Dr Jan Korábečný, University Hospital Hradec Králové; Dr Martin Andrš, University Hospital Hradec Králové; Dr Ondřej Soukup, University Hospital Hradec Králové; Prof. Martina Řezáčová, Medical Faculty of Charles University Prague; Dr Martina Seifrtová, Medical Faculty of Charles University Prague
Centre for Transfer of Biomedical Technologies
DNA repair pathways play a major role in tumor resistance towards chemo- and radiotherapy. Therefore, inhibitors of specific DNA repair pathways might be advantageous when used in combination with DNA-damaging agents, such as ionizing radiation.
Double strand breaks (DSB) are one of the most deleterious lesions in DNA. Unfinished or incorrect DSB repair may lead to a loss of genetic information, genome rearrangement or cell death. Thus, the repair control is necessary for the maintenance of genomic stability. Three phosphatidylinositol 3-kinase like protein kinases (PIKKs): DNA-dependent protein kinase (DNA-PK), ataxia-telangiectasia mutated kinase (ATM) and ATM Rad3-related kinase (ATR) coordinate DNA damage response (DDR), specifically cell cycle arrest, cell survival and DSB repair. Two main pathways are employed in DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). The crucial component of NHEJ is DNA-PK, which consists of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and DNA end binding proteins Ku70/Ku80. Nearly two-thirds of all cancer patients receive some type of radiotherapy or chemotherapy which induce DNA lesions, especially DSBs. Many cancer types are resistant to DNA-damaging anti-cancer therapy and the effect of treatment is attenuated by enhancement of DNA repair processes in tumour cells. Therefore, there have been efforts to inhibit responses to DNA damage (DDR) and DNA repair to enhance the effectiveness of anti-cancer treatment. Blocking DDR has been proposed as a promising therapeutic strategy for cancer treatment and it is the rationale for using PIKKs inhibition in the context of cancer treatment. However, the majority of compounds currently known to specifically inhibit DNA-PK are limited by insufficient selectivity or poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives.
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We have designed and synthesized 52 novel compounds and screened them on the panel of nine cancer and one healthy cell lines alone and in combination with a standard chemotherapy agent doxorubicin. As expected, our compounds alone did not affect the cell growth significantly. However, several compounds greatly enhanced the cytotoxic effect of doxorubicin. Data from the most active compounds are shown in figure 1. The most promising compound is probably K1292. Together with doxorubicin, this compound lowered the number of living cells under 5% compared to control (100%) in three cell lines – HeLa, AGS, and A549. No chemosensitization effect was observed in healthy lung fibroblast MRC-5 cells. K1292 has a simple structure, easily accessible by standard organic synthesis and reasonable aqueous solubility.
Patent application before publication.
Each graph represents viability of 10 cell lines after incubation with selected 5 most efficient inhibitors (single concentration 10 µM) and with their combination with doxorubicin. Concentration of doxorubicin was chosen individually for each cell line according to its sensitivity. Cells were incubated with drugs for 48 hours and stained by WST-1 reagent. Values were calculated as mean of three independent experiments and are expressed as percentage of viability of untreated control cells (100%). Error bars indicate ±SD.