Advanced Insulin Synthesis by One-pot/Stepwise Disulfide Bond Formation Enabled by S-Protected Cysteine Sulfoxide.

An advanced insulin synthesis is presented that utilizes one-pot/stepwise disulfide bond formation enabled by acid-activated S-protected cysteine sulfoxides in the presence of chloride anion. S-chlorocysteine generated from cysteine sulfoxides reacts with an S-protected cysteine to afford S-sulfenylsulfonium cation, which then furnishes the disulfide or reversely returns to the starting materials depending on the S-protection employed and the reaction conditions. Use of S-acetamidomethyl cysteine (Cys(Acm)) and its sulfoxide (Cys(Acm)(O)) selectively give the disulfide under weak acid conditions in the presence of MgCl2 even if S-p-methoxybenzyl cysteine (Cys(MBzl)) and its sulfoxide (Cys(MBzl)(O)) are also present. In contrast, the S-MBzl pair yields the disulfide under more acidic conditions in the presence of a chloride anion source. These reaction conditions allowed a one-pot insulin synthesis. Additionally, lipidated insulin was prepared by a one-pot disulfide-bonding/lipidation sequence.

Novel formulation of antimicrobial peptides enhances antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA).

Antimicrobial peptides (AMPs) have the ability to penetrate as well as transport cargo across bacterial cell membranes, and they have been labeled as exceptional candidates to function in drug delivery. The aim of this study was to investigate the effectiveness of novel formulation of AMPs for enhanced MRSA activity. The strategy was carried out through the formulation of liposomes by thin-layer film hydration methodology, containing phosphatidylcholine, cholesterol, oleic acid, the novel AMP, as well as vancomycin (VCM). Characterization of the AMPs and liposomes included HPLC and LCMS for peptide purity and mass determination; DLS (size, polydispersity, zeta potential), TEM (surface morphology), dialysis (drug release), broth dilution, and flow cytometry (antibacterial activity); MTT assay, haemolysis and intracellular antibacterial studies. The size, PDI, and zeta potential of the drug-loaded AMP2-Lipo-1 were 102.6 ± 1.81 nm, 0.157 ± 0.01, and - 9.81 ± 1.69 mV, respectively, while for AMP3-Lipo-2 drug-loaded formulation, it was 146.4 ± 1.90 nm, 0.412 ± 0.05, and - 4.27 ± 1.25 mV respectively at pH 7.4. However, in acidic pH for both formulations, we observed an increase in size, PDI, and a switch to positive zeta potential, which indicated the pH responsiveness of our liposomal systems. The in vitro drug release studies demonstrated that liposomal formulations released VCM-HCl at a faster rate at pH 6.0 compared to pH 7.4. In vitro antibacterial activity against S. aureus and MRSA revealed that liposomes had enhanced activity at pH 6 compared to pH 7.4. The study revealed that the formulation can potentially be used to enhance activity and penetration of AMPs, thereby improving the treatment of bacterial infections.

Chemical modification of neuropeptide Y for human Y1 receptor targeting in health and disease.

As a very abundant neuropeptide in the brain and widely distributed peptide hormone in the periphery, neuropeptide Y (NPY) appears to be a multisignaling key peptide. Together with peptide YY, pancreatic polypeptide and the four human G protein-coupled receptor subtypes hY1R, hY2R, hY4R and hY5R it forms the NPY/hYR multiligand/multireceptor system, which is involved in essential physiological processes as well as in human diseases. In particular, NPY-induced hY1R signaling plays a central role in the regulation of food intake and stress response as well as in obesity, mood disorders and cancer. Thus, several hY1R-preferring NPY analogs have been developed as versatile tools to unravel the complex NPY/hY1R signaling in health and disease. Further, these peptides provide basic lead structures for the development of innovative drugs. Here, the current research is summarized focusing on the development of differently sized hY1R-preferring NPY analogs as well as their advances with respect to hY1R profiling, potential therapeutic applications and targeted cancer imaging and therapy. Finally, major limitations and innovative strategies for next generation hY1R-preferring NPY analogs are addressed.

Peptide Lipidation - A Synthetic Strategy to Afford Peptide Based Therapeutics.

Peptide and protein aberrant lipidation patterns are often involved in many diseases including cancer and neurological disorders. Peptide lipidation is also a promising strategy to improve pharmacokinetic and pharmacodynamic profiles of peptide-based drugs. Self-adjuvanting peptide-based vaccines commonly utilise the powerful TLR2 agonist PamnCys lipid to stimulate adjuvant activity. The chemical synthesis of lipidated peptides can be challenging hence efficient, flexible and straightforward synthetic routes to access homogeneous lipid-tagged peptides are in high demand. A new technique coined Cysteine Lipidation on a Peptide or Amino acid (CLipPA) uses a 'thiol-ene' reaction between a cysteine and a vinyl ester and offers great promise due to its simplicity, functional group compatibility and selectivity. Herein a brief review of various synthetic strategies to access lipidated peptides, focusing on synthetic methods to incorporate a PamnCys motif into peptides, is provided.

Lipidation of a bioactive cyclotide-based CXCR4 antagonist greatly improves its pharmacokinetic profile in vivo.

The CXCR4 chemokine is a key molecular regulator of many biological functions controlling leukocyte functions during inflammation and immunity, and during embryonic development. Overexpression of CXCR4 is also associated with many types of cancer where its activation promotes angiogenesis, tumor growth/survival, and metastasis. In addition, CXCR4 is involved in HIV replication, working as a co-receptor for viral entry, making CXCR4 a very attractive target for developing novel therapeutic agents. Here we report the pharmacokinetic profile in rats of a potent CXCR4 antagonist cyclotide, MCo-CVX-5c, previously developed in our group that displayed a remarkable in vivo resistance to biological degradation in serum. This bioactive cyclotide, however, was rapidly eliminated through renal clearance. Several lipidated versions of cyclotide MCo-CVX-5c showed a significant increase in the half-life when compared to the unlipidated form. The palmitoylated version of cyclotide MCo-CVX-5c displayed similar CXCR4 antagonistic activity as the unlipidated cyclotide, while the cyclotide modified with octadecanedioic (18-oxo-octadecanoic) acid exhibited a remarkable decrease in its ability to antagonize CXCR4. Similar results were also obtained when tested for its ability to inhibit growth in two cancer cell lines and HIV infection in cells. These results show that the half-life of cyclotides can be improved by lipidation although it can also affect their biological activity depending on the lipid employed.

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.

Synthesis of Antimicrobial Lipopeptides Using the "CLipPA" Thiol-Ene Reaction.

Cysteine Lipidation on a Peptide or Amino acid (CLipPA) technology provides a facile method for the lipidation of unprotected peptides containing a free thiol group by using a "click" radical-initiated thiol-ene reaction to effect addition to a vinyl ester. The methodology is highly versatile, leading to high conversion rates while maintaining excellent chemoselectivity and tolerance for a large variety of peptide substrates and functional groups. Herein we describe the simple general procedure for the synthesis of a focused library of bioactive S-lipidated antimicrobial peptides via late-stage derivatization using solution-phase CLipPA lipidation.