Antitumor Activity And Underlying Mechanisms Of Two Novel MTH1 Inhibitors

With the in-depth research on molecular biology of tumors and related disciplines,molecular targeted drugs are playing an increasingly important role in the clinical treatment of tumors.Unlike traditional cytotoxic drugs,that are associated with poor selectivity,strong toxic side effects and drug resistance,etc.Targeted antitumor drugs target the differences between normal and tumor cells and can achieve high selectivity and low side effects.Therefore,the development of anticancer drugs that selectively kill or inhibit tumor cells has become an important goal in the field of oncology research.Compared to normal cells,overly vigorous metabolism leads to the accumulation of large amounts of reactive oxygen species（ROS）in cancer cells,and high oxidative stress continuously attack DNA molecules,causing severe oxidative damage,of which8-oxo-dGTP（8-oxo-dG）is the most common form of oxidative damage.MTH1（Mut T homolog 1）,a pyrophosphatase,can convert 8-oxo-dGTP into 8-oxo-dGMP,thus effectively scavenging oxidized nucleotides from the nucleotide pool and maintaining genomic stability,and thus is critical for cancer cell survival.MTH1 protein has thus become a new target for cancer therapy,and the development of broad-spectrum and efficient MTH1 inhibitors has become an important approach for new anti-cancer strategies.In previous studies,our laboratory synthesized and screened a batch of potential small molecule MTH1 inhibitors,which had been confirmed to strongly inhibit the activity of the MTH1 enzyme in vitro,and were found to bind intracellular MTH1 protein with high specificity and affinity by cellular thermal shift assay,and the two small molecule inhibitors with better overall performance,501231 and 501221,were selected in this study to further investigate their anticancer activity and underlying mechanisms.First,We found that both 501231 and 501221,two small molecule MTH1inhibitors,significantly inhibited the proliferation ability of human colon cancer cells SW1116,liver cancer cells HepG2 and breast cancer cells MCF-7,causing significant cytotoxicity,but had less effect on human normal colonic epithelial cells NCM460,indicating that both 501231 and 501221 have good cancer cell selective toxicity.We next further explored the underlying anticancer mechanisms of 501231 and 501221.Firstly,we found that drug treatment caused accumulation of a large amount of 8-oxoGuanine（8-oxoG）in cancer cells,and the intracellular levels of 8-oxoG gradually increased in a time and drug concentration-dependent manner,while the ROS inhibitor NAC effectively inhibited the accumulation of 8-oxoG in cancer cells,indicating that the accumulation of 8-oxoG was related to the inhibition of MTH1 protein and the cellular oxidative pressure.Next,we observed that the accumulation of 8-oxoG caused DNA double-strand breaks（DSBs）in cancer cells,resulting in a large amount of DNA damage.The subsequent upregulation of p-Chk1（S317）and p-Chk2（T68）suggested that both DNA damage response（DDR）pathways,ATR-Chk1 and ATM-Chk2,were activated in drug-treated cancer cells,and the upregulation of two senescence markers,p21 and p16,predicted that cancer cells were about to go into cellular senescent.By further testing by flow cytometry,we found that the cancer cells showed a significant G1 phase block,which in turn led to cellular senescence and eventually apoptosis.Meanwhile,we found that ABT-263,a senescent cell scavenger,has synergistic anti-cancer effects with 501231 and 501221,and the combined treatment not only reduced the concentration of the MTH1 inhibitor drugs used,but also further accelerated the rate of apoptosis of cancer cells.However,over-expression of MTH1 protein in cancer cells significantly reduced the cytotoxicity of 501231 and 501221,demonstrating that the anticancer effect of 501231 and 501221 was related to effective inhibition of MTH1protein activity.Finally,we verified that 501231 and 501221 had significant antitumor effects in nude mice by tumor xenograft experiments.In conclusion,this study demonstrated that the novel MTH1 inhibitor 501231 and501221 have good anticancer activity.By effectively inhibiting the activity of MTH1protein in cancer cells,the two small molecule inhibitors caused accumulation of 8-oxoG to produce a large amount of unrepairable DNA damage,resulting in G₁ phase arrest and eventually causing cellular senescence and apoptosis.These findings provide an experimental basis and theoretical data for tumor treatment using MTH1 inhibitors,while the synergistic anti-cancer effects of 501231 and 501221 with ABT-263 provide a new strategy for anticancer therapy.