Molecular Simulations Of MAGUKs-GK Domains And Discovery Of Targeted Small Molecular Inhibitors

Membrane-associated guanylate kinases(MAGUKs)are a family of scaffold proteins that are enriched at the junctions of cells and play an important role in the cell polarity establishment and maintenance,cell adhesion,and cellular signal transduction.This family of proteins contains multiple protein-interacting domains,of which the GK domain binds to multiple proteins in two modes,phosphorylated and non-phosphorylated.However,the physiological function of the GK domain and the molecular mechanism of the binding mode are not clear.Based on this,our paper studies the structural characteristics of different GK domains and dicovery the targeted molecules,which has great significance for understanding its function and discovering new intervention drugs.Large homolog protein(DLG)is an important member of the MAGUK family.The GK domain of DLGs regulates various cellular functions by binding to phosphorylated and non-phosphorylated peptides.In this paper,we studied the interactions of DLG1 and DLG4 with different peptides through molecular dynamics simulations.The results show ed that DLGs-GK proteins have the same binding mode with phosphorylated or non-phosphorylated ligands,dividing into phosphorylation site and hydrophobic site at ?5 fold.In addition,based on binding free energies calculations using MM-GBSA,we predicted that DLG1-GK can bind to the inhibitory peptide GKI-QSF targeting DLG4-GK with similar binding affinity.Subsequently,the Kd value of DLG1-GK/GKI-QSF determined by ITC experiments is 1.20 ± 0.29 ?M,consistenting with the calculation prediction.As a member of the MAGUK family,MAGI2 plays an important role in synapse formation and nerve conduction.The three-dimensional structure of MAGI2-GK was constructed by homologous modeling and the complex structure of MAGI2-GK/p-SAPAP was obtained by molecular docking.Then,the interaction was studied by molecular dynamic simulations.The results indicated that the binding pockets of MAGI2-GK were similar to DLGs-GK,and were mainly divided into phosphorylation sites(Arg134 and Lys137)and hydrophobic interaction sites(Gly167,Thr168,Tyr169,and Tyr174).In addition,we found that the amino acid residue Tyr125 located in the ?1 helix is the key amino acid that distinguishes MAGI2-GK from DLG4-GK,and can be selected as the target amino acid for the development of selective MAGI2-GK small molecule inhibitors.Finally,we screened for small molecule inhibitors targeting MAGI2-GK.The MAGI2-GK protein was prepared by prokaryotic expression and purification,and combined with FITC-labeled p-SAPAP polypeptide,we established and optimized fluorescence polarization assay for compound screening.Then,based on the analysis of the MAGI2-GK binding pocket,we implemented the docking-based virtual screening method.We screened 1.3 million compounds in the Chembridge compound library,and obtained 14 hit candidates for binding affinity testing.We found a small molecule inhibitor 11,but its affinity was weak.On the other side,a lead compound 3a with azaindole scaffold was discovered by screening 435 compounds in the self-built small drug compound library.The compound only showed binding affninity with MAGI2-GK,rather than DLG4-GK,though the structure of GK domains are similar.To optimize the lead by structural modification of the N-position of maleimide and the 3-position of azaindole,14 compounds were synthesized and the most active compound was 3kb(Ki of 0.43 ?M).Finally,we preliminary in vitro evaluated the antitumor activity of 3a,and found that it could significantly inhibit the migration and invasion of gastric cancer cell line MGC803 and affect the EMT process,but had little effect on the proliferation.In summary,binding interfaces between DLGs-GK and MAGI2-GK and their interacting peptides were explored by molecular dynamics simulations.And a new small molecule inhibitor of MAGI2-GK was discovery.Our studies should be significant for the future research on the structure and function of GK domain.