Autocyclized and oxidized forms ofSCR7 induce cancer cell death by inhibiting nonhomologousDNAend joining in a LigaseIVdependent manner

Abstract

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 (C H N OS), whereas its oxidized form, SCR7-pyrazine, possesses a different molecular formula (C H N 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.

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

“…In our previous study, we have reported that SCR7 can improve the effect of radiation in mice tumor and cancer cell lines. 17,18 In this study, SCR130 used in combination with radiation showed elevated levels of 53BP1 and γH2AX foci in cancer cells. This was further confirmed using neutral comet assay.…”

“…Among that, SCR130 exhibited 20-fold better cytotoxicity as compared with SCR7. 17,18 Moreover, SCR130 specifically inhibited Ligase IV-mediated joining with no effect on Ligase I activity. Although at equivalent concentrations SCR130 was insensitive to Ligase III-mediated joining, it showed an effect at four-fold higher concentration (data not shown).…”

“…17 Furthermore, different forms of SCR7 (autocyclized and pyrazine) can inhibit Ligase IV-mediated end-joining both in vitro and in vivo. 18 Although both the forms exhibited comparable cytotoxicity in cell lines, the oxidized form, SCR7-pyrazine (SCR7-P) displayed nonspecific effects within cells at higher concentrations. Consistent to this, the water-soluble version of SCR7-P exhibited anticancer effect, by inhibiting the activity of all three ligases.…”

“…18 Although attempts have been made for improving the bioavailability of SCR7 by encapsulation in a pluronic copolymer micelle, the improvement achieved was limited. [18][19][20] In addition, sodium salt of SCR7-Pyrazine (Na-SCR7-P, water-soluble version) was also characterized, although it inhibited the end-joining of all three Ligases. 21 Besides, several research groups have reported a 2 to 19 fold increase in genome editing after administration of SCR7 ex vivo and in vivo together with CRISPR-Cas9 constructs.…”

A Universal Approach to Target Various HBB Gene Mutations in Hematopoietic Stem/Progenitor Cells for Beta-Thalassemia Gene Therapy by CRISPR/Cas9 and the rAAV6 Vector

“…To further improve HDR e ciency, we tested two different small-molecule compounds, Scr7 and L755507. Scr7 is a speci c DNA ligase IV inhibitor that can improve HDR e ciency by inhibiting the nonhomologous endjoining (NHEJ) pathway [26], and L-755507 is a selective β3 adrenergic receptor partial agonist that enhances CRISPR-mediated HDR e ciency [21]. Two of these small-molecule compounds have been proven to improve the HDR ratio in mammalian cells, mouse embryonic stem cells and pluripotent stem cells in other studies [22,27].…”