Identification of GDF15 peptide fragments inhibiting GFRAL receptor signaling.

Growth differentiation factor 15 (GDF15) is believed to be a major causative factor for cancer-induced cachexia. Recent elucidation of the central circuits involved in GDF15 function and its signaling through the glial cell-derived neurotrophic factor family receptor α-like (GFRAL) has prompted the interest of targeting the GDF15-GFRAL signaling for energy homeostasis and body weight regulation. Here, we applied advanced peptide technologies to identify GDF15 peptide fragments inhibiting GFRAL signaling. SPOT peptide arrays revealed binding of GDF15 C-terminal peptide fragments to the extracellular domain of GFRAL. Parallel solid-phase peptide synthesis allowed for generation of complementary GDF15 peptide libraries and their subsequent functional evaluation in cells expressing the GFRAL/RET receptor complex. We identified a series of C-terminal fragments of GDF15 inhibiting GFRAL activity in the micromolar range. These novel GFRAL peptide inhibitors could serve as valuable tools for further development of peptide therapeutics towards the treatment of cachexia and other wasting disorders.

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.

Stabilisation of nucleic acid secondary structures by oligonucleotides with an additional nucleobase; synthesis and incorporation of 2'-deoxy-2'-C-(2-(thymine-1-yl)ethyl)uridine.

A nucleoside with two nucleobases is incorporated into oligonucleotides. The synthetic building block, 2'-deoxy-2'-C-(2-(thymine-1-yl)ethyl)uridine, 2, is prepared from uridine via 5',3'-TIPDS-protected 2'-deoxy-2'-C-allyluridine by an oxidative cleavage of the allyl group, a Mitsunobu reaction for the introduction of thymine and appropriate deprotection reactions. This compound is converted into a DMT-protected phosphoramidite and incorporated once into a 13-mer oligodeoxynucleotide sequence, once in an isosequential LNA-modified oligodeoxynucleotide and four times in the middle of a 12-mer oligodeoxynucleotide. These sequences are mixed with different complementary DNA and RNA sequences in order to study the effect of the additional nucleobase in duplexes, in bulged duplexes and in three-way junctions. The first additional thymine is found to be well-accommodated in a DNA-RNA duplex, whereas a DNA-DNA duplex was slightly destabilised. A three-way junction with the additional thymine in the branching point is found to be stabilised in both a DNA-DNA and a DNA-RNA context but destabilised where the modified LNA-sequence is used. In a Mg2+-containing buffer, however, the relative stability of the three-way junctions is found to be opposite with especially the LNA-modified DNA-DNA complex being significantly stabilised by the additional nucleobase.