Multivalent interactions based on supramolecular self-assembly and peptide-labeled quantum dots for imaging GPCRs.

摘要

Multivalent interactions are very common in nature, such as influenza virus infecting epithelial cells, clearance of pathogens by antibody-mediated attachment to macrophages, etc. In an effort to mimic nature, we utilized a bottom-up approach to develop various multivalent self-assembling systems based on coiled-coil peptides. We synthesized several pairs of coiled-coil peptides, also called leucine-zippers, and tethered them to PAMAM dendrimers to form leucine-zipper dendrimers (LZDs). We conjugated natural leucine-zipper peptides Fos/Jun to the PAMAM dendrimer to make D0Fos4 and D0Jun4, and studied the interactions between these LZDs and their cognate peptide target, either Jun or Fos. Our experiments showed that the LZD D 0Fos4 can non-covalently assemble four copies of Jun, and that this approach can be further used as a new method for the rapid non-covalently assembling of multimeric ligands. We also pursued the multivalent target of GPCRs with a leucine-zipper assembly, and surprisingly found the Fos/Jun complex can potentially be used as a molecular switch to target GPCRs with controlled ligand activity. In a related project for bio-material design based on self-assembly of LZDs, we synthesized a different pair of LZDs, D0Ez4 and D0Kz4, and established that they can assemble at neutral pH to form helical fibrils which display higher order self-organized structures, providing a new methodology for bio-material design.; In another effort for studying multivalent interactions, we covalently conjugated three copies of the F23, mini-protein that binds the HIV-1 capsid protein, to a trimesic acid and obtained a trivalent inhibitor, Tri-F23. This trivalent inhibitor showed enhanced binding in ELISA against gp120, but was not significantly more effective preventing HIV entry. This methodology provides a new strategy for developing multivalent inhibitors for preventing HIV-1 infection at the entry level.; In a related area, we are developing imaging agents based on photoluminescent nanoparticles (quantum dots) that can detect GPCRs on whole cells and at the single molecule level. To this end, a new method was developed for biocompatible amphphilic polymers to coat quantum dots. This amphiphilic polymer facilitates rapid quantum dot conjugation to any ligand that has a free thiol or engineered cysteine. Several GPCR targeted peptides have been utilized for imaging receptors on whole cells and as single molecules. These efforts will guide the rational design of multivalent ligands for targeting GPCRs and other cell surface proteins.