D002166:Camptothecin