Half-Life Extending Modifications of Peptide YY3-36 Direct Receptor-Mediated Internalization.

Peptide YY3-36 (PYY3-36) is an endogenous ligand of the neuropeptide Y2 receptor (Y2R), on which it acts to reduce food intake. Chemically modified PYY3-36 analogues with extended half-lives are potential therapeutics for the treatment of obesity. Here we show that the common half-life extending strategies PEGylation and lipidation not only control PYY3-36's pharmacokinetics but also affect central aspects of its pharmacodynamics. PEGylation of PYY3-36 inhibited endocytosis by increasing receptor dissociation rates (koff), which reduced arrestin-3 (Arr3) activity. This is the first link between Arr3 recruitment and Y2R residence time. C16-lipidation of PYY3-36 had a negligible impact on Y2R signaling, binding, and endocytosis. In contrast, C18acid-lipidation minimized endocytosis, which indicated a decreased internalization through non-arrestin-related mechanisms. We propose a temporal model that connects the properties and position of the half-life extender with receptor Gi versus Arr3 signaling bias. We believe that this will be important for future design of peptide therapeutics.

Optimisation of in silico derived 2-aminobenzimidazole hits as unprecedented selective kappa opioid receptor agonists.

Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treatment of various pains. Pharmacophore based mining of databases led to the identification of 2-aminobenzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR and MOR. A short SAR exploration with the objective of identifying more polar and hence less brain penetrant agonists is described herewith. Modeling studies of the recently published structures of KOR, DOR and MOR are used to explain the receptor selectivity. The synthesis, biological evaluation and SAR of novel benzimidazole derivatives as KOR agonists are described. The in vivo proof of principle for anti-nociceptive effect with a lead compound from this series is exemplified.

The identification of GPR3 inverse agonist AF64394; the first small molecule inhibitor of GPR3 receptor function.

The identification of the novel and selective GPR3 inverse agonist AF64394, the first small molecule inhibitor of GPR3 receptor function, is described. Structure activity relationships and syntheses based around AF64394 are reported.

Machine-Learning-Guided Peptide Drug Discovery: Development of GLP-1 Receptor Agonists with Improved Drug Properties.

Peptide-based drug discovery has surged with the development of peptide hormone-derived analogs for the treatment of diabetes and obesity. Machine learning (ML)-enabled quantitative structure-activity relationship (QSAR) approaches have shown great promise in small molecule drug discovery but have been less successful in peptide drug discovery due to limited data availability. We have developed a peptide drug discovery platform called streaMLine, enabling rigorous design, synthesis, screening, and ML-driven analysis of large peptide libraries. Using streaMLine, this study systematically explored secretin as a peptide backbone to generate potent, selective, and long-acting GLP-1R agonists with improved physicochemical properties. We synthesized and screened a total of 2688 peptides and applied ML-guided QSAR to identify multiple options for designing stable and potent GLP-1R agonists. One candidate, GUB021794, was profiled in vivo (S.C., 10 nmol/kg QD) and showed potent body weight loss in diet-induced obese mice and a half-life compatible with once-weekly dosing.

Uncovering CNS access of lipidated exendin-4 analogues by quantitative whole-brain 3D light sheet imaging.

Peptide-based drug development for CNS disorders is challenged by poor blood-brain barrier (BBB) penetrability of peptides. While acylation protractions (lipidation) have been successfully applied to increase circulating half-life of therapeutic peptides, little is known about the CNS accessibility of lipidated peptide drugs. Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method to visualize whole-brain 3D distribution of fluorescently labelled therapeutic peptides at single-cell resolution. Here, we applied LSFM to map CNS distribution of the clinically relevant GLP-1 receptor agonist (GLP-1RA) exendin-4 (Ex4) and lipidated analogues following peripheral administration. Mice received an intravenous dose (100 nmol/kg) of IR800 fluorophore-labelled Ex4 (Ex4), Ex4 acylated with a C16-monoacid (Ex4_C16MA) or C18-diacid (Ex4_C18DA). Other mice were administered C16MA-acylated exendin 9-39 (Ex9-39_C16MA), a selective GLP-1R antagonist, serving as negative control for GLP-1R mediated agonist internalization. Two hours post-dosing, brain distribution of Ex4 and analogues was predominantly restricted to the circumventricular organs, notably area postrema and nucleus of the solitary tract. However, Ex4_C16MA and Ex9-39_C16MA also distributed to the paraventricular hypothalamic nucleus and medial habenula. Notably, Ex4_C18DA was detected in deeper-lying brain structures such as dorsomedial/ventromedial hypothalamic nuclei and the dentate gyrus. Similar CNS distribution maps of Ex4_C16MA and Ex9-39_C16MA suggest that brain access of lipidated Ex4 analogues is independent on GLP-1 receptor internalization. The cerebrovasculature was devoid of specific labelling, hence not supporting a direct role of GLP-1 RAs in BBB function. In conclusion, peptide lipidation increases CNS accessibility of Ex4. Our fully automated LSFM pipeline is suitable for mapping whole-brain distribution of fluorescently labelled drugs.

Design and optimization of quinazoline derivatives as melanin concentrating hormone receptor 1 (MCHR1) antagonists.

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays a role in metabolic and CNS disorders. The modeling-supported design, synthesis and multi-parameter optimization (biological activity, solubility, metabolic stability, hERG) of novel quinazoline derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified. Clusters of refined hMCHR1 homology models derived from the X-ray structure of the ß2-adrenergic receptor, including extracellular loops, were developed and used to guide the design.

Design and optimization of quinazoline derivatives as melanin concentrating hormone receptor 1 (MCHR1) antagonists: part 2.

Melanin concentrating hormone receptor 1 (MCHR1) antagonists have potential for the treatment of obesity and several CNS disorders. In the preceding article, we have described a novel series of quinazolines as MCHR1 antagonists and demonstrated in vivo proof of principle with an early lead. Herein we describe the detailed SAR and SPR studies to identify an optimized lead candidate having good efficacy in a sub-chronic DIO model with a good cardiovascular safety window.

Lipidated PrRP31 metabolites are long acting dual GPR10 and NPFF2 receptor agonists with potent body weight lowering effect.

Prolactin-releasing peptide (PrRP) is an endogenous neuropeptide involved in appetite regulation and energy homeostasis. PrRP binds with high affinity to G-protein coupled receptor 10 (GPR10) and with lesser activity towards the neuropeptide FF receptor type 2 (NPFF2R). The present study aimed to develop long-acting PrRP31 analogues with potent anti-obesity efficacy. A comprehensive series of C18 lipidated PrRP31 analogues was characterized in vitro and analogues with various GPR10 and NPFF2R activity profiles were profiled for bioavailability and metabolic effects following subcutaneous administration in diet-induced obese (DIO) mice. PrRP31 analogues acylated with a C18 lipid chain carrying a terminal acid (C18 diacid) were potent GPR10-selective agonists and weight-neutral in DIO mice. In contrast, acylation with aliphatic C18 lipid chain (C18) resulted in dual GPR10-NPFF2R co-agonists that suppressed food intake and promoted a robust weight loss in DIO mice, which was sustained for at least one week after last dosing. Rapid in vivo degradation of C18 PrRP31 analogues gave rise to circulating lipidated PrRP metabolites maintaining dual GPR10-NPFF2R agonist profile and long-acting anti-obesity efficacy in DIO mice. Combined GPR10 and NPFF2R activation may therefore be a critical mechanism for obtaining robust anti-obesity efficacy of PrRP31 analogues.

Discovery of novel, orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists.

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.

Beta-arrestin-based Bret2 screening assay for the "non"-beta-arrestin binding CB1 receptor.

CB1 receptor (CB1R) antagonists have been demonstrated to be effective in treating obesity and related disorders. This study has been focused on establishing a beta-arrestin 2-based screening assay for the CB1R using BRET2 technology. When the existing BRET2 screening platform was applied to the CB1R, the authors discovered that the receptor interacted weakly with beta-arrestin 2, resulting in unsatisfactory assay performance. To enhance the beta-arrestin binding capacity, they replaced the C-terminal tail of the CB1R with tails from either the V2 or BRS3 receptors, both of which interact strongly with beta-arrestin 2. Using this chimeric approach, the authors screened a small compound library and identified 21 antagonist and inverse agonist hits with IC50 and EC50 values ranging from 0.3 nM to 7.5 microM. Both primary and secondary screening were performed with Z'>0.5, suggesting that the assay is a robust and cost-effective alternative to existing cell-based assays.