ABSTRACT
Tumor targeting anticancer drug conjugates that contain a tumor recognition motif (homing device) are of high current relevance. Cryptophycins, naturally occurring cytotoxic cyclo-depsipeptides, have been modified by total synthesis to provide analogues suitable for conjugation to peptide-based homing devices. An array of functionalized ß(2)-amino acids was synthesized and incorporated into cryptophycins. All analogues proved to be highly active in the cytotoxicity assay using the human cervix carcinoma cell line KB-3-1 and its multidrug-resistant subclone KB-V1. Conformational analysis of cryptophycin-52 and two synthetic analogues was performed by NMR and MD methods to obtain information on the influence of the unit C configuration on the overall conformation. An azide-functionalized cryptophycin was connected by CuAAC to an alkyne-containing fluorescently labeled cyclic RGD-peptide as the homing device for internalization studies. Confocal fluorescence microscopy proved integrin-mediated internalization by endocytosis and final lysosomal localization of the cryptophycin prodrug.
Subject(s)
Depsipeptides/metabolism , Endocytosis , Oligopeptides/metabolism , Cell Line, Tumor , Depsipeptides/chemical synthesis , Depsipeptides/chemistry , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Female , Humans , Inhibitory Concentration 50 , Microscopy, Confocal , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Protein ConformationABSTRACT
The human pathogen Helicobacter pylori that may cause different gastric diseases exploits integrins for infection of gastric cells. The H. pylori protein CagL present on the outer region of the type IV secretion pilus contains an RGD sequence (-Arg-Gly-Asp-) that enables binding to cells presenting integrins α5ß1 and αVß3. This interaction can be inhibited with conformationally designed cyclic RGD peptides derived from the CagL epitope -Ala-Leu-Arg-Gly-Asp-Leu-Ala-. The inhibition of the CagL-αVß3 interaction by different RGD peptides strongly suggests the importance of the RGD motif for CagL binding. CagL point mutants (RAD, RGA) show decreased affinity to integrin αVß3. Furthermore, structure-activity relationship studies with cyclic RGD peptides in a spatial screening approach show the distinct influence of the three-dimensional arrangement of RGD motif on the ability to interfere with this interaction. Resulting from these studies, similar structural requirements for the CagL epitope as previously suggested for other ligands of integrin αVß3 are proposed.
