Design,Optimization And Mechanism Study Of Inhibitors Against Targets In Colorectal Cancer

Nowadays,cancer becomes one of the major diseases that threaten the health of human being.Colorectal cancer is one of the leading causes of cancer deaths among adults in the world.Currently,therapeutic agents in the treatment of metastatic colorectal cancer are fluorouracil that are toxic with severe adverse reactions.Therefore,new agents with less toxic and less adverse reactions are urgent in clinical.At the same time,our deep understanding of the pathogenesis of cancer and emergence of newer molecular targets for colorectal cancer has created a powerful impact.It has been proved that the initial identification of the adenomatous polyposis coli gene as a related gene of the colorectal cancer was described very early.Mutation of APC is the early event in colorectal cancer.And mutant truncated APC which constitutively activates Asef is found to cause the aberrant migration behavior of colorectal cancer cells.Therefore,inhibitors blocking the interaction between APC and Asef can be a plausible approach for the development of new therapies for the treatment of colorectal cancer.Besides,Jinjin Zou,et al.found that protein kinase CK2alpha was overexpressed in colorectal cancer and modulated cell proliferation and invasion via regulating EMT-related genes.Lin KY,et al.found that overexpression of nuclear protein kinase CK2 alpha catalytic subunit?CK2alpha?as a poor prognosticator in human colorectal cancer.Both APC and CK2?are involved in wnt signal pathway,and kinases are currently important anti-cancer targets.The inhibitors of kinases are mainly ATP-competitive with low selectivity.The binding sites easily become drug resistance.Therefore,to find new allosteric site for designing allosteric inhibitors will be another choice for inhibiting the activity of CK2,which may further inhibit the invasion of colorectal cancer cells.The co-crystal structure of APC-ARM complexed with Asef-ABR-SH3 shows that their interaction is mostly mediated through the conserved Asef peptide spanning the region from residue 180 to residue 194?SSSHHYSHPGGGGEQLAINELISDG?.In the present study,we chose Asef170-194 as the beginning peptide and found a seven-peptide with an IC₅₀ of7.97?M by designing peptides from length,mutation and combination optimization.Then we tried to characterize the recognition mechanism of APC-ARM with its peptides.Among the best peptides,the structure of three peptides in complex with APC-ARM were solved by us.Based on the co-crystal,we designed and synthesized a series of new peptides with new capping group and new amino acids.Finally,we obtained MAI-150 with an IC₅₀ of 1.09?M.Results of ITC experiment showed MAI-150 bound to APC with the stoichiometry of 1:1and Kd?dissociation constant?value of 289 nM.The structure of MAI-150 in complex with APC was also solved by us.Based on the structures and activities of peptides with APC,we further optimized a new series of peptidomimetics and finally got MAI-203 with a Kd of 39nM.After analysis,we characterized the recognition mechanism of APC-ARM and MAI-203,and then we also analyzed the mechanism of MAI-203 inhibiting APC/Asef complex by GEF activity assay,and proposed some hints for this process.Furthermore,we investigated the cellular functions of MAI-203,we found MAI-203inhibited the interaction of APC and Asef overexpressed in 293T.MAI-203 could also decrease the GTP-bound CDC42 in SW480,but did not affect GTP-bound Rac1 as the same as the expression of both Rac1 and CDC42.Besides,we also found MAI-203 inhibited the migration of SW480 and HCT116.For CK2,we successfully predicted and validated a new allosteric site combiningcomputational Allosite and mutation analysis.Five residues in this new predicted site were mutated,with I78C and I78W inhibiting CK2?activity,V31R,K75E,I82C and P109C increasing CK2?activity.Furthermore,multiple sequence alignments of CK2?with the other family kinases showed the diversity of the residues in the new predicted site.The residues of this site are not conserved and can be targeted with rational drug design for specific inhibitors or be studied further for the discovery of their biological relevance.In conclusion,we performed an extensively methods and techniques of medicinal chemistry,chemical biology and structural biology to design a series of peptide inhibitors,and the complexes of APC/peptides have been successfully solved and the mechanism of the peptides inhibiting the APC/Asef interaction have been well analyzed yet.Furthermore,we explored its bioactivities and functions in human colorectal cell lines preliminarily.These results will help us to better understand the biology of APC/Asef in migration and provide important information for the drug design and clinical research.Besides,we found a new allosteric site in CK2,which may be targeted for the discovery of new allosteric inhibitors.