Design and evaluation of fluorescent and non-fluorescent bivalent ligands to explore their mode of action at neuropeptide Y receptors

Recent advances in understanding G protein coupled receptor (GPCR) pharmacology include accumulating evidence that GPCRs can form homo- and hetero- meric complexes, and that these complexes may affect physiological function and ligand pharmacology. However, the continuing controversy over the significance of such complexes has arisen, in part, due to a limited availability of appropriate molecular tools and ligands that would allow for a more comprehensive investigation of oligomer pharmacology. One avenue to exploit dimer selective pharmacology has been the development of bivalent ligands. However, a number of mechanisms, not all of which involve receptor dimerization, can often explain the properties of bivalent compounds. The four cloned members of the neuropeptide Y receptor (YR) family mediate the responses to neuropeptide Y (NPY) and related peptides, pancreatic polypeptide (PP) and peptide YY (PYY), through shared Gi/o protein coupling pathways, and facilitate the physiological roles these peptides play in appetite regulation. The bivalent anti-parallel peptide dimer GR231118 (Ile-Glu-Pro-Dap-Tyr-Arg-Leu-Arg-Tyr-NH2; where Glu and Dap are bound to Dap and Glu, respectively, in the corresponding peptide via lactam bridges) is a Y1R antagonist, distinguished by a much higher affinity for the Y1R than equivalent monomer peptides based on the NPY C-terminus, such as BVD15 (Ile-Asn-Pro-Ile-Tyr-Arg-Leu-Arg-Tyr-NH2). This thesis explores the mode of action of GR231118-based analogues at the Y1R and the Y4R, by using solid phase peptide synthesis (SPPS) to produce novel peptides, high content imaging assays to monitor ligand binding and function, and fluorescence correlation spectroscopy (FCS) to probe ligand-receptor complex stoichiometry. Chapter 3 uses SPPS to create fluorescently tagged YR peptide ligands, as well as the development of novel cyclic and alanine scan GR231118 derivatives, to help elucidate structure-activity relationships (SAR) at the Y1R and Y4R. Chapter 4 then examines the pharmacology of fluorescent and non-fluorescent BVD15 and GR231118 based ligands in high content imaging assays. Ligand function was investigated using bimolecular fluorescence complementation (BiFC) YR β-arrestin2 recruitment assays and ligand affinities were measured using saturation or competition binding assays based on high content imaging. The resultant SARs at the Y1R indicated that the diaminopropionic acid cyclic moiety of GR231118 is the optimal size for high affinity and Y1R selectivity over the Y4R. An alanine scan of GR231118, focused on a single arm of the peptide dimer, demonstrated that the second [Tyr5] residue in the peptide contributed significantly (10 fold) to the high Y1R affinity of GR231118. This decrease in affinity, from [Tyr5] to [Ala5] substitution, was not observed at the Y4R, revealing a Y1R selective interaction. In Chapter 5, FCS in combination with photon counting histogram (PCH) analysis investigated fluorescent ligand binding to the Y1R and its impact on receptor oligomerisation. FCS and PCH are subcellular resolution imaging and analysis techniques that allow the concentration, diffusion co-efficient and molecular brightness of fluorescently tagged species to be measured within the defined confocal volume (~0.3 fL). Using these methods, the interactions of Cy5-labelled GR231118 dimer and BVD15 monomeric derivatives with the Y1R were successfully quantified via the determination of slowed ligand diffusion upon binding to the larger receptor protein complexes in living cells. Molecular brightness PCH analysis provided evidence for Y1R oligomerisation following treatment with both labelled and unlabelled GR231118 dimer, but not labelled BVD15 monomeric analogues. In conclusion, the high Y1R binding affinity of the bivalent ligand GR231118, in part, derives from an extended contact interface with the Y1R binding site, involving residues in the second arm of the peptide dimer. However, these high affinity dimeric peptides are also capable of promoting Y1R oligomerisation in living cells. Novel fluorescent GR231118 and BVD15 derivatives will provide useful tools for assessing Y1R pharmacology in whole cells using imaging based methodologies