title: Screening of Small-Molecule Inhibitors of Protein-Protein Interaction with Capillary Electrophoresis Frontal Analysis.
abstract: A simple and effective method for identifying inhibitors of protein-protein interactions (PPIs) was developed by using capillary electrophoresis frontal analysis (CE-FA). Antiapoptotic B-cell-2 (Bcl-2) family member Bcl-XL protein, a 5-carboxyfluorescein labeled peptide truncated from the BH3 domain of Bid (F-Bid) as the ligand, and a known Bcl-XL-Bid interaction inhibitor ABT-263 were employed as an experimental model for the proof of concept. In CE-FA, the free ligand is separated from the protein and protein-ligand complex to permit the measurement of the equilibrium concentration of the ligand, hence the dissociation constant of the protein-ligand complex. In the presence of inhibitors, formation of the protein-ligand complex is hindered, thereby the inhibition can be easily identified by the raised plateau height of the ligand and the decayed plateau of the complex. Further, we proposed an equation used to convert the IC50 value into the inhibition constant Ki value, which is more useful than the former for comparison. In addition, the sample pooling strategy was employed to improve the screening throughput more than 10 times. A small chemical library composed of synthetic compounds and natural extracts were screened with the method, two natural products, namely, demethylzeylasteral and celastrol, were identified as new inhibitors to block the Bcl-XL-Bid interaction. Cell-based assay was performed to validate the activity of the identified compounds. The result demonstrated that CE-FA represents a straightforward and robust technique for screening of PPI inhibitors.
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Lingling Road 345, Shanghai 200032, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Lingling Road 345, Shanghai 200032, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Lingling Road 345, Shanghai 200032, China.
authors.forename: Mi
authors.initials: M
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Lingling Road 345, Shanghai 200032, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Lingling Road 345, Shanghai 200032, China.
authors.forename: Renxiao
authors.initials: R
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Lingling Road 345, Shanghai 200032, China.|ShanghaiTech University , Yueyang Road 319, Shanghai 200031, China.
authors.forename: Jingwu
authors.initials: J
authors.lastname: Kang
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D051023:BH3 Interacting Domain Death Agonist Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001667:Binding, Competitive; D019075:Electrophoresis, Capillary / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D019073:HT29 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D053978:Pentacyclic Triterpenes; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism; D014315:Triterpenes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D051020:bcl-X Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D051023:BH3 Interacting Domain Death Agonist Protein; D005452:Fluoresceins; D008024:Ligands; D053978:Pentacyclic Triterpenes; D054852:Small Molecule Libraries; D013449:Sulfonamides; D014315:Triterpenes; D051020:bcl-X Protein; C045132:4-carboxyfluorescein; C050414:celastrol; C528561:navitoclax