Screening Of Small Molecule Inhibitors Targeting BLM Helicase And Study Of Their Anti-prostate Cancer Mechanis

The Bloom Syndrome Protein（BLM）helicase is a member of the Rec Q helicase family（RECQ1,BLM,WRN,RECQ4,RECQ5）,which unwinds secondary nucleic acid structures like duplexes,Holliday junctions,G-quadruplexes,and DNA displacement loops as a 3’-5’ATP-driven DNA helicase.BLM is a crucial component of DNA damage repair（DDR）pathways,helping to maintain genomic integrity through homologous recombination repair（HRR）,telomere maintenance,and replication stress reduction.Mutations in BLM cause the autosomal recessive disease Bloom syndrome（BS）.Multiple malignancies,including prostate,breast,and lung cancers,are more common in BS patients.Prostate cancer（PCa）is a major disease threatening men’s health with high morbidity and mortality.Recent studies have indicated that the BLM gene is the most important gene among seven high-risk PCa genes,and the high expression of BLM in PCa tissues is positively correlated with the malignant degree of the tumor.Previous work has shown that BLM overexpression promotes PC3 cell proliferation,and knockdown of BLM promotes PCa apoptosis and inhibits PCa cell proliferation.In brief,BLM is considered a potential and promising target for cancer treatment.In order to obtain new anticancer drugs,many researchers use BLM helicase to screen for or design small molecule inhibitors.However,there are still few reports on specific anticancer drugs targeting BLM helicase,so it is of great theoretical value and scientific significance to develop new inhibitors of BLM helicase and analyze the mechanism of anticancer specificity and effectiveness of small molecule inhibitors targeting BLM helicase.The research results are as follows:1.The high expression of BLM helicase in PCa tissues and cells suggested that BLM helicase might be an anti-prostate cancer target.We used the BLM antibody to analyze the expression of BLM helicase in 60 PCa tissues,30 hyperplasia tissues,and 30 paracancer tissues.The results showed that the expression of BLM helicase in PCa was significantly higher than that in hyperplasia and paracancer tissues and PCa.In PCa tissue samples,the high expression of BLM was significantly or extremely significantly associated with clinical stage and Gleason grade but not with age,Gleason score,lymph node metastasis,or distant metastasis.The expression of BLM helicase in multiple PCa cell lines was significantly higher than that in normal prostate cells.2.BLM helicase inhibitor AO/854 was screened.Using BLM helicase as the target,30 small molecule compounds possibly targeting BLM were screened from a library of 200,000 small molecule compounds by molecular docking technology.The 30 small-molecule compounds were further studied by the fluorescence polarization technique.The results showed that the small molecule compound AO/854 significantly reduced the DNA binding activity of BLM^642-1290helicase,and the relative DNA binding activity of BLM^642-1290helicase was reduced to 31.5%after 20μmol/L AO/854 treatment.The results of extracellular fluorescence polarization,ATPase activity,and EMSA showed that AO/854,as a competitive inhibitor,disrupted the binding of BLM helicase to DNA and inhibited the chain uncoupling activity and ATPase activity of BLM^642-1290helicase.The results of the intracellular activity assay showed that AO/854 inhibited the intracellular unwinding process.The results of circular dichroism showed that AO/854 destroyed the secondary structure of the BLM^642-1290helicase.3.AO/854 cause DNA damage in PC3 cells,activate the DNA damage repair pathway,activate or restore the downstream P53 pathway,and then cause a series of changes in cellular biological functions.We conducted a series of cell,animal,and molecular experiments to investigate the effect of AO/854 on PCa and its mechanism.The results showed that AO/854could inhibit the proliferation,migration,and invasion of PC3.Flow cytometry was used to detect the effects of AO/854 on the cell cycle and apoptosis of PC3 cells.The results showed that AO/854 could shorten the S phase,prolong the G0/G1 phase,and induce apoptosis in PC3 cells.The results of the comet assay and WB experiments showed that AO/854 can induce DNA damage,upregulate p-Chk1 and p-Chk2,and activate the DDR pathway.To explore the anti-tumor mechanism of AO/854,we used proteomics to investigate the differences in protein expression of PC3 cells after AO/854 treatment.After AO/854 treatment,multiple differential proteins were associated with BLM helicase and involved in multiple cancer-related pathways,among which the P53 signaling pathway was the most obvious.The verification by WB concluded that AO/854 was able to up regulate P53 protein expression in PC3cells and reactivate or restore wild-type P53 function in PC3 cells.In conclusion,we can conclude that AO/854,an inhibitor of BLM,causes DNA damage and activates the DNA damage repair pathway and downstream P53-dependent apoptosis in PC3cells.At the cell level,this was verified at the animal level.4.The BLM helicase inhibitors can enhance the sensitivity of PCa cells to cisplatin（CDDP）Based on the critical role of BLM helicase in DNA damage repair and the mechanism by which CDDP can cause DNA damage,we explored whether BLM helicase inhibitors could enhance the sensitivity of PCa cells to CDDP.We examined the synergistic cytotoxicity of AO/854 and CDDP in PCa cells by the CCK8 assay.Comet assays,indirect immunofluorescence,and WB experiments were used to test the effects of AO/854 and CDDP alone and in combination on DNA damage.The effect of AO/854 and CDDP on apoptosis was examined by flow cytometry.The verification was verified at the animal level.The experimental results showed that AO/854 and cisplatin showed synergistic cytotoxicity in several PCa cell lines,and the synergistic effect between AO/854 and cisplatin was most obvious in PC3 cells.AO/854 can significantly increase the proportion of apoptosis in PC3 cells caused by cisplatin by enhancing co-DNA double-strand breaks（DSBs）induced by cisplatin.We verified the experimental results at the cell level through animal experiments.In summary,we established a high-throughput screening method and obtained a new BLM helicase inhibitor,which could activate the P53 signaling pathway and cause some changes in cell cycle,proliferation and metastasis.This study provided a new idea for drug research targeting BLM helicase.