Computer-Aided Design And Activity Detection Of The BH3-Mimetic Peptides

Beclin 1 is an important protein in mammals that can regulate autophagy.In mice and humans,when the BH3 domain of Beclin 1 interacts with proteins such as Bcl-2and Bcl-XL in the Bcl-2 family,the activation of Beclin 1 on autophagy will be inhibited.Researches showed that L116 and F123 of the BH3 domain in Beclin 1 are essential for the binding to Bcl-XL.When they mutate to alanine,Beclin 1 will lost its affinity to Bcl-XL.In our previous study,we obtained two new peptide sequences(MP1:LSRRMKVTGDLW,MP2: LSRRIKVTGDLF)with better binding ability to Bcl-XL than the wild-type(WT)BH3 peptide.In this study,computer aided design and optimization protocol was performed again on the basis of the optimal mutant MP1 to obtain more potential BH3-mimetic peptides and the activities of the virtual screened BH3-mimetic peptides were verified by cell proliferation experiment and Western Blot experiment.This study provides theoretical and material basis for the research of BH3-mimetic peptides anticancer drugs.This paper consists of four parts.The first part introduces the basic knowledge of cancer and autophagy,including related regulatory genes,occurrence process and detection methods of autophagy.The basic knowledge of Bcl-XL and Beclin 1 proteins involved in this study is introduced,including their family and structure information.Then,the CADD methods used in this paper are briefly introduced.Finally,the main research significance and research content of this topic are expounded.In the second part of the paper,we design the BH3-mimetic peptides based on a single mutation.MP1 was used as a template to mutate amino acids in BH3 peptide except L116 and F123.The 3D structures of the resulting peptides sequences were constructed by homologous modeling and site-specific mutation.Then,the binding abilities to Bcl-XL of the BH3-mimetic peptides were evaluated by molecular docking,MD simulation and binding free energy calculation and filter based on the evaluation results.Finally,three BH3-mimetic peptides that named S113 P MP1,T119 Mse MP1and S113 I MP1 with potential binding ability were obtained.In the third part of the paper,we designed the BH3-mimetic peptides using double mutations.In this part,using MP1 as a template,the amino acids at L112 and R115 were double mutated and the 3D structures of these peptide sequences were obtained with homologous modeling method.Then,the binding abilities to Bcl-XL of the BH3-mimetic peptides were evaluated by molecular docking,MD simulation and binding free energy calculation and filter based on the evaluation results.Finally,three BH3-mimetic peptides that named L112F＿R115K MP1,L112F＿R115L MP1 and L112F＿R115T MP1 with potential binding ability were obtained.In the fourth part,the activities of the screened BH3-mimetic peptides were verified.The results of cell proliferation experiment showed that T119 Mse MP1 and L112F＿R115K MP1 showed better inhibitory activity to K562 than MP1 and WT.Western Blot results showed the BH3-mimetic peptide named T119 Mse MP1 had better autophagy induction ability than MP1 and WT.