Preliminary Evaluation Of The Pharmacological Activity Of The TRPC6 Novel Inhibitor BP3112 Against Hepatocellular Carcinoma

Hepatocellular carcinoma is a malignant tumor with high morbidity and mortality.With the increasing number of patients with liver disease,the research of liver cancer has also received increasing attention.A large number of studies have shown that classical transient receptor potential channel 6（TRPC6）plays a key role in the initiation and progression of hepatocellular carcinoma;TRPC6 is involved in the proliferation of hepatoma cells and other aspects of biological behavior by regulating[Ca²⁺]_i levels.Therefore,therapeutic strategies targeting TRPC6 could be a promising treatment for liver cancer.The development of novel TRPC6-specific inhibitors will help explore the specific mechanism of action of TRPC6 in the development of hepatocellular carcinoma and provide more treatment options,which is expected to become a new strategy for the treatment of liver cancer in the future.In this paper,the TRPC6 small molecule inhibitor3-[（1-methyl-1H-indole-2-carbonyl）amino]propionate butyl ester（BP3112）was fully synthesized,and its in vivo and in vitro anti-hepatocellular carcinoma activity was carefully evaluated,and the anti-hepatocellular carcinoma mechanism was preliminarily elucidated,in order to provide data for the research and development of anti-hepatocellular carcinoma drugs.Methods:1.The compound BP3112 was fully synthesized by acylation reaction,hydrolysis reaction and esterification reaction,and compound BP3112 was identified by ¹H NMR and ¹³C NMR.2.The affinity and binding specificity of compound BP3112 and commonly used inhibitor SKF96365 on TRPC6 were determined by microthermophoresis,and the effects of BP3112 and SKF96365 on the proliferation,cycle and apoptosis of hepatoma cells in vitro and the blocking effect on TRPC6 channel were determined by thiazolyl blue tetrazolium bromide method,flow cytometry and dual-wavelength ion imaging experiments.3.A model of subcutaneous transplantation tumors in nude mice was established,and24 mice with successful modeling were randomly divided into 4 groups,and 6 mice/group were administered intraperitoneally,respectively,the model group（normal saline）,BP3112low-dose group（25 mg/kg）,BP3112 high-dose group（50 mg/kg）,and SKF96365 group（50 mg/kg）,and the tumor suppression rate of BP3112 and SKF96365 on subcutaneous tumors in nude mice was determined.The effects of BP3112 and SKF96365 on lung and kidney tissue morphology were evaluated by HE staining,the expression of TRPC6 and Ki67 in tumor tissues was determined by immunohistochemistry,and the anti-liver cancer activity of BP3112 and SKF96365 was comprehensively evaluated.4.CRISPR/Cas9 technology constructed stable transfer lines of HepG2 and HCCLM3cells knocked out by TRPC6,and further explored the mechanism of BP3112 inhibition of liver cancer through TRPC6 targets by PCR,Western blot,thiazolyl blue tetrazolium bromide method and flow cytometry.Results:1.The synthesized compound was identified as butyl3-[（1-methyl-1H-indole-2-carbonyl）amino]propionate,（named BP3112）.2.Preliminary evaluation of BP3112 anti-liver cancer activity in vitro:The results of microthermal phoresis showed that BP3112 and TRPC6 binding constant was 2.65±0.25μM and did not bind to TRPC3 and TRPC7,SKF 96365 and TRPC6 binding constant was308.32±115.79μM.The binding constants with TRPC3 and TRPC7 were 3.95±1.27μM and 3.42±0.95μM,respectively.BP3112 specifically bound to TRPC6 than the commonly used inhibitor SKF96365.The results of thiazolyl blue tetrazolium bromide method showed that BP3112 and SKF96365 significantly inhibited the proliferation of HepG2 and HCCLM3 in hepatoma cells,and were dose-dependent and time-dependent,the 48-hour half-inhibitory concentrations of BP3112 were 27.05±5.34μM and 1.97±1.79μM,respectively,and the 48-hour half-inhibitory concentrations of SKF96365 were 33.44±4.56μM and 9.58±2.02μM,respectively.The flow cytometry results showed that BP3112and SKF96365 had a significant effect on the cycle of HepG2 and HCCLM3 cells,causing cell arrest in G1 stage and apoptosis of hepatoma cells.The results of dual-wavelength imaging experiments showed that BP3112 could inhibit calcium influx in vitro,with an inhibition rate of 74.78±0.77%at 40μM.3.Preliminary evaluation of the anti-liver cancer activity of BP3112 in vivo:BP3112can inhibit HepG2 xenograft tumors in vivo with concentration-dependence,and the inhibition rates of subcutaneous tumors in nude mice in the 50 mg/kg BP3112 and positive drug SKF 96365 administration groups were 77.5%and 42.1%,respectively.The results of HE staining showed that BP3112 and the positive drug SKF96365 had less influence on lung and kidney tissue morphology.The immunohistochemical results showed that the administration of BP3112 and SKF96365 significantly reduced the expression of Ki67 in tumor tissues,and the effect of SKF96365 was weaker than that of BP3112,indicating that BP3112 had a stronger anti-hepatoma cell proliferation effect in vivo.The administration of BP3112 and SKF96365 significantly reduced the expression of TRPC6 in tumor tissues,and the effect of SKF 96365 was weaker than that of BP3112,indicating that BP3112 had a stronger effect of inhibiting TRPC6 expression in vivo.4.Preliminary study on the anti-liver cancer mechanism of BP3112 targeting TRPC6:PCR results showed that BP3112 could inhibit the m RNA expression of TRPC6 in hepatoma cells.Western blot results showed that BP3112 could specifically inhibit the protein expression of TRPC6 in hepatoma cells without affecting TRPC3/7,and BP3112could also inhibit nuclear translocation and protein expression of activated T nuclear factor c1（NFAT c1）and protein expression of Cyclin D1 in concentration-dependence.CRISPR/Cas9 technology successfully constructed two TRPC6 knockout stable transfer cells,with a knockout rate of more than 80%.Western blot results showed that BP3112administration of TRPC6 knockout stable transfer cells could not further downregulate TRPC6 and Cyclin D1 levels.The results of flow cytometry showed that BP3112administration of TRPC6 knockout stable transfer cells could not further cause cell cycle G1 phase arrest.The results of thiazolyl blue tetrazolium bromide method showed that BP3112 administration of TRPC6 knockout stable transfer cells could not further inhibit cell proliferation.Further studies showed that TRPC6 is a specific target for BP3112 to cause decreased expression of Cyclin D1 protein,cell cycle G1 phase arrest,and cell proliferation inhibition.In summary,the small molecule inhibitor BP3112 can specifically bind to TRPC6 to produce significant in vivo and in vitro anti-liver cancer activities,and the mechanism of action is to inhibit the nuclear translocation and protein expression of downstream NFAT c1by inhibiting TRPC6 channel function and protein expression,so that the key protein Cyclin D1 protein that regulates the G1 phase of the cell cycle is down-regulated,thereby affecting the proliferation,cycle and apoptosis of hepatoma cells.BP3112 is expected to be further developed as a novel drug targeting TRPC6 for the treatment of liver cancer.