Cardiogenesis Promoting Activity Evaluation Screen Of Small Molecules From Quinazolines Library And Related Molecular Events Research

Embryonic stem cells(ES cells) can maintain undifferentiated in vitro and differentiate into different cell types from ectoderm or mesoderm or endoderm when giving the right culture conditions.Since many promising studies have shown the therapeutic potential of differentiated derivatives of ES cells in ameliorating a range of diseases in animal models,ES cells have played a gradual important role in regenerative medicine & drug discovery.The pharmaceutical industry is beginning to use ESC derived models for specialized screens,and for the evaluation of the biological activities of focused libraries built around a lead compound.It is likely that drug-like molecules will be discovered which themselves will play a role in driving differentiation in vitro.Drug candidates based on such leads could also have a future in the clinical treatment of degenerative disease by modulating the regeneration of tissue in vivo.Cardiovascular disease has threatened human health for a long time,especially once heart has been damaged,it can't be regenerated again.There are promising studies have shown,myocardial cells differentiated from ES cells in vitro have the same myocardial characteristics and function as normal heart[11-13],in addition to their potential for application in regenerative medicine,ES cells have important roles in cardiovascular drug discovery.Recent studies have identified novel small molecules that mediate cell-fate acquisition and the differentiation behavior of a number of stem cell types.A cell-based screen of chemical libraries was carried out to identify small molecules that control the differentiations of ES cells.Small molecules from chemical library possessing a desired activity were reported in this paper.Because of its wide range of biological activities, Quinazoline has been attracted people's attention.As a inhibitor of epidermal growth factor receptor(EGFR) or EGF receptor tyrosine kinase,it shows anti-cancer activity and can be used in cancer treatment,including lung cancer,stomach cancer and gall bladder cancers.Cause stem cells and tumor cells share a lot of molecular signaling pathway in differentiation and proliferation,a series of quinazolines using combinatorial chemistry were synthesized and evaluated for their cardiac differentiation activities throngh P19 and ES cells.For the primary screen,stably transfected with cardiac-specific atrial natriuretic factor(ANF) gene promoter-driven luciferase reporter gene,expression of which could limited to developing cardiomyocyte,mouse embryonic carcinoma(EC) cell line P19 was used for the screen of quinazolines for their ability to induce cardiac differentiation through quantitative fluorescence detecting.After treating with compounds in 0.10,0.50 and 1.00μM respectively for 3 days and without for 4 days,detect the fluorescence intensity through ELIASA(negative control:DMSO/positive control:0.001μM RA). Out of thirty-one quinazolines identified,165,62,D39 and Z34 have the inducible effects on differentiation of P19 cells into cardiomyocytes in vitro.Especially,62 could remarkably induce murine ES cells directionally differentiate into cardiomyocytes at a concentration of 0.1μM.To confirm that these compounds are general cardiomyogenesis inducing agents,we analyzed their effects on undifferentiated mouse ESCs.The four quinazolines were added into mouse ESCs during all stages of differentiation,microscopic observations were conducted to monitor beating embryonic bodies(EBs),and to determine the beating rate on d 5+7.Among these four compounds,62 could remarkably induce murine ES cells directionally differentiate into cardiomyocytes at a concentration of 0.1μM by calculating the total percentage of beating EBs during the course of differentiation,and the beating areas of EBs could stain with cardiac-specificα-actinin protein through immunofluorescence after compounds treatment.MTT test examine the toxic effects of quinazoline for ES cells,and 62 showed almost no toxicity,which turns out that 62 could be selected as a target compound for the follow-up mechanism research.First of all,we tested the inhibition effect on EGFR tyrosine kinase of 62 compared to Iressa,and the inhibition was not manifest,indicating differentiated induction of 62 may not be entirely through EGFR to carry out.besides the results of the computer-aided design support this conclusion.So we haved explore the mechanism of 62 during ESCs differentiation with compound 193,which shows no obvious differentiated induction effect,as negative control.After detected the protein expressions of Oct-4,JP2,p38, p-p38,NF-κB,we found the expression of differentiated flagging protein Oct-4 was markedly decreased,indicating 62 have significant role in inducing ESCs differentiation. At the same time,JP2,which expressed a lot in adult heart,has a remarked increase, while the most part of nuclear transcription factor NF-κB transferred into nuclear.The two phenomenon reveled these two protein and their related signaling pathway play an important role in the differentiated induction of 62.In the controversy,the expression level of MAPK family protein p-p38 actually decreased during the differentiation course,and 62 may not achieved induction through this pathway.Summary1.Compound 62 of quinazoline has a strong ability to induct ESCs differentiate into cardiomyocytes,while 0.10μM is the ideal concentration.2.the mechanism of 62 is possibly related to the protein JP2 and nuclear transcription factor NF-κB,while EGFR tyrosine kinase and MAPK family of p-p38 may not closely associated.