α/Sulfono-γ-AA peptide hybrids agonist of GLP-1R with prolonged action both in vitro and in vivo.

Peptides are increasingly important resources for biological and therapeutic development, however, their intrinsic susceptibility to proteolytic degradation represents a big hurdle. As a natural agonist for GLP-1R, glucagon-like peptide 1 (GLP-1) is of significant clinical interest for the treatment of type-2 diabetes mellitus, but its in vivo instability and short half-life have largely prevented its therapeutic application. Here, we describe the rational design of a series of α/sulfono-γ-AA peptide hybrid analogues of GLP-1 as the GLP-1R agonists. Certain GLP-1 hybrid analogues exhibited enhanced stability (t 1/2 > 14 days) compared to t 1/2 (<1 day) of GLP-1 in the blood plasma and in vivo. These newly developed peptide hybrids may be viable alternative of semaglutide for type-2 diabetes treatment. Additionally, our findings suggest that sulfono-γ-AA residues could be adopted to substitute canonical amino acids residues to improve the pharmacological activity of peptide-based drugs.

Recombinant Expression and Stapling of a Novel Long-Acting GLP-1R Peptide Agonist.

Owing to their pleiotropic metabolic benefits, glucagon-like peptide-1 receptor (GLP-1R) agonists have been successfully utilized for treating metabolic diseases, such as type 2 diabetes and obesity. As part of our efforts in developing long-acting peptide therapeutics, we have previously reported a peptide engineering strategy that combines peptide side chain stapling with covalent integration of a serum protein-binding motif in a single step. Herein, we have used this strategy to develop a second generation extendin-4 analog rigidified with a symmetrical staple, which exhibits an excellent in vivo efficacy in an animal model of diabetes and obesity. To simplify the scale-up manufacturing of the lead GLP-1R agonist, a semisynthesis protocol was successfully developed, which involves recombinant expression of the linear peptide followed by attachment of a polyethylene glycol (PEG)-fatty acid staple in a subsequent chemical reaction step.

The activity of sulfono-γ-AApeptide helical foldamers that mimic GLP-1.

Existing long α-helix mimicking necessitates the retention of most natural amino acid residues to maintain their biological activity. Here, we report the exploration of helical sulfono-γ-AApeptides with entire unnatural backbones for their ability to structurally and functionally mimic glucagon-like peptide 1 (GLP-1). Our findings suggest that efficient construction of novel GLP-1 receptor (GLP-1R) agonists could be achieved with nanomolar potencies. In addition, the resulting sulfono-γ-AApeptides were also proved to display remarkable stability against enzymatic degradation compared to GLP-1, augmenting their biological potential. This alternative strategy of α-helix mimicking, as a proof of concept, could provide a new paradigm to prepare GLP-1R agonists.

New Generation Oxyntomodulin Peptides with Improved Pharmacokinetic Profiles Exhibit Weight Reducing and Anti-Steatotic Properties in Mice.

Oxyntomodulin (OXM) is an intestinal peptide hormone that activates both glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptors. The natural peptide reduces body weight in obese subjects and exhibits direct acute glucoregulatory effects in patients with type II diabetes. However, the clinical utility of OXM is limited due to its lower in vitro potency and short in vivo half-life. To overcome these issues, we developed stapled, long-acting, and highly potent OXM analogs with balanced activities at both GLP-1 and GCG receptors. The lead molecule O14 exhibits potent and long-lasting effects on glucose control, body weight loss, and reduction of hepatic fat reduction in DIO mice. Importantly, O14 significantly reversed hepatic steatosis; reduced liver weight, total cholesterol, and hepatic triglycerides; and improved markers of liver function in a nonalcoholic steatohepatitis (NASH) mouse model. A symmetrical version of the peptide was also shown to be more efficacious and long-lasting in controlling glucose than semaglutide and the clinical candidate cotadutide in wild-type mice, highlighting the utility of our designs of the dual agonist as a potential new therapy for diabetes and liver diseases.

Engineering a Potent, Long-Acting, and Periphery-Restricted Oxytocin Receptor Agonist with Anorexigenic and Body Weight Reducing Effects.

The effects of oxytocin on food intake and body weight reduction have been demonstrated in both animal models and human clinical studies. Despite being efficacious, oxytocin is enzymatically unstable and thus considered to be unsuitable for long-term use in patients with obesity. Herein, a series of oxytocin derivatives were engineered through conjugation with fatty acid moieties that are known to exhibit high binding affinities to serum albumin. One analog (OT-12) in particular was shown to be a potent full agonist at the oxytocin receptor (OTR) in vitro with good selectivity and long half-life (24 h) in mice. Furthermore, OT-12 is peripherally restricted, with very limited brain exposure (1/190 of the plasma level). In a diet-induced obesity mouse model, daily subcutaneous administration of OT-12 exhibited more potent anorexigenic and body weight reducing effects than carbetocin. Thus, our results suggest that the long-acting, peripherally restricted OTR agonist may offer potential therapeutic benefits for obesity.

Engineering PEG-fatty acid stapled, long-acting peptide agonists for G protein-coupled receptors.

G protein-coupled receptors (GPCRs) play a key role in signal transduction and human pathophysiological processes. Family B GPCRs are activated by a number of secreted peptide hormones, and engineering of these peptide ligands in order to improve stability and half-life, and therefore clinical efficacy has proven successful for drug discovery. In this chapter we discuss a novel peptide engineering strategy that combines peptide side chain stapling with covalent incorporation of a serum protein binding motif in a single step. The application of this approach to the enhancement of the helicity and stability of GLP-1R peptide agonists, resulting in their improved in vitro potencies, in vivo half-lives and ultimately efficacies, will be described. Discussion of the stapling technology and target selection rationale, peptide engineering and final biological characterization of the long-acting agonists will also be provided.

Antimicrobial random peptide cocktails: a new approach to fight pathogenic bacteria.

Antibiotic resistance in bacteria has become a serious threat to public health, and therefore there is an urgent need to develop new classes of antimicrobial agents. Nowadays, natural antimicrobial peptides (AMPs) and their synthetic derivatives are considered as promising alternatives to traditional antibiotics. The broad molecular diversity of AMPs, in terms of sequences and structures, suggests that their activity does not depend on specific features of amino acid sequence or peptide conformation. We therefore selected two common properties of AMPs, (high percentage of hydrophobic and cationic amino acids), to develop a novel approach to synthesize random antimicrobial peptide mixtures (RPMs). Instead of incorporating a single amino acid at each coupling step, a mixture of hydrophobic and cationic amino acids in a defined proportion is coupled. This results in a mixture that contains up to 2n sequences, where n is the number of the coupling step, of random peptides with a defined composition, stereochemistry, and controlled chain length. We have discovered that RPMs of hydrophobic and cationic α-amino acids, such as phenylalanine and lysine, display strong and broad antimicrobial activity towards Gram-negative, Gram-positive, clinically isolated antibiotic resistant "superbugs", and several plant pathogenic bacteria. This review summarizes our efforts to explore the mode of action of RPMs and their potential as bioactive agents for multiple applications, including the prevention of biofilm formation and degradation of mature biofilm (related to human health), reduction of disease severity in plant bacterial disease models (related to crop protection), and inhibition of bacterial growth in milk (related to food preservation). All our findings illustrate the effectiveness of RPMs and their great potential for various applications.

Using Chemical Synthesis to Probe Structure-Activity Relationships of the Glycoactive Bacteriocin Glycocin F.

Glycocin F, a bacteriocin produced by Lactobacillus plantarum KW30, is glycosylated with two N-acetyl-d-glucosamine sugars, and has been shown to exhibit a rapid and reversible bacteriostasis on susceptible cells. The roles of certain structural features of glycocin F have not been studied to date. We report here the synthesis of various glycocin F analogues through solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), allowing us to probe the roles of different structural features of this peptide. Our results indicate that the bacteriostatic activity of glycocin F is controlled by the glycosylated interhelical loop, while the glycosylated flexible tail appears to be involved in localizing the peptide to its cellular target.

Total chemical synthesis of glycocin F and analogues: S-glycosylation confers improved antimicrobial activity.

Glycocin F (GccF) is a unique diglycosylated bacteriocin peptide that possesses potent and reversible bacteriostatic activity against a range of Gram-positive bacteria. GccF is a rare example of a 'glycoactive' bacteriocin, with both the O-linked N-acetylglucosamine (GlcNAc) and the unusual S-linked GlcNAc moiety important for antibacterial activity. In this report, glycocin F was successfully prepared using a native chemical ligation strategy and folded into its native structure. The chemically synthesised glycocin appeared to be slightly more active than the recombinant material produced from Lactobacillus plantarum. A second-generation synthetic strategy was used to prepare 2 site selective 'glyco-mutants' containing either two S-linked or two O-linked GlcNAc moieties; these mutants were used to probe the contribution of each type of glycosidic linkage to bacteriostatic activity. Replacing the S-linked GlcNAc at residue 43 with an O-linked GlcNAc decreased the antibacterial activity, while replacing O-linked GlcNAc at position 18 with an S-linked GlcNAc increased the bioactivity suggesting that the S-glycosidic linkage may offer a biologically-inspired route towards more active bacteriocins.

Structure activity relationship study on the peptide hormone preptin, a novel bone-anabolic agent for the treatment of osteoporosis.

Preptin is a 34-residue pancreatic hormone shown to be anabolic to bone in vitro and in vivo. The bone activity of preptin resides within the (1-16) N-terminal fragment. Due to its peptidic nature, the truncated fragment of preptin is enzymatically unstable; however it provides an attractive framework for the creation of stable analogues using various peptidomimetic techniques. An alanine scan of preptin (1-16) was undertaken which showed that substitution of Ser at position 3 or Pro at position 14 did not inhibit the proliferative activity of preptin in primary rat osteoblasts (bone-forming cells). Importantly, Ser-3 to Ala substitution also showed a significant activity on osteoblast differentiation in vitro and increased the formation of mineralised bone matrix. Additional modifications with non-proteinogenic amino acids at position 3 improved the stability in liver microsomes, but diminished the osteoblast proliferative activity. In addition, to provide greater structural diversity, a series of macrocyclic preptin (1-16) analogues was synthesised using head-to-tail and head-to-side chain macrolactamisation as well as ring-closing metathesis. However, a detrimental effect on osteoblast activity was observed upon macrocyclisation.

Short Anabolic Peptides for Bone Growth.

Loss of bone occurs in the age-related skeletal disorder, osteoporosis, leading to bone fragility and increased incidence of fractures, which are associated with enormous costs and substantial morbidity and mortality. Recent data indicate that osteoporotic fractures are more common than other diseases, which usually attract public attention (e.g., heart attack and breast cancer). The prevention and treatment of this skeletal disorder are therefore of paramount importance. Majority of osteoporosis medications restore skeletal balance by reducing osteoclastic activity, thereby reducing bone resorption. These agents, however, do not regenerate damaged bone tissue, leaving limited options for patients once bone loss has occurred. Recently, attention has turned to bone-anabolic agents. Such agents have the ability to increase bone mass and strength, potentially reversing structural damage. To date, only one bone-anabolic drug is available in the market. The discovery of more novel, cost-effective bone anabolic agents is therefore a priority to treat those suffering from this disabling condition. Short peptides offer an important alternative for the development of novel bone-anabolic agents given their high target binding specificity, which translates into potent activity with limited side effects. This review summarizes attempts in the identification of bone-anabolic peptides, and their development for promoting bone growth.

Synthesis and in vitro bone cell activity of analogues of the cyclohexapeptide dianthin G.

The cyclohexapeptide natural product dianthin G promotes osteoblast (bone-forming cell) proliferation in vitro at nanomolar concentrations, and is therefore considered a promising candidate for the treatment of osteoporosis. An N(α)-methyl amide bond scan of dianthin G was performed to probe the effect of modifying amide bonds on osteoblast proliferation. In addition, to provide greater structural diversity, a series of dicarba dianthin G analogues was synthesised using ring closing metathesis. Dianthin G and one novel dicarba analogue increased the number of human osteoblasts and importantly they did not increase osteoclast (bone-resorbing cell) differentiation in bone marrow cells.

Synthesis of Natural Cyclopentapeptides Isolated from Dianthus chinensis.

The first syntheses of the naturally occurring cyclic peptides dianthin I (1), pseudostellarin A (2), and heterophyllin J (3) are described. The linear protected peptide precursors were prepared efficiently via Fmoc-solid-phase synthesis and subsequently cyclized in solution under dilute conditions. The structures of the synthetic cyclopentapeptides were confirmed by NMR spectroscopy and mass spectrometry and were in agreement with the literature data reported for the natural products.