| BACKGROUNDHedgehog signaling pathway plays an important role in embryonic development,differentiation,microenvironmental stability,tissue repair and other physiological processes.In recent years,Hedgehog signaling pathway was reported to be abnormally activated in many cancers,which was related to the cell proliferation,differentiation,apoptosis,angiogenesis,invasion and metastasis.Hedgehog signaling pathway is composed of Hedgehog(Hh)ligand,Ptched(Ptch)protein,smoothened(SMO)protein,glioma associated oncogene homolog(Gli)transcription factor and its downstream targets.SMO protein,the signal switch protein of Hedgehog signaling pathway,is an intermediate transduction target of Hedgehog signaling pathway,with a huge intracellular multimolecular network downstream including suppressor of fused(Sufu)、protein kinase A(PKA)、glycogen synthase kinase 3(GSK3)and dual-specificity tyrosine phosphorylation-regulated kinase-1A(DYRK1)which would modify Gli protein regulating the expression of the target genes.Vismodegib and NVP-LDE-225 are the only two SMO inhibitors used in clinic,but serious side effects and drug resistance limited their use.Therefore,development of novel SMO inhibitors with high selectivity and high performance is very important.OBJECTIVESTo design and synthesize a series of novel SMO protein inhibitors with high selectivity,low toxicity and evaluate their biological activities in vitro and in vivo.METHODS1.To design and synthesize a series of novel 8-chloro-3-(5-amino-2-chlorophenyl)-[1,2,4]triazolo[4,3-a]pyridine derivatives(A1-A20).2.Schrodinger was used to predict the pharmacokinetic properties for absorption,distribution,metabolism,and excretion of compounds(A1-A20)in vivo.3.Dual luciferase reporter assay was used to evaluate the effect of compounds(A1-A20)on SMO protein.4.Molecular docking was used to simulate the interaction between the SMO protein and compounds A3、A14、A15、A17.5.Surface Plasmon Resonance(SPR)experiment was used to evaluate the binding strength between compounds A3、A14、A15、A17 and SMO protein.6.The fluorescent confocal technique was used to evaluate the effect of compounds A3 and A14 on blocking localization of SMO to primary cilia.7.The MTT assay was used to evaluate the effect on proliferation of compounds(A1-A20)on human colon cancer cell lines(HCT-15,RKO,HCT-116,SW-620,HT-29),human breast cancer cell lines(MDA-MB-468。MDA-MB-231,MCF-7,T47D),human non-small cell lung cancer cell line(A549),human glioma cell line(U251)and human cervical cancer cell lines(Hela,AN3-CA).8.Clonogenic assay was used to evaluate the effect of compound A14 on colony formation capability of tumor cells.9.The effect of compound A14 on the cell cycle and apoptosis of tumor cell lines HCT-15,RKO,HCT-116,SW-620 and HT-29 was detected by flow cytometry.The expression of cell cycle related and apoptosis related proteins were detected by western-blot.10.Scratch wound healing assay was used to examine the effect of compound A14 on migration ability of tumor cells.11.The effect of compound A14 on the expression of Hedgehog signaling pathway proteins in tumor cells was detected by western-blot.12.Immunofluorescence detection was used to investigate the effect of compound A14 on localization of SMO protein in tumor cell lines HT-29 and RKO.13.The effect of compound A14 on proliferation of HT-29 was evaluated in vivo.RESULTS1.Twenty novel 8-chloro-3-(5-amino-2-chlorophenyl)-[1,2,4]triazolo[4,3-a]pyridine derivatives(A1-A20)were designed and synthesized,and the structures of them were confirmed by 1H-NMR,13C-NMR,and HR-ESI-MS.2.Schrodinger prediction results showed that the pharmacokinetics of ADME of compounds(A1-A20)were in accordance with the Lipinski’s rules.3.Dual luciferase assay results showed that compounds(A1-A20)could significantly inhibit the activity of SMO protein.Compound A3,A14,A15 and A17 could inhibit the activity of SMO protein with IC50 values of 101 ± 14.7 nM,95.8±1.6 nM,93±7.3 nM and 92.6±12.9 nM,respectively.4.Molecular docking experiment showed that compounds A3、A14、A15 and A17 could bind to the ligand domain of the SMO protein receptor(PDB,5L7I)through the hydrogen bond.5.SPR results showed that the equilibrium dissociation constants(KD)between the compound A3,A14,A15 and A17 and SMO were 1.21 ×10-5 mol/L,1.36×10-5 mol/L,1.03×10-8 mol/L and 1.06×10-8 mol/L,respectively.6.In the fluorescence confocal experiment,compound A3 and A14 could block the localization of cytoplasmic SMO protein to primary cilia.7.The results of MTT experiment showed that compounds(A1-A20)could inhibit the proliferation of HCT-15,RKO,HCT-116,SW-620,HT-29,MDA-MB-468,MDA-MB-231,MCF-7,T47D,A549,U251,HeLa,and AN3-CA cancer cells with IC50 values range from 6.2±1.5 μM to 96.4 ±11.2μM.8.Compound A14 could inhibit the cologenic growth of HCT-15,RKO,HCT-116,SW-620 and HT-29 with IC50 values of 12.05 ± 2.35 μM,3.53± 0.34 μM,18.20 ±3.80 μM,1 1.65 ±0.29 μM,and 11.75 ±0.35μM,respectively.9.The results of cell cycle experiment showed that compound A14 could induce cell cycle arrest of HCT-15,RKO,HCT-116,SW-620 and HT-29 at G0/G1 phase and induce cell apoptosis.The results of western-blot showed that compound A14 could up-regulate the expression of p53 and p21,and inhibit the expression of E2F1 in RKO and HT-29 cells.The expression of p-AKT and p-Erkl/2 were decreased.Except for HOT-116,Bax was down-regulated in other four cancer cells.10.Compound A14 could inhibit the healing of the scratched area of HT-29 and RKO cells.11.The results of western-blot showed that the expression of Glil was down-regulated in HCT-15,HCT-116 and HT-29 after treating with compound A14 for 72 h.Shh but not Ptch2 and SMO were down-regulated in HT-29 cells.12.The results of the fluorescence confocal experiment showed that compound A14 could block the localization of SMO protein to primary cilia in HT-29 and RKO cells.13.Compound A14 had no obvious toxicity in vivo,and its effect on proliferation in nude mice was in a concentration-dependent manner.CONCLUSIONThese results of our research showed that the novelly designted and synthesized 8-chloro-3-(5-amino-2-chlorophenyl)-[1,2,4]triazolo[4,3-a]pyridine derivatives(A1-A20)could specifically inhibit the activity of SMO protein.The characteristic of this series of compounds showed that they could enter into the ligand domain of SMO protein and bind to SMO protein through the hydrogen bond,with the equilibrium dissociation constant values less than 1×10-5 mol/L.Although not affecting the expression of SMO protein,they could significantly affect the localization of SMO proteins.Compounds(A1-A20)produced significant and anti-tumor effects with low cytotoxicity to human normal cells.Among them,compound A14 showed particularly effective against colon cancer,and could significantly block the cell cycle of colon cancer cells at G0/G1 phase,induce cell apoptosis,and inhibit cell migration.Compound A14 had no obvious toxicity and inhibited the proliferation of HT-29 in nude mice in a concentration-dependent manner in vivo. |
