Identification and characterization of mercaptopyrimidine-based small molecules as inhibitors of nonhomologous DNA end joining.

Mercaptopyrimidine derivatives are heterocyclic compounds with potent biological activities including antiproliferative, antibacterial, and anti-inflammatory properties. The present study describes the synthesis and characterization of several mercaptopyrimidine derivatives through condensation of 5,6-diamino-2-mercaptopyrimidin-4-ol with various heterocyclic and aromatic aldehydes. Previous studies have shown that SCR7, synthesized from 5,6-diamino-2-mercaptopyrimidin-4-ol, induced cytotoxicity by targeting cancer cells by primarily inhibiting DNA Ligase IV involved in nonhomologous end joining, one of the major DNA double-strand break repair pathways. Inhibition of DNA repair pathways is considered as an important strategy for cancer therapy. Due to limitations of SCR7 in terms of IC50 in cancer cells, here we have designed, synthesized, and characterized potent derivatives of SCR7 using 5,6-diamino-2-mercaptopyrimidin-4-ol as the starting material. Several synthesized imine compounds exhibited significant improvement in inhibition of end joining and cytotoxicity up to 27-fold lower concentrations than SCR7. Among these, two compounds, SCR116 and SCR132, showed increased cancer cell death in a Ligase IV-dependent manner. Treatment with the compounds also led to reduction in V(D)J recombination efficiency, cell cycle arrest at G2/M phase, accumulation of double-strand breaks inside cells, and improved anti-cancer potential when combined with γ-radiation and radiomimetic drugs. Thus, we describe novel inhibitors of NHEJ with higher efficacy and potential, which can be developed as cancer therapeutics.

SCR7, an inhibitor of NHEJ can sensitize tumor cells to ionization radiation.

Nonhomologous end joining (NHEJ), one of the major DNA double-strand break repair pathways, plays a significant role in cancer cell proliferation and resistance to radio and chemotherapeutic agents. Previously, we had described a small molecule inhibitor, SCR7, which inhibited NHEJ in a DNA Ligase IV dependent manner. Here, we report that SCR7 potentiates the effect of γ-radiation (IR) that induces DNA breaks as intermediates to eradicate cancer cells. Dose fractionation studies revealed that coadministration of SCR7 and IR (0.5 Gy) in mice Dalton's lymphoma (DLA) model led to a significant reduction in mice tumor cell proliferation, which was equivalent to that observed for 2 Gy dose when both solid and liquid tumor models were used. Besides, co-treatment with SCR7 and 1 Gy of IR further improved the efficacy. Notably, there was no significant change in blood parameters, kidney and liver functions upon combinatorial treatment of SCR7 and IR. Further, the co-treatment of SCR7 and IR resulted in a significant increase in unrepaired DSBs within cancer cells compared to either of the agent alone. Anatomy, histology, and other studies in tumor models confirmed the cumulative effects of both agents in activating apoptotic pathways to induce cytotoxicity by modulating DNA damage response and repair pathways. Thus, we report that SCR7 has the potential to reduce the side effects of radiotherapy by lowering its effective dose ex vivo and in mice tumor models, with implications in cancer therapy.

Identification and characterization of novel SCR7-based small-molecule inhibitor of DNA end-joining, SCR130 and its relevance in cancer therapeutics.

Targeting DNA repair with small-molecule inhibitors is an attractive strategy for cancer therapy. Majority of DNA double-strand breaks in mammalian cells are repaired through nonhomologous end-joining (NHEJ). It has been shown that small-molecule inhibitors of NHEJ can block efficient repair inside cancer cells, leading to cell death. Previously, we have reported that SCR7, an inhibitor of NHEJ can induce tumor regression in mice. Later studies have shown that different forms of SCR7 can inhibit DNA end-joining in Ligase IV-dependent manner. Recently, we have derivatized SCR7 by introducing spiro ring into core structure. Here, we report the identification of a novel inhibitor of NHEJ, named SCR130 with 20-fold higher efficacy in inducing cytotoxicity in cancer cell lines. SCR130 inhibited DNA end-joining catalyzed by rat tissue extract. Specificity analysis revealed that while SCR130 was specific to Ligase IV, it showed minimal or no effect on Ligase III and Ligase I mediated joining. Importantly, SCR130 exhibited the least cytotoxicity in Ligase IV-null cell line as compared with wild type, confirming Ligase IV-specificity. Furthermore, we demonstrate that SCR130 can potentiate the effect of radiation in cancer cells when used in combination with γ-radiation. Various cellular assays in conjunction with Western blot analysis revealed that treatment with SCR130 led to loss of mitochondrial membrane potential leading to cell death by activating both intrinsic and extrinsic pathways of apoptosis. Thus, we describe a novel inhibitor of NHEJ with higher efficacy and may have the potential to be developed as cancer therapeutic.

Autocyclized and oxidized forms of SCR7 induce cancer cell death by inhibiting nonhomologous DNA end joining in a Ligase IV dependent manner.

Nonhomologous DNA end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals. Previously, we have described a small molecule inhibitor, SCR7, which can inhibit NHEJ in a Ligase IV-dependent manner. Administration of SCR7 within the cells resulted in the accumulation of DNA breaks, cell death, and inhibition of tumor growth in mice. In the present study, we report that parental SCR7, which is unstable, can be autocyclized into a stable form. Both parental SCR7 and cyclized SCR7 possess the same molecular weight (334.09) and molecular formula (C18 H14 N4 OS), whereas its oxidized form, SCR7-pyrazine, possesses a different molecular formula (C18 H12 N4 OS), molecular weight (332.07), and structure. While cyclized form of SCR7 showed robust inhibition of NHEJ in vitro, both forms exhibited efficient cytotoxicity. Cyclized and oxidized forms of SCR7 inhibited DNA end joining catalyzed by Ligase IV, whereas their impact was minimal on Ligase III, Ligase I, and T4 DNA Ligase-mediated joining. Importantly, both forms inhibited V(D)J recombination, although the effect was more pronounced for SCR7-cyclized. Both forms blocked NHEJ in a Ligase IV-dependent manner leading to the accumulation of DSBs within the cells. Although cytotoxicity due to SCR7-cyclized was Ligase IV specific, the pyrazine form exhibited nonspecific cytotoxicity at higher concentrations in Ligase IV-null cells. Finally, we demonstrate that both forms can potentiate the effect of radiation. Thus, we report that cyclized and oxidized forms of SCR7 can inhibit NHEJ in a Ligase IV-dependent manner, although SCR7-pyrazine is less specific to Ligase IV inside the cell.