An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

Differential receptor selectivity of the FGF15/FGF19 orthologues determines distinct metabolic activities in db/db mice.

Fibroblast growth factors (FGF) 19, 21 and 23 are characterized by being endocrinely secreted and require co-receptor α-klotho or ß-klotho (BKL) for binding and activation of the FGF receptors (FGFR). FGF15 is the rodent orthologue of human FGF19, but the two proteins share only 52% amino acid identity. Despite the physiological role of FGF21 and FGF19 being quite different, both lower blood glucose (BG) when administered to diabetic mice. The present study was designed to clarify why two human proteins with distinct physiological functions both lower BG in db/db mice and if the mouse orthologue FGF15 has similar effect to FGF19 and FGF21. Recombinant human FGF19, -21 and a mouse FGF15 variant (C110S) were expressed and purified from Escherichia coli While rhFGF19 (recombinant human fibroblast growth factor 19) and rhFGF21 (recombinant human fibroblast growth factor) bound FGFRs in complex with both human and mouse BKL, rmFGF15CS (recombinant mouse fibroblast growth factor 15 C110S) only bound the FGFRs when combined with mouse BKL. Recombinant hFGF21 and rhFGF19, but not rmFGF15CS, increased glucose uptake in mouse adipocytes, while rhFGF19 and rmFGF15CS potently decreased Cyp7a1 expression in rat hepatocytes. The lack of effect of rmFGF15CS on glucose uptake in adipocytes was associated with rmFGF15CS's inability to signal through the FGFR1c/mouse BKL complex. In db/db mice, only rhFGF19 and rhFGF21 decreased BG while rmFGF15CS and rhFGF19, but not rhFGF21, increased total cholesterol. These data demonstrate receptor- and species-specific differential activity of FGF15 and FGF19 which should be taken into consideration when FGF19 is used as a substitute for FGF15.

Kir6.2-dependent high-affinity repaglinide binding to beta-cell K(ATP) channels.

1. The beta-cell K(ATP) channel is composed of two types of subunit - the inward rectifier K(+) channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N-terminus (Kir6.2DeltaN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2DeltaN14/SUR1 channels. All binding experiments are performed on membranes in ATP-free buffer at 37 degrees C. 2. Repaglinide was found to bind with low affinity (K(D)=59+/-16 nM) to SUR1 alone, but with high affinity (increased approximately 150-fold) when SUR1 was co-expressed with Kir6.2 (K(D)=0.42+/-0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. 3. Repaglinide bound with low affinity (K(D)=51+/-23 nM) to SUR1 co-expressed with Kir6.2DeltaN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild-type channels. 4. In whole-cell patch-clamp experiments inhibition of Kir6.2DeltaN14/SUR1 currents by both repaglinide and nateglinde is abolished. 5. The results suggest that Kir6.2 causes a conformational change in SUR1 required for high-affinity repaglinide binding, or that the high-affinity repaglinide-binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1.

Differential interactions of nateglinide and repaglinide on the human beta-cell sulphonylurea receptor 1.

Repaglinide and nateglinide represent a new class of insulin secretagogues, structurally unrelated to sulphonylureas, that were developed for the treatment of type 2 diabetes. The inhibitory effect of these drugs was investigated on recombinant wild-type and mutant Kir6.2/SUR1 channels expressed in HEK293 cells. Nateglinide and repaglinide dose-dependently inhibited whole-cell Kir6.2/SUR1 currents with half-maximal inhibitory concentration (IC(50)) values of 800 and 21 nmol/l, respectively. Mutation of serine 1237 in SUR1 to tyrosine (S1237Y) abolished tolbutamide and nateglinide block, suggesting that these drugs share a common point of interaction on the SUR1 subunit of the ATP-sensitive K(+) channel. In contrast, repaglinide inhibition was unaffected by the S1237Y mutation (IC(50) = 23 nmol/l). Radioligand binding studies revealed a single high-affinity binding site for [(3)H]repaglinide on membranes prepared from HEK293 cells expressing wild-type (equilibrium dissociation constant [K(D)] = 0.40 nmol/l) or mutant (K(D) = 0.31 nmol/l) Kir6.2/SUR1 channels. Nateglinide and tolbutamide displaced [(3)H]repaglinide binding to wild-type channels with IC(50) values of 0.7 and 26 micro mol/l, respectively, but produced <10% displacement of [(3)H]repaglinide bound to mutant channels. This is consistent with the idea that binding of nateglinide and tolbutamide, but not repaglinide, is abolished by the SUR1[S1237Y] mutation and that the binding site for repaglinide is not identical to that of nateglinde/tolbutamide. These results are discussed in terms of a conformational analysis of the drug molecules.