Alessia Belgi

Minimization of Human Relaxin-3 Leading to High-Affinity Analogues with Increased Selectivity for Relaxin-Family Peptide 3 Receptor (RXFP3) over RXFP1

Relaxin-3 is a neuropeptide that is implicated in the regulation of stress responses and memory. The elucidation of its precise physiological role(s) has, however, been hampered by cross-activation of the relaxin-2 receptor, RXFP1, in the brain. The current study undertook to develop analogues of human relaxin-3 (H3 relaxin) that can selectively bind and activate its receptor, RXFP3. We developed a high-affinity selective agonist (analogue 2) by removal of the intra-A chain disulfide bond and deletion of 10 residues from the N terminus of the A chain. Further truncation of this analogue from the C terminus of the B chain to Cys(B22) and addition of an Arg(B23) led to a high-affinity, RXFP3-selective, competitive antagonist (analogue 3). Central administration of analogue 2 in rats increased food intake, which was blocked by prior coadministration of analogue 3. These novel RXFP3-selective peptides represent valuable pharmacological tools to study the physiological roles of H3 relaxin/RXFP3 systems in the brain and important leads for the development of novel compounds for the treatment of affective and cognitive disorders.

Dicarba α-conotoxin Vc1.1 analogues with differential selectivity for nicotinic acetylcholine and GABAB receptors.

Conotoxins have emerged as useful leads for the development of novel therapeutic analgesics. These peptides, isolated from marine molluscs of the genus Conus, have evolved exquisite selectivity for receptors and ion channels of excitable tissue. One such peptide, α-conotoxin Vc1.1, is a 16-mer possessing an interlocked disulfide framework. Despite its emergence as a potent analgesic lead, the molecular target and mechanism of action of Vc1.1 have not been elucidated to date. In this paper we describe the regioselective synthesis of dicarba analogues of Vc1.1 using olefin metathesis. The ability of these peptides to inhibit acetylcholine-evoked current at rat α9α10 and α3β4 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes has been assessed in addition to their ability to inhibit high voltage-activated (HVA) calcium channel current in isolated rat DRG neurons. Their solution structures were determined by NMR spectroscopy. Significantly, we have found that regioselective replacement of the native cystine framework with a dicarba bridge can be used to selectively tune the cyclic peptides innate biological activity for one receptor over another. The 2,8-dicarba Vc1.1 isomer retains activity at γ-aminobutyric acid (GABAB) G protein-coupled receptors, whereas the isomeric 3,16-dicarba Vc1.1 peptide retains activity at the α9α10 nAChR subtype. These singularly acting analogues will enable the elucidation of the biological target responsible for the peptides potent analgesic activity.

Structure and function relationship of murine insulin-like peptide 5 (INSL5): free C-terminus is essential for RXFP4 receptor binding and activation.

Insulin-like peptide 5 (INSL5) is a member of insulin/relaxin superfamily of peptides. It has recently been identified as the cognate ligand for the G-protein-coupled receptor, RXFP4. Although the complete physiological role of this naturally occurring peptide is still under investigation, there is evidence that it acts to both stimulate appetite and activate colon motility. This suggests that both agonists and antagonists of the peptide may have potential therapeutic applications. To further investigate the physiological role of this peptide and because of the ready availability of the mouse as an experimental animal, the preparation of mouse INSL5 was undertaken. Because of its complex structure and the intractable nature of the two constituent chains, different solid phase synthesis strategies were investigated, including the use of a temporary B-chain solubilizing tag. Unfortunately, none provided significantly improved yield of purified mouse INSL5 which reflects the complexity of this peptide. In addition to the native peptide, two mouse INSL5 analogues were also prepared. One had its two chains as C-terminal amides, and the other contained a europium chelate monolabel for use in RXFP4 receptor assays. It was found that the INSL5 amide was substantially less potent than the native acid form. A similar observation was made for the human peptide acid and amide, highlighting the necessity for free C-terminal carboxylates for function. Two additional human INSL5 analogues were prepared to further investigate the necessity of a free C-terminal. The results together provide a first insight into the mechanism whereby INSL5 binds to and activates RXFP4.

Dicarba Analogues of α-Conotoxin RgIA. Structure, Stability, and Activity at Potential Pain Targets

α-Conotoxin RgIA is both an antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype and an inhibitor of high-voltage-activated N-type calcium channel currents. RgIA has therapeutic potential for the treatment of pain, but reduction of the disulfide bond framework under physiological conditions represents a potential liability for clinical applications. We synthesized four RgIA analogues that replaced native disulfide pairs with nonreducible dicarba bridges. Solution structures were determined by NMR, activity assessed against biological targets, and stability evaluated in human serum. [3,12]-Dicarba analogues retained inhibition of ACh-evoked currents at α9α10 nAChRs but not N-type calcium channel currents, whereas [2,8]-dicarba analogues displayed the opposite pattern of selectivity. The [2,8]-dicarba RgIA analogues were effective in HEK293 cells stably expressing human Cav2.2 channels and transfected with human GABAB receptors. The analogues also exhibited improved serum stability over the native peptide. These selectively acting dicarba analogues may represent mechanistic probes to explore analgesia-related biological receptors.

Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide.

The Chemical Synthesis of Insulin: From the Past to the Present

Insulin is a circulating peptide hormone that is best known as a critical regulator of glucose levels. It consists of two peptide chains (A and B) that are held together by two disulfide bonds and a third within the A-chain. The first mile- stone step towards its chemical synthesis was made by Sanger and colleagues who were the first to determine the primary sequence of sheep insulin as well as the precise pairings of the disulfide bonds. However, it was more than ten years be- fore the first total chemical synthesis of bovine insulin was achieved which was via conventional solution phase synthesis of the two individual chains followed by their random combination and folding in solution. Later in that same year, Merri- field applied his newly-developed solid phase peptide synthesis (SPPS) methodology to synthesize bovine insulin within a few days. However the combination of the A- and B-chains invariably led to a low yield of correctly folded peptide. A stepwise disulfide bond formation approach was then introduced by Sieber and co-workers in their complex fragment syn- thesis of human insulin. In an attempt to overcome the limitations of these approaches, alternative protocols have been subsequently developed, one of which was optimized in our laboratory in which regioselective stepwise formation of the three disulfide bonds is achieved via the use of cysteine S-protecting groups cleaved in different chemical conditions. This review describes the historical course of the advancement of the chemical synthesis of insulin with a particular emphasis on the difficulty and limitations of its assembly.

Minimum Active Structure of Insulin-like Peptide 5

Insulin-like peptide 5 (INSL5) is a complex two-chain peptide hormone constrained by three disulfide bonds in a pattern identical to insulin. High expression of INSL5 in the colon suggests roles in activation of colon motility and appetite control. A more recent study indicates it may have significant roles in the regulation of insulin secretion and β-cell homeostasis. This peptide thus has considerable potential for the treatment of eating disorders, obesity, and/or diabetes. However, the synthesis of INSL5 is extremely challenging either by chemical or recombinant means. The A-chain is very poorly soluble and the B-chain is highly aggregating in nature which, together, makes their postsynthesis handling and purification very difficult. Given these difficulties, we have developed a highly active INSL5 analogue that has a much simpler structure with two disulfide bonds and is thus easier to assemble compared to native INSL5. This minimized peptide represents an attractive new mimetic for investigating the functional role of INSL5.

Structure and activity of contryphan-Vc2: Importance of the d-amino acid residue

Abstract In natural proteins and peptides, amino acids exist almost invariably as l‐isomers. There are, however, several examples of naturally‐occurring peptides containing d‐amino acids. In this study we investigated the role of a naturally‐occurring d‐amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d‐amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d‐chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane‐mediated mechanism of action for this peptide. Graphical abstract Figure. No caption available. HighlightsContryphan‐Vc2 contains a natural d‐amino acid residue.Both d‐ and l‐analogues exhibit the same bioactivity.Both analogues interact with lipid membranes.Possible membrane‐mediated mechanism of action.

Solid Phase Synthesis of an Analogue of Insulin, A0:R glargine, That Exhibits Decreased Mitogenic Activity

Numerous analogues of insulin have been prepared over the past three decades for use in diabetic therapy. However, only two long-acting insulins have been approved for clinical use. One is Levemir (Novo Nordisk) and the other is Lantus (Sanofi-Aventis). Glargine (commercial name: Lantus) is characterized by a substitution of Gly in place of Asn at the C terminus of the A-chain and addition of two Arg residues to the C terminus of the B-chain. Despite the clinical advantages of glargine, it is not without concern that its increased affinity for the IGF-1 receptor may correlate with increased mitogenic activity. Recently, a systematic study of modified analogues of glargine showed that placement of an extra Arg residue at the N terminus of the A-chain conferred improved insulin:IGF-1 receptor selectivity without significant loss of pharmacological profile. However, as it is difficult to prepare such an analogue in high yield by recombinant DNA methods, we undertook its chemical assembly by our refined solid phase synthesis method. We describe herein its chemical preparation and biological activity in both insulin receptor binding assays and DNA synthesis assays. The synthetic analogue, A0:R glargine, showed slightly reduced affinity for IR-B (twofold) compared to native insulin. In stimulating DNA synthesis, A0:R glargine was slightly less potent compared to insulin or glargine. This result ultimately confirms the previous report that A0:R glargine has a lower potency in mitogenic assays compared to glargine. This glargine analogue thus could be a potential lead compound for drug design and development for the treatment of diabetes.

Insulin in motion: The A6-A11 disulfide bond allosterically modulates structural transitions required for insulin activity

The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin’s ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues.