Discovery And Mechanism Study Of Selective Cyclic Peptide Inhibitors Of Bcl-2 Family Proteins

Bcl-2 family proteins are crucial regulatory factors of mitochondrial apoptosis,which can be classified into anti-apoptotic proteins,pro-apoptotic proteins and BH3-only proteins according to their functions.Bcl-2 and Bcl-XL are important anti-apoptotic proteins that impact the development and drug resistance of various cancers.Two classes of inhibitors,linear peptides and small molecule compounds,have been developed based on the structural information of Bcl-2 and Bcl-XL.However,linear peptides are difficult to enter clinical trials because of their instability and low membrane penetration efficiency,while small molecule inhibitors face the challenge of Bcl-XL-dependent platelet toxicity,and only Venetoclax(ABT199)has been approved by the FDA at present.But Venetoclax has faced the challenge of resistance mutation of G101V site of Bcl-2 in patients.Therefore,it is urgent to screen new specific inhibitors targeting Bcl-2 and to overcome the above secondary drug resistance.In collaboration with Professor Chuanliu Wu’s team from Xiamen University,we used phage display technology to identify a cyclic peptide named Cpl that binds both Bcl-2 and BclXL but preferentially binds to Bcl-2.Based on the structural biological information,we elucidated the mechanism of selectivity and resistance of cyclic peptide to the two proteins and then optimized and screened out the Cp3 targeting Bcl-XL.Firstly,we studied the selectivity mechanism of cyclic peptides to the two proteins.We obtained the complex structure of Bcl-2 with Cpl and Bcl-XL with Cp1 at the resolution of 1.95 A and 2.0 A,respectively.Unlike the reported ligand binding mode,Cpl binds to the ligand-binding pocket of both proteins in a novel mode that causes little conformational changes in the α3 and α4 helices.In addition,the amino acid interaction between Cpl and Bcl-2,Cpl and Bcl-XL is conserved.The difference is that the D111 residue of Bcl-2 forms a hydrogen bond with the G6 residue of Cpl,while the corresponding residue of Bcl-XL is A104 residue that cannot form a hydrogen bond with Cp1.Meanwhile,the surface plasmon resonance experiment showed that the affinity of Bcl-2 with Cp1 decreased from 0.79 μM to 5.9 μM due to the D111A site mutation.These results indicate that the difference in D111/A104 residue between Bcl-2 and Bcl-XL is one of the key factors to the selectivity of Cp1 to Bcl-2.Secondly,we investigated whether cyclic peptides can overcome clinical drug resistance mutations.We obtained the complex structure of the Bcl-2 mutant containing the G101V resistant mutation with Cpl at a resolution of 1.85 A.It has been reported that the G101V mutation of Bcl-2 in patients causes the conformational change in E152 residue,resulting in a steric effect with Venetoclax and a 180-fold decrease in affinity of Bcl-2 and Venetoclax,and leading to drug resistance.However,our co-crystalization structure showed that the distance between Cp1 and V101,Cp1 and E152 residues of Bcl-2-G101 V was 7.7 (?) and 9.9 (?),respectively,which did not cause steric effect and was expected to overcome drug resistance.Consistent with this,the surface plasmon resonance experiment showed no significant difference between the affinity of Cp1 and Bcl-2 before and after the mutation,with KD values of 0.79 μM and 1.14 μM,respectively.Based on the above structural information,we optimized and screened the selective cyclic peptide 3 targeting Bcl-XL and obtained the complex structure of Bcl-XL with Cp3 at a resolution of 1.4 A.Corresponds to the first part,the P6 residue of Cp3 interacts face to face with the A104 residue of Bcl-XL,but has a steric effect with the D111 residue of Bcl-2.Therefore,we proposed the selectivity "switch" hypothesis,where the cyclic peptide can not only show preferential selectivity of Bcl-2 by forming a hydrogen bond with D111 residue through G6 residue but can also display selectivity of Bcl-XL by forming an aspect interaction with A 104 residue through P6 residue.Finally we verified the pro-apoptotic effect of TAT modified cyclic peptide on tumor cells.Flow cytometry showed that Cp l-TAT and Cp3-TAT both inhibited the growth of Jurcat.E14 and Daudi tumor cells,and the inhibitory effect of Cp3-TAT on Daudi cells was superior to that of marketed drug Venetoclax at the same concentration.In addition,Annexin V-FITC/PI double staining experiment showed that Cp1-TAT and Cp3-TAT significantly increased the percentage of apoptosis,indicating that Cpl-TAT and Cp3-TAT inhibit cell proliferation by promoting apoptosis.In conclusion,our work solved the complex structures of Bcl-2-Cp1,Bcl-XL-Cp1 and Bcl-XL-Cp3.revealed the binding mode and selectivity mechanism of cyclic peptide to the two proteins,and provided structural basis for the selectivity and affinity optimization of cyclic peptide;We solved the structure of the Bcl-2-G 101 V-Cp1 complex,demonstrated that cyclic peptides can overcome clinical resistance mutations;We demonstrated that cyclic peptides inhibit cell proliferation by promoting cell apoptosis.This study introduces an innovative way for cyclic peptides to selectively inhibit Bcl-2 or Bcl-XL by regulating protein-protein interactions.which will benefit the development of next generation Bcl-2 targeting drugs.