Discovery Of Small Molecule Inhibitors Of Tyrosine Kinase RET

The REarranged during Transfection(RET)receptor protein was one of the first Receptor tyrosine kinases(RTKs)that play a role in neoplasia.Expressed primarily in neural crest-derived and urogenital cells,RET is thought to be involved in the signal transduction required for proliferation,migration,differentiation and survival of neural crest cells,kidney organogenesis and spermatogenesis.Aberrant RET expression or mutation was found in multiple human diseases such as papillary thyroid carcinoma(PTC),multiple endocrine neoplasia type 2(MEN 2),an inherited cancer syndrome characterised by medullary thyroid carcinoma(MTC),pheochromocytoma,and parathyroid adenomas.RET aberrant fusions,KIF5B-RET and CCDC6-RET,contribute approximately 1.2%to 2.0%patients in Lung adenocarcinoma(LADC).Among them,KIF5B-RET is the major subtype,which presents in approximately 70%to 90%of cases reported to date.Meanwhile,CCDC6-RET presents in approximately 10%to 25%.There's no specific small molecule inhibitor of RET reported till now.It's an urgent need to develop highly potent and selective RET inhibitors.In this study,to discovery RET inhibitors,we firstly developped a targeted differential cytotoxic screening platform containing six kinds of Ba/F3 cells which stably transfected RET fusion genes including CCDC6-RET,KIF5B-RET,CCDC6-RET[V804M],KIF5B-RET[V804M],CCDC6-RET[M918T]and KIF5B-RET[M918T],respectively.With this platform in hand,we carried out a large-scale anti-RET activity screen using an in-house compound library with 750 small molecules,followed by a counter screen to exclude the general cytotoxicity using wild-type Ba/F3 cells.Finally,we successfully identified five potential specific RET inhibitors with good anti-RET activity and selectivity,including HZX125,YZY002,ZT043,LXY027,and LXY052.Their antiproliferative IC50 values against CCDC6-RET Ba/F3 were 12.29 nmol/L,34.58 nmol/L,54.39 nmol/L,20.66 nmol/L and 53.72 nmol/L,respectively.And the IC50 values against KIF5B-RET Ba/F3 were 12.66 nmol/L,32.73 nmol/L,74.14 nmol/L,85.70 nmol/L and 74.29 nmol/L,respectively.While,the positive control compound Cabozantinib exhibited antiproliferative IC50 values of 226.5 nmol/L and 301.8 nmol/L against CCDC6-RET Ba/F3 and KIF5B-RET Ba/F3,respectively.The molecular level experiments further confirmed that they had significant inhibitory effect on RET signaling.Among the five compounds,HZX125 could completely inhibit the phosphorylation of RET at a concentration of 0.1 ?mol/L,while the other four were active at the concentration of 0.5 ?mol/L.Additionally,our FACS data showed that YZY002 induced CCDC6-RET Ba/F3 cell death via apoptosis pathway.In summary,in this thesis we have established a RET-targeted screening platform and identified five potent and selective small molecule inhibitors of RET.These inhibitors will serve as good starting-points for medicinal development of RET-targeted theraphy,which have potential applications for RET-related cancers.