| Cancer is the first health threat to human death diseases,of which the primary biological properties behave highly metastatic and invasive compared to normal cells,leading to very difficult treat and high mortality.More than 90%patients with cancer died of tumor metastasis,and the other survival could hardly ever survive more than five years.Therefore,it is the great key of cancer treatment to control cancer proliferation and metastasis.Adenomatous polyposis coli APC is an important tumor suppressor protein.Cancer patients’ APC genes generally tend to appear gene mutation,expressed as a truncated APC.Truncated APC no longer make a normal physiological function,but selectively combine with APC-stimulated guanine nuclotide exchange factor Asef and make it activated,thereby promoting cancer cell proliferation,invasion and metastasis.Thus,the interaction between the truncated APC and Asef plays a vital role in tumor mutation and proliferation,of which inhibitors have become a new significant target for cancer therapy.Contraposing the APC/Asef interaction inhibitors,the research on this topic is based on known active skeleton structure and computer-aided drug design including comparative moleular field analysis,docking operation analysis and so on.Dihydro-pyrazol-morpholine was designed and filtered out as a basic skeleton,which mignt develop a potent therapeutic small molecule inhibitor having a specific inhibition for interactions of APC/Asef.Meanwhile,the build-related 3D-QSAR model analysed a quantitative structure-activity relationship group of homologues skeleton with a good prediction and correlation.25 target compounds were designed in this research,which were all first synthesized and made correct structural characterization by utilizing 1H NMR,ESI-MS,IR and 13C NMR.Meanwhile,single crystal X-ray diffraction structure analysis further identified nine compounds to determine the specific type of bone structure as representative of target compounds.As for biological activity,Interaction Inhibitions between APC and Asef were estimated by Fluorescence Polarization Immunoassay and Native-PAGE experiments.Wherein the most potent compound A7 exhibited excellent inhibitory activities with IC50 value 0.22±0.01 μM.Besides,it is potent in the cell antiproliferative activity in six tested cancer cell lines(MCF-7,HeLa,A549,HepG2,HCT116 and SW620),with most effective antiproliferation in the HCT116.The best behaved active compound was also compound A7 with IC50 value 0.34±0.01μM,while the positive control drug,Regorafenib having the IC50 value remained 1.15±0.04 μM.In addition,compound A7 was further proved to induce HCT116 apoptosis by flow cytometry with Annexin V-FITC/PI Apoptosis Detection Kit and Laser Scaing Confocal Microscopy,from number and morphology,respectively.Meanwhile,Mitochondrial Membrane Potential experiment results showed the compound A7 induced early HCT116 apoptosis echoed with Annexin V-FITC/PI Apoptosis.Moreover,compound A7 was visually expressed to inhibit HCT116 invasion and metastasis effectively by Transwell Invasion Assay the,and made safety evaluation by cytotoxicity experiment.In conclusion,all compounds were scrutinized to make virtual model by 3D quantitative structure-activity relationship,providing a guideline to design and optimize more effective interaction inhibitors between APC and Asef,of which compound A7 was expected to be a novel potential anti-cancer lead compound. |
