Discovery And Structure-Activity Relationship Study Of Novel Inducing Neuroblastoma Differentiation Regulators And WSB-1 Inhibitors

Malignant tumor is one of the most serious diseases that threaten human life and health.In recent years,a variety of molecule-targeted treatment,tumor immunotherapy,cell therapy and other treatment strategies for cancer have been developed,which to a certain extent improves the quality of life of patients and prolongs the survival period.However,malignant tumor is a complex disease controlled by many factors and the pathogenesis is very complex.The prevention and treatment of tumor still faces many challenges.Therefore,it is still the hot spot for tumor treatment to find new target and new mechanism,which has important research value and social significance.According to the intervention of neuroblastoma and tumor metastasis,on the one hand,the phenotypic screening model for inducing Neuro2a cell differentiation was used.Compound E22,a leading molecule for Neuro2a cell differentiation,was found in our compound library.On the other hand,based on the phenotypic screening method（scratch wound model）and mechanism exploration for anti-tumor metastasis,compound W15,a novel WSB1 inhibitor with anti-tumor metastasis activity,was found from the internal compound library.Furthermore,we carried out a systematic study on the structure-activity relationship.The specific work is as follows:Discovery and optimization of novel inducing neuroblastoma differentiation regulators:Differentiation induction therapy of neuroblastoma is considered to be an effective therapeutic strategy,but only 13-cis RA is used as differentiation induction agent in high-risk neuroblastoma.Therefore,in this part of work,based on the internal compound library of the research group（22 compounds with diverse structures and drug-like character）,Neuro2a was used in phenotypic screening,and the average neurite length was used as the evaluation index of induced differentiation.Compound E22 with novel structure and unique mechanism of action was found,which compared with the blank group,average neurite length increased by 55%,equivalent to the positive compound 13-cis RA（average neurite length increased by 69%）.With further structure optimization,six series of 25 compounds were designed and synthesized in this study through the strategies of bioisosterism,scaffold hopping and cyclization,and the inducing differentiation activity of the compound was evaluated by differentiation rate,average neurite length and maximum neurite length.As a result,more than 10 compounds showed better inducing differentiation activity than positive control,and most of them showed good cell activity with IC₅₀ below 5μM.Interestingly,a series of VI compounds obtained through the cyclization strategy have significantly improved their inducing differentiation activity.Especially compound 1-95 and 1-97,respectively,showed a differentiation rate of 11.73 and 9.71,which were 5.6 and 4.6 times of 13-cis RA.Ten compounds were selected for cell cycle investigation,and it was found that these compounds could cause G0/G1 arrest,which further indicated that these compounds could induce cell differentiation.Metabolic studies showed that high active compounds 1-91,1-95 and 1-97 showed excellent whole blood stability and medium liver microsome stability in vitro.Discovery and optimization of leading compound targeting WSB1 protein under hypoxia microenvironment:Tumor metastasis is closely related to tumor hypoxia microenvironment,and hypoxia of tumor often contributes to more metastatic and invasive tumors.WSB-1 is the key protein of tumor metastasis under hypoxia,which is closely related to tumor metastasis.It is of great research value to develop specific WSB1 inhibitors.Therefore,in this part of work,compound W15 found by phenotypic screening before was used as the lead compound.First,four series of 33 compounds were designed and synthesized in this part through the strategies of bioisosterism,scaffold hopping under the guidance of rational drug design principles.The results of the activity tested by wound healing assay showed that some of the compounds have good anti-metastasis activity in SW1990 and H460,and have less cytotoxicity(IC₅₀>20μM).In SW1990 and H460,there are six compounds with better anti-metastasis activity than W15respectively.Representative compounds 2-18 and 2-51 can significantly suppress the degradation of RhoGDI₂ and ubiquitination of MORF4L1 by WSB1 and show superior to compound W15.In addition,compound 2-18 can significantly inhibit the migration of WSB1 overexpressed KHOS cells（KHOS-WSB1）,and its anti-tumor metastasis activity is similar to that of positive.Therefore,compound 2-18 has a clear WSB1 pathway inhibition and anti-tumor metastasis activity,and worthy to further research.In summary,according to the abnormal differentiation of neuroblastoma and WSB1 protein under hypoxia microenvironment,we found the highly active leading compound 1-95,1-97 to induce differentiation of neruo2a and specific WSB1 inhibitor2-18 to suppress tumor migration through the rational drug design based on phenotypic screening,bioisosterism and scaffold hopping,which provided the basis for differentiation inducing therapy of neuroblastoma and anti-tumor metastasis therapy targeting hypoxia microenvironment.