| The majority of bioactive proteins ?exert their biological activity through relatively small regions of their folded surfaces, so their actions could in principle be reproduced by much smaller ?designer? molecules that retain these localized bioactive surfaces, but have improved pharmacodynamic and kinetic properties?. In most cases these bioactive sites are discontinuous in nature, meaning that more than one sequentially separated regions of the protein sequence are involved. We showed previously that the use of CLIPS-scaffolds can turn inactive linear peptides into bioactive FSH protein mimics via fixation of their secondary structure. However, for the complete mimicry of discontinuous protein binding sites including all the different parts of the binding site, more advanced scaffold technologies are needed. We propose the design and synthesis of a novel family of synthetic (hybrid) scaffolds, termed ?CLIPS/CLICK? scaffolds, for the functional reconstruction of discontinuous (antibody) binding sites on proteins. The scaffold molecules are designed to assemble various different peptide fragments (2-3, linear and/or cyclic) that constitute the discontinuous binding site. The combined CLIPS/CLICK-technology has a number of important advantages over existing scaffolds: 1. There is no need for difficult protection/deprotection strategies in addressing the various reactive sites. 2. Assembly of the scaffolded peptides proceeds under extremely mild conditions (aqueous solution, room temperature, pH~7.8). 3. CLIPS and CLICK-chemistries are extremely time- and product-efficient and convert linear peptides in 2 steps (1st step: <15 min.; 2nd step: 2-3h!!!) into double-looped peptides structures in high yield. This project should yield a generic set of synthetic scaffolds that was specifically designed for the reconstruction of the sequentially separated beta1-beta3-loop region observed in various members of the cys-knot growth factor family (FSH, hCG, LH, VEGF, etc.). However, the unique combi of CLIPS-technology and CLICK-chemistry has broad and general applicability and can be easily made available for the design and synthesis of related scaffolds that can aid to reconstruct differently shaped discontinuous and/or conformation protein binding sites (e.g. design of HIV type-I vaccin). |
