RESUMO
Recombinant peptide synthesis allows for large-scale production of peptides with therapeutic potential. However, access to dicarba peptidomimetics via sidechain-deprotected sequences becomes challenging with exposed Lewis basicity presented by amine and sulfur-containing residues. Presented here is a combination of strategies which can be used to deactivate coordinative residues and achieve high-yielding Ru-catalyzed ring-closing metathesis. The chemistry is exemplified using α-conotoxin EpI, a native bicyclic disulfide-containing sequence isolated from the marine conesnail Conus episcopatus. Replacement of the loop I disulfide with E/Z-dicarba bridges was achieved with high conversion via solution-phase ring-closing metathesis of the unprotected linear peptide after simple chemoselective oxidation and ion-exchange masking of problematic functionality. Metathesis was also attempted in green solvent choices to further improve the sustainability of dicarba peptide synthesis.
Assuntos
Conotoxinas , Cistina , Peptídeos/química , Dissulfetos/química , Oxirredução , Conotoxinas/químicaRESUMO
Ru-Alkylidene catalysed olefin metathesis generates metabolically stable cystine bridge peptidomimetics with defined geometry. Deleterious coordinative bonding to the catalyst by sulfur-containing functionality found in cysteine and methionine residues can be negated by in situ and reversible oxidation of thiol and thioether functionality, as disulfides and S-oxides respectively, to facilitate high yielding ring-closing and cross metathesis of bioorthogonally protected peptides.
Assuntos
Cisteína , Metionina , Cisteína/química , Metionina/química , Peptídeos/química , Cistina/química , RacemetioninaRESUMO
Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.
Assuntos
Conotoxinas , Caramujo Conus , Receptores Nicotínicos , Camundongos , Humanos , Animais , Conotoxinas/farmacologia , Caramujo Conus/metabolismo , Peçonhas , Receptores Nicotínicos/metabolismo , Peptídeos/metabolismo , Antagonistas Nicotínicos/farmacologiaRESUMO
The mechanisms by which insulin activates the insulin receptor to promote metabolic processes and cellular growth are still not clear. Significant advances have been gained from recent structural studies in understanding how insulin binds to its receptor. However, the way in which specific interactions lead to either metabolic or mitogenic signalling remains unknown. Currently there are only a few examples of insulin receptor agonists that have biased signalling properties. Here we use novel insulin analogues that differ only in the chemical composition at the A6-A11 bond, as it has been changed to a rigid, non-reducible C=C linkage (dicarba bond), to reveal mechanisms underlying signaling bias. We show that introduction of an A6-A11 cis-dicarba bond into either native insulin or the basal/long acting insulin glargine results in biased signalling analogues with low mitogenic potency. This can be attributed to reduced insulin receptor activation that prevents effective receptor internalization and mitogenic signalling. Insight gained into the receptor interactions affected by insertion of an A6-A11 cis-dicarba bond will ultimately assist in the development of new insulin analogues for the treatment of diabetes that confer low mitogenic activity and therefore pose minimal risk of promoting cancer with long term use.
Assuntos
Insulina , Receptor de Insulina , Dissulfetos , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Mitógenos/metabolismo , Mitógenos/farmacologia , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismoRESUMO
Several analgesic α-conotoxins have been isolated from marine cone snails. Structural modification of native peptides has provided potent and selective analogues for two of its known biological targets-nicotinic acetylcholine and γ-aminobutyric acid (GABA) G protein-coupled (GABAB) receptors. Both of these molecular targets are implicated in pain pathways. Despite their small size, an incomplete understanding of the structure-activity relationship of α-conotoxins at each of these targets has hampered the development of therapeutic leads. This review scrutinises the N-terminal domain of the α-conotoxin family of peptides, a region defined by an invariant disulfide bridge, a turn-inducing proline residue and multiple polar sidechain residues, and focusses on structural features that provide analgesia through inhibition of high-voltage-activated Ca2+ channels. Elucidating the bioactive conformation of this region of these peptides may hold the key to discovering potent drugs for the unmet management of debilitating chronic pain associated with a wide range of medical conditions.
Assuntos
Analgésicos/química , Conotoxinas/química , Peptídeos/química , Peptidomiméticos/química , Analgesia , Analgésicos/uso terapêutico , Animais , Dissulfetos/química , Humanos , Peptídeos/uso terapêutico , Peptidomiméticos/uso terapêutico , Conformação ProteicaRESUMO
The development of fast-acting and highly stable insulin analogues is challenging. Insulin undergoes structural transitions essential for binding and activation of the insulin receptor (IR), but these conformational changes can also affect insulin stability. Previously, we substituted the insulin A6-A11 cystine with a rigid, non-reducible C=C linkage ("dicarba" linkage). A cis-alkene permitted the conformational flexibility of the A-chain N-terminal helix necessary for high-affinity IR binding, resulting in surprisingly rapid activity in vivo Here, we show that, unlike the rapidly acting LysB28ProB29 insulin analogue (KP insulin), cis-dicarba insulin is not inherently monomeric. We also show that cis-dicarba KP insulin lowers blood glucose levels even more rapidly than KP insulin, suggesting that an inability to oligomerize is not responsible for the observed rapid activity onset of cis-dicarba analogues. Although rapid-acting, neither dicarba species is stable, as assessed by fibrillation and thermodynamics assays. MALDI analyses and molecular dynamics simulations of cis-dicarba insulin revealed a previously unidentified role of the A6-A11 linkage in insulin conformational dynamics. By controlling the conformational flexibility of the insulin B-chain helix, this linkage affects overall insulin structural stability. This effect is independent of its regulation of the A-chain N-terminal helix flexibility necessary for IR engagement. We conclude that high-affinity IR binding, rapid in vivo activity, and insulin stability can be regulated by the specific conformational arrangement of the A6-A11 linkage. This detailed understanding of insulin's structural dynamics may aid in the future design of rapid-acting insulin analogues with improved stability.
Assuntos
Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Insulina/análogos & derivados , Insulina/farmacologia , Animais , Glicemia/metabolismo , Linhagem Celular , Cristalografia por Raios X , Cisteína/química , Cisteína/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Células NIH 3T3 , Conformação Proteica , Estabilidade Proteica , Receptor de Insulina/metabolismo , TermodinâmicaRESUMO
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.
Assuntos
Dissulfetos/química , Insulina/química , Insulina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , EstereoisomerismoRESUMO
Certain peptide folds, owing to a combination of intrinsic stability and resilience to amino acid substitutions, are particularly effective for the display of diverse functional groups. Such "privileged scaffolds" are valuable as starting points for the engineering of new bioactive molecules. We have identified a precursor peptide expressed in the venom gland of the marine snail Conus victoriae, which appears to belong to a hitherto undescribed class of molluscan neuropeptides. Mass spectrometry matching with the venom confirmed the complete mature peptide sequence as a 31-residue peptide with a single disulfide bond. Solution structure determination revealed a unique peptide fold that we have designated the single disulfide-directed ß hairpin (SDH). The SDH fold is highly resistant to thermal denaturation and forms the core of several other multiple disulfide-containing peptide folds, including the inhibitor cystine knot. This elementary fold may offer a valuable starting point for the design and engineering of new bioactive peptides.
Assuntos
Venenos de Moluscos/química , Neuropeptídeos/química , Peptídeos/química , Caramujos/química , Animais , Cisteína , Espectrometria de Massas , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
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 α3ß4 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 peptide's 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 peptide's potent analgesic activity.
Assuntos
Conotoxinas/química , Receptores de GABA/química , Receptores Nicotínicos/química , Sequência de Aminoácidos , Animais , Conotoxinas/genética , Conotoxinas/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Neurônios/química , Neurônios/efeitos dos fármacos , Oócitos/química , Oócitos/efeitos dos fármacos , Ratos , Receptores Nicotínicos/genética , Xenopus/genética , Ácido gama-Aminobutírico/químicaRESUMO
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.
Assuntos
Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/antagonistas & inibidores , Relaxina/química , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células CHO , Colágeno/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Injeções Intraventriculares , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Relaxina/farmacologia , Pele/citologia , Relação Estrutura-AtividadeRESUMO
Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However, the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains--including the notoriously poorly soluble A-chain--followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.