SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV - PubMed (original) (raw)

SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV

George E Greco et al. DNA Repair (Amst). 2016 Jul.

Abstract

DNA ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA ligase inhibitors with differing selectivity for the three human DNA ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA ligase IV, inhibit the repair of DSBs by the DNA ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA ligases I and III than DNA ligase IV. Furthermore, SCR7-R failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA ligase IV.

Keywords: DNA double strand break repair; DNA ligase inhibitors; Human DNA ligases; Non-homologous end-joining.

Copyright © 2016 Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1

Structures of SCR7 derivatives. In (A) is shown the structure of SCR7 as reported by Srivastava et al. [11]. The structure of the major product generated by the synthesis protocol described by Srivastava et al. [11] and by a different synthesis method [20] (SCR7-G) and the compound sold as SCR7 by XcessBio (SCR7-X) are shown. Our structure determination of the SCR7 provided by Dr. Sathees Raghavan (SCR7-R) and the pan human DNA ligase inhibitor (L189) described by Chen et al. [2] are also shown.

Fig. 2

Fig. 2

Purified human DNA ligases. Five pmoles of each DNA ligase were electrophoresed in an SDS-containing 10% polyacrylamide gel and then stained with Coomassie Brilliant Blue. Lane 1, LigI; lane 2, LigIIIβ; lane 3, LigIIIα/XRCC1; lane 4, LigIV/XRCC4.

Fig. 3

Fig. 3

Effects of SCR7 derivatives on nick ligation by LigI, LigIIIα/XRCC1 and LigIV/XRCC4 using nicked, fluorescent DNA substrate. DNA ligation reactions with a labeled, nicked DNA substrate (0.2 pmol) were carried as described in Materials and Methods. LigI (0.05 pmol, Panel A), LigIIIα/XRCC1 (0.05 pmol, Panel B) and LigIV/XRCC4 (0.2 pmol, Panel C) were incubated with SCR7 derivatives; SCR7-G (triangle); SCR7-R (circle); SCR7-X (square). Data are the mean ± standard deviation of three independent experiments.

Fig. 4

Fig. 4

Effects of SCR7 derivatives on nick ligation by LigI, LigIIIα/XRCC1 and LigIV/XRCC4 using radioactively-labeled nicked DNA substrate. DNA ligation reactions with a nicked, fluorescent DNA substrate (0.2 pmol) were carried as described in Materials and Methods. LigI (0.1 pmol, Panel A), LigIIIα/XRCC1 (0.1 pmol, Panel B) and LigIV/XRCC4 (1.1 pmol, Panel C) were incubated with SCR7 derivatives; SCR7-G (triangle); SCR7-R (circle); SCR7-X (square). Data are the mean ± standard deviation of three independent experiments.

Fig. 5

Fig. 5

Effects of SCR7 derivatives on double-strand ligation by LigIIIα/XRCC1 and LigIV/XRCC4. (A) Schematic structure of the DNA substrate for double-strand ligation. The 5′ terminus is 32P-phosphorylated. (B) The DNA substrate (0.2 pmol) was incubated with either LigIIIα/XRCC1 (0.2 pmol) or LigIV/XRCC4 (1 pmol) in the absence or presence of the indicated concentrations of SCR7 derivatives prior to electrophoresis in 7 M urea-containing 12.5% polyacrylamide gels. The products resulting from ligation of both strands (ds-ligated) and only one strand (ss-ligated) as well as the original substrate (unligated) are indicated. (C) The results of the inter-molecular ligation assays are shown graphically. LigIIIα/XRCC1 (open symbols) and LigIV/XRCC4 (closed symbols) incubated with either SCR7-R (circles) or SCR7-X (squares). Ligation efficiency was calculated by setting the reactions with no inhibitor as 100%. Data are the mean ± standard deviation of three independent experiments.

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