Virtual Screening And Anti-cancer Activity Of Allosteric Inhibitors Targeting Protein Kinase CK2

Protein kinase（Casein Kinase 2,CK2）is a serine/threonine kinase that is a key regulator of many cellular processes,and the over-expressed CK2 have been found in a variety of cancers.Hence,CK2 has been considered as an valuable target for anti-cancer drug discovery.However,the development of selective inhibitors remains challenging because of the highly conserved ATP-binding pocket（orthosteric site）of kinase family.By targeting the much more diversified allosteric site relative to the highly conserved ATP-binding pocket,allosteric inhibitors might avoid the drawbacks of most conventional ATP-competitive inhibitors with their enhanced selectivity and reduced toxicity.Therefore,targeting the allosteric sites of CK2 offering an excellent perspective to develop efficient and selective allosteric CK2 inhibitors.TheaD allosteric site of CK2 is a deep and hydrophobic U-shaped cavity with high structural flexibility and partially opened unique conformation,making it an important target site for the study of highly selective CK2 allosteric inhibitors.Here we discovered the novel allosteric inhibitors through the combing structure-based virtual screening and biological evaluation methods.This study provide the construction of pharmacophore model based on CK2αD pocketConstruction of pharmacophore model based on receptor structure is an important method in drug design.Molecular dynacmics simulation was performed to elucidate the binding mode of CK2αwith ligand（PDB ID:5ORJ）.And key residues of protein kinase CK2aD site were revealed,including hydrophobic residues Phe121,Tyr125,Ile133,Tyr136,Met221 and Met225,and polar residues Asn118,Pro159 or Val162.Furthermore,the pharmacophore model based on CK2αD pocket was constructed by using discovery Studio 4.0.Two donors and two hydrophobics features were identified as the pharmacophore model.Validation results showed that two donors matched with the amino group of 10 known active ligands,and pointed to the skeleton carbonyl group of Pro159 and Val162,as well as the carbonyl group on the side chain of Asn118.Two hydrophobics features were related to the chloro group,alkylbenzene or methoxy group of the biphenyl and fell into the hydrophobic cavity consisting of Ile133,Tyr136,Met221 and Met225.This result showed that the pharmacophore model is reliable and can be used to guide the receptor based virtual screening.Small molecular fragment compounds have the advantages of small molecular weight,simple scaffolds and a variety of chemical spaces for further optmization.Therefore,the well validated pharmacophore model was used as a 3D query to screen chembridge?fragment library and then retained molecules were subjected to filter based on binding affinity prediction,molecular docking and interaction-moding analysis.Then six compound with high Gold＿fitness score,good binding affinity,can interact with Asn118,Pro159 or Val162 and embed the pocket composed of Phe121,Tyr125,Ile133,Tyr136,Met221 and Met225 were obtained for evaluation of biological activity.Integrating a method of molecular biology and cell biology were employed to test the activity of kinase inhibition and anticancer of the six theoretical hits.Results of ADP-Glo^TM kinase assay indicated that compound 3 exhibited the highest CK2 kinase inhibitory activity(IC₅₀=13.1μM),but compound 4 which share the similar structure with compound 3 showed IC₅₀>1000μM.Molecular dynamics simulation results indicated that the steric hindrance effect of naphthalene substituents on compound 4 is the structural factor that leads to the difference of their inhibitory activities.The results showed that compound 3,4,5 and 6 inhibited the cell proliferation,migration and invasion of A549,especially compound 3 was a new skeleton small molecule which have both high kinase inhibitory and anti-canceractivity.The IC₅₀ values of this compound against A549 was 23.1μm.Cell migration and invasion experiments in A549showed that it inhibited 47%cell migration at 40μm and 28%cell invasion at 20μm with 36 h.To sum up,based on theaD allosteric site of CK2,pharmacophore model,molecular docking,affinity prediction and interaction mode analysis were carried out for virtual screening study point at chembridge?fragment library.Combined with the biological activity evaluation of theoretical hits,we obtained a new skeleton leading compound which have both high kinase inhibitory and anti-cancercell proliferation,migration and invasion activity.This study will be helpful for the structural optimization design of leading compound and provided a new idea for the development of potent anti-cancer drugs targeting CK2α.