Design Of Inhibitors Targeting ATG5 Protein

With the development of drug design field,the design of traditional drug molecular targets such as kinases and ion channels has approached saturated.Pharmaceutical scientists hope to find more drug targets to provide more possibilities for drug design.Protein-protein interaction（PPIs）targets have gradually come into focus,which involved in regulating DNA replication,transcription,translation and transmembrane signal transduction,transformation,autophagy and other cellular functions.Autophagy is an important way for cells to degrade intracellular useless proteins and organelles.In recent years,the research progress of autophagy has highlighted its importance under physiological and pathological conditions.Study has shown that protein-protein interaction network in the autophagy pathway regulates the autophagy pathway in a complex and controllable way.The ATG5 protein is a PPIs protein in the autophagy pathway.The ternary complex formed with ATG16 and ATG12 is the key to the extension of the autophagosome membrane.The current research on the ATG5 protein still remains at the mechanism and genome level.There are few pharmacological studies about ATG5 protein.Therefore,it is a very novel and challenging work to study related drugs targeting ATG5 protein.In the first part,peptide inhibitors was designed for ATG5 protein.Molecular dynamics simulation was performed on existing ATG5-TECAIR inhibited peptide,combined with HTRF assay data,binding mode of the peptide was determined.Then explored the length of peptide through assay method of HTRF and computational method of free energy decomposition.After optimization,IC₅₀ value of peptide has increased by about four times.At present,the highest IC₅₀ value of this series of peptides can reach 28 n M.Next,we explored the membrane permeability of wild-type peptides 16a10 by adding membrane penetrating peptides and fluorescent group FITC.The results of fluorescence confocal microscopic experiment showed that membrane penetrating peptides were the key for wild-type peptides 16a10 to enter the membrane.In another part of the study of membrane permeability,we attempted to make hydrophobic mutations on the protein binding surface of the peptide.Interestingly,we found that some peptides also had inhibitory activity of about 5μM to the Atg5-Atg16complex,although the inhibitory activity was not high,but initially showed a tendency to inhibit autophagy in cells.The second part of this work is the designing of small molecular inhibitors targeting ATG5 protein.Firstly,alanine scanning was used to predict the“hot spot”residues on the ATG5 protein.These hot residues laid the foundation for the structural modification of small molecules and the exploration of the length of peptides and the most suitable residues for each site subsequently.Subsequently,molecular docking and molecular dynamics simulation methods was used to determine the binding mode of the lead compound TS13 in the pocket of ATG5.By means of similarity search,shape matching and virtual screening,the first round of searching for 1367 listed drugs was completed,and the drug nitrofurnyl hydrazone with a completely different backbone from TS13 was obtained.In the second round of screening,we the structures of nitrofuren hydrazone and the lead compound TS13 was superimposed to produce pharmacophores model for the hydrogen bond acceptor of TS13,the hydrogen bond donor of nitrofuren hydrazone,and the hydrophobic aromatic ring which both structures are owned.The shape and pharmacophores model was used to search more than three million compounds in the chemdiv database,and a second round of virtual screening was carried out.Finally,the compound TC25 was obtained.Its activity was equivalent to TS13 and the PAINS structure in TS13 was abandoned.Reduced the difficulty of chemical synthesis.