Exploring the signaling space of a GPCR using bivalent ligands with a rigid oligoproline backbone.

G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward ß-arrestin-1 and ß-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting ß-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling.

Distance-Dependent Cellular Uptake of Oligoproline-Based Homobivalent Ligands Targeting GPCRs-An Experimental and Computational Analysis.

Tumor targeting with bivalent radiolabeled ligands for GPCRs is an attractive means for cancer imaging and therapy. Here, we studied and compared the distance dependence of homobivalent ligands for the human gastrin-releasing peptide receptor (hGRP-R) and the somatostatin receptor subtype II (hSstR2a). Oligoprolines were utilized as molecular scaffolds to enable distances of 10, 20, or 30 Å between two identical, agonistic recognition motifs. In vitro internalization assays revealed that ligands with a distance of 20 Å between the recognition motifs exhibit the highest cellular uptake in both ligand series. Structural modeling and molecular dynamics simulations support an optimal distance of 20 Å for accommodating ligand binding to both binding sites of a GPCR dimer. Translation of these findings to the significantly higher complexity in vivo proved difficult and showed only for the hGRP-R increased tumor uptake of the bivalent ligand.

Oligoprolines as Molecular Entities for Controlling Distance in Biological and Material Sciences.

Nature utilizes large biomolecules to fulfill tasks that require spatially well-defined arrangements at the molecular level such as electron transfer, ligand-receptor interactions, or catalysis. The creation of synthetic molecules that enable precise control over spacing and functionalization provides opportunities across diverse disciplines. Key requirements of functionalizable oligomeric scaffolds include the specific control of their molecular properties where the correct balance of flexibility and rigidity must be maintained in addition to the prerequisite of defined length. These molecules must ideally be equally applicable in aqueous and organic environments, they must be easy to synthesize in a controlled stepwise fashion, and they must be easily modified with a palette of chemical appendages having diverse functionalities. Oligoproline, a peptidic polymer comprised of repeating units of the amino acid proline, is an ideal platform to meet such challenges. Oligoproline derives its characteristic rigidity and well-defined secondary structure from the innate features of proline. It is the only naturally occurring amino acid that has its side-chain cyclized to its α-amino group, generating often-populated trans and cis conformers around the tertiary amide bonds formed in proline oligomers. Oligoprolines are widely applied to define distance on the molecular level as they are capable of serving as both a "molecular ruler" with a defined length and as a "molecular scaffold" with precisely located and predictably oriented substitutions along the polymeric backbone. Our investigations focus on the use of oligoproline as a molecular scaffold. Toward this end, we have investigated the role of solvent upon helical structure of oligoproline, and the effect that substituents on the pyrrolidine ring and the oligomer termini have on the stability of the helix. We have also further explored the molecular characteristics of oligoproline through spectroscopic and crystallographic methods. All of these structural insights laid the basis for implementation of oligoproline in materials science and chemical biology. Within this Account, we highlight the value of oligoprolines for applications in distinctly different research areas. Toward materials chemistry, we have utilized oligoprolines for the size-controlled generation of noble metal nanoparticles, and to probe the role of spatial preorganization of π-systems for molecular self-assembly. Within the biological realm, we have applied oligoprolines to probe the role of distance on G-protein coupled receptor-mediated ligand uptake by cancerous cells and to investigate the effects of charge preorganization on the efficacy of cationic cell-penetrating peptides.

Hybrid bombesin analogues: combining an agonist and an antagonist in defined distances for optimized tumor targeting.

Radiolabeled hybrid ligands with defined distances between an agonist and an antagonist for the gastrin-releasing peptide receptor were found to have excellent tumor-targeting properties. Oligoprolines served as rigid scaffolds that allowed for tailoring distances of 10, 20, and 30 Å between the recognition elements. In vitro and in vivo studies revealed that the hybrid ligand with a distance of 20 Å between the recognition elements exhibits the highest yet observed tumor cell uptake and retention time in prostate cancer cells.