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  • br Introduction DNA ligation is


    Introduction DNA ligation is required during DNA replication and to complete almost all DNA repair events. In human cells, the DNA ligases encoded by three LIG genes are responsible for joining interruptions in the phosphodiester backbone [1]. These enzymes have distinct but overlapping functions in cellular DNA metabolism. Interestingly, Gossypol synthesis expression levels are frequently dysregulated in cancer. For example, the steady state levels of DNA ligase I (LigI) are usually elevated in cancer cell lines and tumor specimens [2], [3]. This is presumed to reflect the increased proliferation that is a characteristic of cancer cells. In addition, a significant fraction of cancer cell lines have elevated levels of DNA ligase IIIα (LigIIIα) and reduced levels of DNA ligase IV (LigIV) [2]. Notably, this reciprocal change in DNA ligase levels has been shown to result in abnormal repair of DNA double-strand breaks in leukemia, breast cancer and neuroblastoma, with increased levels of LigIIIα correlating with reduced survival [4], [5], [6]. Given their dysregulation in cancer and almost ubiquitous involvement in DNA transactions, DNA ligases are potential therapeutic targets for the development of novel anti-cancer agents. There have been several attempts to identify DNA ligase inhibitors by screening of synthetic chemical and natural product libraries that have met with limited success. These have mainly involved radioactive-based assays and the screening of a relatively small number of compounds [7], [8], [9]. A series of small molecule inhibitors with differing specificities for the three human DNA ligases were identified by a structure-based approach using the atomic resolution structure of the DNA binding domain of human DNA ligase I complexed with nicked DNA [2], [10]. As expected, several of these inhibitors were cytotoxic and, at subtoxic concentrations, they potentiated cell killing by DNA damaging agents [2]. Unexpectedly, this enhancement of cytotoxicity occurred in malignant cells, but not their non-neoplastic counterparts [2]. In further studies, a LigI/III inhibitor L67 was found to synergistically increase the cytotoxicity of a PARP inhibitor by inhibiting LigIIIα in therapy-resistant chronic myeloid leukemia and breast cancer cells lines with abnormal DNA repair characterized by elevated levels of LigIIIα and PARP-1 [5], [6]. Using molecular modeling to predict the structure of the DNA ligase IV DNA binding domain with L189, the inhibitor of all three human DNA ligases identified in the previous structure-based approach [2], Raghavan and colleagues reported the identification of a derivative of L189, which they called SCR7 [11]. SCR7 appeared to selectively inhibit the repair of DSBs by the non-homologous end-joining (NHEJ) pathway in a DNA ligase IV-dependent manner as well as to both reduce tumor growth and increase the efficacy of DSB-inducing therapeutic modalities [11]. In attempting to synthesize SCR7 by the published procedure [11], we encountered problems with the synthesis procedures and discovered discrepancies in the reported structure of SCR7. Using three different preparations of SCR7, we found that it is a DNA ligase inhibitor with greater activity against DNA ligases I and III than DNA ligase IV and that it fails to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay.
    Materials and methods
    Discussion Raghavan and colleagues described the synthesis and characterization of a compound, SCR7, that inhibited DNA ligase IV and blocked DNA ligase IV-dependent NHEJ in extracts and cell-based assays [11]. Furthermore, they reported that this compound reduced the growth of tumor xenografts, both in combination with genotoxic agents used clinically and as a single agent [11]. In this study, we have found that the published synthesis protocol does not generate the compound with the structure described by Raghavan and colleagues and that SCR7 provided by Dr. Raghavan (SCR7-R) also does not have the structure described [11]. We have determined the structure of SCR7-R and SCR7-G, the compounds generated by the synthesis protocol described by Raghavan and colleagues [11]. Furthermore, our analysis of commercially available SCR7 (from XcessBio and designated SCR7-X here) revealed that it has the same structure as SCR7-G rather than the structure listed by XcessBio.