Abstract Conjugates of therapeutic oligonucleotides (ONs) including peptide conjugates, provide a potential solution to the major challenge of specific tissue delivery faced by this class of drugs. Conjugations are often positioned terminal at the ONs, although internal placement of other chemical modifications are known to be of critical importance. The introduction of internal conjugation handles in chemically modified ONs require highly specialized and expensive nucleoside phosphoramidites. Here, we present a method for synthesizing a library of peptide–siRNA conjugates by conjugation at internal phosphorous positions via sulfonylphosphoramidate modifications incorporated into the sense strand. The sulfonylphosphoramidate modification offers benefits as it can be directly incorporated into chemically modified ONs by simply changing the oxidation step during synthesis, and furthermore holds the potential to create multifunctionalized therapeutic ONs. We have developed a workflow using a novel pH-controlled amine-to-amine linker that yields peptide–siRNA conjugates linked via amide bonds, and we have synthesized conjugates between GLP1 peptides and a HPRT1 siRNA as a model system. The in vitro activity of the conjugates was tested by GLP1R activity and knockdown of the HPRT1 gene. We found that conjugation near the 3′-end is more favorable than certain central internal positions and different internal conjugation strategies were compared.

Glucagon-like peptide-1 (GLP-1) and leptin signal recent feeding and long-term energy stores, respectively, and play complementary roles in the modulation of energy balance. Previous work using single-cell techniques in mice revealed the existence of a population of leptin receptor (Lepr)-containing dorsomedial hypothalamus (DMH) neurons marked by the expression of GLP-1 receptor (Glp1r; LepRGlp1r neurons) that play important roles in the control of feeding and body weight by leptin. Here, we demonstrate the existence of a population of LepRGlp1r neurons in the DMHs of nonhuman primates (NHPs), suggesting the potential translational relevance of these neurons. Consequently, we developed a GLP-1R/LepR dual agonist and demonstrated the physiological activity of both components in vivo using leptin-deficient and Lepr-deficient murine models. We further found roles for LepRGlp1r neurons in mediating the dual agonist's efficacy on food intake and body weight loss. Ablating Lepr in Glp1r-expressing neurons (LeprGlp1rKO mice) abrogated the suppression of food intake by the dual agonist. Furthermore, reactivation of Glp1r expression in Lepr neurons on an otherwise Glp1r-null background (Glp1rLeprRe mice) was sufficient to permit the suppression of food intake and body weight by the dual agonist. Hence, LepRGlp1r neurons represent targets for a GLP-1R/LepR dual agonist that potently reduces food intake and body weight.

IntroductionInsulin icodec is a novel, long-acting insulin analog designed to cover basal insulin requirements with once-weekly subcutaneous administration. Here we describe the molecular engineering and the biological and pharmacological properties of insulin icodec.Research design and methodsA number of in vitro assays measuring receptor binding, intracellular signaling as well as cellular metabolic and mitogenic responses were used to characterize the biological properties of insulin icodec. To evaluate the pharmacological properties of insulin icodec in individuals with type 2 diabetes, a randomized, double-blind, double-dummy, active-controlled, multiple-dose, dose escalation trial was conducted.ResultsThe long half-life of insulin icodec was achieved by introducing modifications to the insulin molecule aiming to obtain a safe, albumin-bound circulating depot of insulin icodec, providing protracted insulin action and clearance. Addition of a C20 fatty diacid-containing side chain imparts strong, reversible albumin binding, while three amino acid substitutions (A14E, B16H and B25H) provide molecular stability and contribute to attenuating insulin receptor (IR) binding and clearance, further prolonging the half-life. In vitro cell-based studies showed that insulin icodec activates the same dose-dependent IR-mediated signaling and metabolic responses as native human insulin (HI). The affinity of insulin icodec for the insulin-like growth factor-1 receptor was proportionately lower than its binding to the IR, and the in vitro mitogenic effect of insulin icodec in various human cells was low relative to HI. The clinical pharmacology trial in people with type 2 diabetes showed that insulin icodec was well tolerated and has pharmacokinetic/pharmacodynamic properties that are suited for once-weekly dosing, with a mean half-life of 196 hours and close to even distribution of glucose-lowering effect over the entire dosing interval of 1 week.ConclusionsThe molecular modifications introduced into insulin icodec provide a novel basal insulin with biological and pharmacokinetic/pharmacodynamic properties suitable for once-weekly dosing.Trial registration numberNCT02964104.

Expression of recombinant proteins traditionally require a cellular system to transcribe and translate foreign DNA to a desired protein. The process requires special knowledge of the specific cellular metabolism in use and is often time consuming and labour intensive. A cell free expression system provides an opportunity to express recombinant proteins without consideration of the living cell. Instead, a cell free system relies on either a cellular lysate or recombinant proteins to carry out protein synthesis, increasing overall production speed and ease of handling. The one-pot cell free setup is commonly known as an in vitro transcription/translation reaction (IVTT). Here we focused on a PURE (Protein synthesis Using Recombinant Elements) IVTT system based on recombinant proteins from Escherichia coli. We evaluated the cell free system's ability to express functional insulin analogues compared to Saccharomyces cerevisiae, a well-established system for large scale production of recombinant human insulin and insulin analogues. Significantly, it was found that correct insulin expression and folding was governed by the inherent properties of the primary amino acids sequence of insulin, whereas the eukaryotic features of the expression system apparently play a minor role. The IVTT system successfully produced insulin analogues identical in structure and with similar insulin receptor affinity to those produced by yeast. In conclusion we demonstrate that the PURE IVTT system is highly suited for expressing soluble molecules with higher order features and multiple disulphide bridges.

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A series of 2-thiohydantoins were prepared as somatostatin subtype 4 (sst(4)) ligands. Reaction of a N-substituted-L-tryptophan methyl ester with an isothiocyanate in the presence of triethylamine readily afforded the target compounds. The 2-thiohydantoins were evaluated for binding affinities in cell lines expressing somatostatin receptor subtypes 2A (sst(2A)) and 4 (sst(4)). Compared to the thiourea NNC-26-9100 (3), all 2-thiohydantoins demonstrated lower binding affinities at sst(4). Incorporation of the thiourea moiety into the more rigid 2-thiohydantoin nucleus leads to a loss of conformational freedom and may prevent optimal interaction with sst(4).

AIMS/HYPOTHESIS:There is controversy with respect to molecular characteristics of insulin analogues. We report a series of experiments forming a comprehensive characterisation of the long acting insulin analogues, glargine and detemir, in comparison with human insulin, IGF-1, and the super-mitogenic insulin, X10.METHODS:We measured binding of ligands to membrane-bound and solubilised receptors, receptor activation and mitogenicity in a number of cell types.RESULTS:Detemir and glargine each displayed a balanced affinity for insulin receptor (IR) isoforms A and B. This was also true for X10, whereas IGF-1 had a higher affinity for IR-A than IR-B. X10 and glargine both exhibited a higher relative IGF-1R than IR binding affinity, whereas detemir displayed an IGF-1R:IR binding ratio of ≤ 1. Ligands with high relative IGF-1R affinity also had high affinity for IR/IGF-1R hybrid receptors. In general, the relative binding affinities of the analogues were reflected in their ability to phosphorylate the IR and IGF-1R. Detailed analysis revealed that X10, in contrast to the other ligands, seemed to evoke a preferential phosphorylation of juxtamembrane and kinase domain phosphorylation sites of the IR. Sustained phosphorylation was only observed from the IR after stimulation with X10, and after stimulation with IGF-1 from the IGF-1R. Both X10 and glargine showed an increased mitogenic potency compared to human insulin in cells expressing many IGF-1Rs, whereas only X10 showed increased mitogenicity in cells expressing many IRs.CONCLUSIONS:Detailed analysis of receptor binding, activation and in vitro mitogenicity indicated no molecular safety concern with detemir.

GPR39 is an orphan member of the ghrelin receptor family that recently was suggested to be the receptor for obestatin, a peptide derived from the ghrelin precursor. Here, we compare the effect of obestatin to the effect of Zn2+ on signal transduction and study the effect of obestatin on food intake. Although Zn2+ stimulated inositol phosphate turnover, cAMP production, arrestin mobilization, as well as cAMP response element-dependent and serum response element-dependent transcriptional activity in GPR39-expressing cells as opposed to mock-transfected cells, no reproducible effect was obtained with obestatin in the GPR39-expressing cells. Moreover, no specific binding of obestatin could be detected in two different types of GPR39-expressing cells using three different radioiodinated forms of obestatin. By quantitative PCR analysis, GPR39 expression was readily detected in peripheral organs such as duodenum and kidney but not in the pituitary and hypothalamus, i.e. presumed central target organs for obestatin. Obestatin had no significant and reproducible effect on acute food intake in either freely fed or fasted lean mice. It is concluded that GPR39 is probably not the obestatin receptor. In contrast, the potency and efficacy of Zn2+ in respect of activating signaling indicates that this metal ion could be a physiologically relevant agonist or modulator of GPR39.

The turn-inducing sequence Ala-Aib introduced into positions 31 and 32 of neuropeptide Y (NPY) and its analogues has been identified as the key structure for Y(5)-receptor selectivity. Analogues of NPY and PP/NPY chimera containing the motif Ala-Aib were prepared; these peptides turned out to be selective for the Y(5)-receptor. The affinity of the NPY-based peptides was in the range of 6-150 nM, while the affinity of three (Ala-Aib)-containing PP/NPY chimera was in the range of 0.2-0.9 nM. The circular dichroism spectra of the Aib analogues in aqueous solution were all characteristic of an alpha helix; however, they had different intensities of the two negative bands at 220 and 208 nm. Affinity and selectivity for the Y(5)-receptor were correlated with the ratio of the ellipticity at 220 nm versus the one at 208 nm (R), which indicates the presence of a pronounced helix (R > 1) versus a less stabile one (R < 1). When R was in the range 0.74-0.96, the affinity at the Y(5)-receptor was in the range >5 nM, while there was complete loss of affinity at the Y(4)-receptor. R > 1.15 was associated with very high affinity at the Y(5)-receptor and weak affinity at the Y(4)-receptor. These results suggest that the selectivity of the Ala(31)-Aib(32) motif for the Y(5)-receptor derives from a specific conformation that must be correlated with the bioactive conformation of NPY at this subtype.