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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.

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 IC

Apoptosis or programmed cell death is a highly regulated process, which eliminates unwanted and damaged cells. Inhibition of apoptosis is a hallmark of cancer cells. BCL2 family proteins are known to play a vital role in the regulation of apoptosis. Overexpression of BCL2, an antiapoptotic protein, provides the advantage of prolonged survival to cancer cells. Over the years, several BCL2 inhibitors have been investigated extensively for their anticancer potential. However, most of them were abolished before clinical use due to their side effects. Previously, we had identified and characterized a novel BCL2 inhibitor, Disarib, with the potential to eliminate tumor cells in a BCL2 specific manner leading to reduction in tumor burden in multiple mouse models. Notably, a head-to-head comparison of Disarib to ABT199, the only FDA approved BCL2 inhibitor revealed that Disarib is as potent as ABT199. Recent studies using mice revealed that Disarib did not invoke significant side effects in mice. In the present study, we have investigated the acute toxicity of Disarib in Wistar rats. The bioavailability studies following exposure of Disarib in Wistar rats revealed its maximum availability in serum at 24 h following oral administration. Acute toxicity analysis revealed that even a dose as high as 2000 mg/kg of Disarib did not cause significant toxicity in rats. There was no significant variation in blood parameters or kidney and liver functions following administration of Disarib. Histological analysis of different tissues from Disarib treated groups revealed standard architecture with no observable cellular damage. Importantly, exposure to Diasrib did not result in genotoxicity as determined by micronucleus assay. Further, solubility assays revealed that besides DMSO, Disarib is also soluble in alcohol. While the high acidic condition can increase the solubility of Disarib, even a lower percentage of alcohol with acidic conditions can improve its solubility. Thus, the toxicological profile in the current study revealed no significant side effects when Disarib was administered orally to rats.

In the present day, it is possible to incorporate targeted mutations or replace a gene using genome editing techniques such as customisable CRISPR/Cas9 system. Although induction of DNA double-strand breaks (DSBs) by genome editing tools can be repaired by both non-homologous end joining (NHEJ) and homologous recombination (HR), the skewness of the former pathway in human and other mammals normally result in imprecise repair. Scientists working at the crossroads of DNA repair and genome editing have devised new strategies for using a specific pathway to their advantage. Refinement in the efficiency of precise gene editing was witnessed upon downregulation of NHEJ by knockdown or using small molecule inhibitors on one hand, and upregulation of HR proteins and addition of HR stimulators, other hand. The exploitation of cell cycle phase differences together with appropriate donor DNA length/sequence and small molecules has provided further improvement in precise genome editing. The present article reviews the mechanisms of improving the efficiency of precise genome editing in several model organisms and in clinics.

Small molecule inhibitors targeting DNA repair pathways in cancer cells is a novel and promising approach in cancer therapy, which can improve current therapeutic regimen. Although various attempts have been made for designing inhibitors against DNA damage response and repair proteins, reports on Nonhomologous End Joining (NHEJ) inhibitors are limited. Of the several chemical moieties identified, SCR7 and its oxidized form are novel and potent DNA Ligase IV inhibitors involved in the abrogation of DNA end joining thereby leading to cell death. In the present study, we have synthesized sodium salt of SCR7 to generate a water-soluble version of the molecule, referred to as water-soluble SCR7 (WS-SCR7). WS-SCR7 inhibits NHEJ in Ligase IV dependent manner, with a subtle effect on Ligase III at higher concentration. No effect on Ligase I mediated joining was observed. WS-SCR7 shows cytotoxicity in cancer cell lines, leading to induction of apoptosis in a dose-dependent manner.

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.

In the present day, it is possible to incorporate targeted mutations or replace a gene using genome editing techniques such as customisable CRISPR/Cas9 system. Although induction of DNA double-strand breaks (DSBs) by genome editing tools can be repaired by both non-homologous end joining (NHEJ) and homologous recombination (HR), the skewness of the former pathway in human and other mammals normally result in imprecise repair. Scientists working at the crossroads of DNA repair and genome editing have devised new strategies for using a specific pathway to their advantage. Refinement in the efficiency of precise gene editing was witnessed upon downregulation of NHEJ by knockdown or using small molecule inhibitors on one hand, and upregulation of HR proteins and addition of HR stimulators, other hand. The exploitation of cell cycle phase differences together with appropriate donor DNA length/sequence and small molecules has provided further improvement in precise genome editing. The present article reviews the mechanisms of improving the efficiency of precise genome editing in several model organisms and in clinics.

The CRISPR-Cas system currently stands as one of the best multifaceted tools for site-specific genome engineering in mammals. An important aspect of research in this field focusses on improving the specificity and efficacy of precise genome editing in multiple model systems. The cornerstone of this mini-review is one of the extensively investigated small molecule inhibitor, SCR7, which abrogates NHEJ, a Ligase IV-dependent DSB repair pathway, thus guiding integration of the foreign DNA fragment via the more precise homology directed repair during genome editing. One of our recent studies sheds light on properties of different forms of SCR7. Here, we give a succinct account on the use of SCR7 and its different forms in CRISPR-Cas system, highlighting their chemical properties and biological relevance as potent efficiency-enhancing CRISPR tools.

Repair of DNA double-strand breaks (DSBs) and its regulation are tightly integrated inside cells. Homologous recombination, nonhomologous end joining and microhomology mediated end joining are three major DSB repair pathways in mammalian cells. Targeting proteins associated with these repair pathways using small molecule inhibitors can prove effective in tumors, especially those with deregulated repair. Sensitization of cancer to current age therapy including radio and chemotherapy, using small molecule inhibitors is promising and warrant further development. Although several are under clinical trial, till date no repair inhibitor is approved for commercial use in cancer patients, with the exception of PARP inhibitors targeting single-strand break repair. Based on molecular profiling of repair proteins, better prognostic and therapeutic output can be achieved in patients. In the present review, we highlight the different mechanisms of DSB repair, chromatin dynamics to provide repair accessibility and modulation of inhibitors in association with molecular profiling and current gold standard treatment modalities for cancer.

Conventional cancer treatment modalities such as radiation and chemotherapy, cause cancer cell death by inducing DNA damage, particularly DNA strand breaks. Over the years, newer avenues have emerged for overcoming radio/chemoresistance by harnessing repair proteins as targets for small molecule inhibitors. Analysis of genome-wide expression data in cancer subtypes and understanding synthetic lethal interactions among repair pathways are important stepping-stones. Several inhibitors targeting DNA strand break repair proteins have yielded good effects in preclinical studies, and have the potential to be developed as therapeutics in cancer as monotherapy or in combination with radiation and chemotherapy. Furthermore, these small molecule inhibitors can aid in precise genome editing (using CRISPR) by harnessing the differential levels of repair inside cells. Shifting the repair balance towards homology-directed repair using inhibitors of NHEJ or stimulators of HR has yielded promising effects alongside CRISPR in cells and several disease models. In short, DNA strand break repair inhibitors are the forerunners in cancer therapy and genome editing, working in concert with the established artillery in the field.

International audience; 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 (C18H14N4OS), whereas its oxidized form, SCR7-pyrazine, possesses a different molecular formula (C18H12N4OS), 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.