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.

Figure 1

Flow chart summarizing our biochemical screening strategy for TDP1 inhibitors. (A) The 352,260-compound NIH Molecular Libraries Small Molecule Repository (MLSMR) was screened at 8 concentrations against TDP1 by quantitative high throughput screening (qHTS) (31), which led to the identification of 986 positive hits (results accessible online at PubChem AID 485290). (B) Typical qHTS concentration-response for a representative positive compound. (C) The entire set of positives hits was subsequently tested against endogenous human TDP1 from whole cell extracts (WCE) leading to the identification of 10 compounds that can be categorized in 2 chemical groups (dashed rectangles). (D) Two analogs, each representing one chemical group, were selected and further tested.

Figure 2

Whole Cell Extract (WCE) TDP1 assay. (A) Schematic representation of the 14-mer single-stranded TDP1 substrate bearing a 3’-phosphotyrosine (N14Y). In the presence of WCE, endogenous TDP1 excises the terminal tyrosine to generate a 14-mer 3’-phosphate DNA product (N14P). (B) Representative gel showing the concentration-dependent appearance of the N14P product in the presence of hTDP1 WCE. This reaction is specific of TDP1 because it is absent with WCE from TDP1 knockout cells (−/−TDP1). WCE concentrations were from 900 µg/ml in 3-fold decrements. (C) Representative gel showing the concentration-dependent inhibition of TDP1 by positive hits (horizontal brackets). Because of the specificity of the TDP1 reaction in WCE, 10 consecutive loadings of 14 compounds tested at 3 concentrations were performed on the same gel.

Figure 3

Differential kinetics of TDP1 reactions in the presence of different buffers. (A) Lineweaver-Burk double reciprocal plot obtained for recombinant TDP1 in the presence HTS buffer or WCE buffer. (B) Intersecting curves in the origin area of the Lineweaver-Burk double-reciprocal plot presented in (A). (C) Concentration response inhibitory curves obtained for NCGC00183964 in HTS and WCE buffers. (D) Correlation between recombinant TDP1 (REC) and endogenous TDP1 (WCE) inhibition by the compounds presented in Figure 1 and Supplementary Table S1. The regression line is represented by a solid line, and dashed lines correspond to 95% confidence interval.

Figure 4

TDP2 counter-screening assay. (A) Schematic representation of the catalytic reaction carried out by recombinant (REC) TDP1 and TDP2. Both enzymes excise a terminal tyrosine residue from single-stranded oligonucleotides but with an opposite polarity: 3’-tyrosine for TDP1 and 5’-tyrosine for TDP2. (B) Representative gel showing enzyme concentration-dependent cleavage reactions for TDP1 and TDP2. REC TDP1 and REC TDP2 concentrations are from 160 pM and 1600 pM in 2-fold decrements, respectively. (C) Representative gels showing concentration-dependent inhibition of recombinant TDP1 (upper gels) and TDP2 (lower gels) by JLT048 and NCGC00183674. (D) Concentration-response curves for JLT048 (left panel) and NCGC00183674 (right panel) with REC TDP1 (solid circles) or REC TDP2 (open circles).

Figure 5

Cellular survival curves in the presence of CPT and various concentrations of JLT048 (A), NCGC00183674 (B) and veliparib (C) in hTDP1 cells.