Neurotensin (NT) is an endogenous neuropeptide involved in a variety of central and peripheral neuromodulatory effects. Here we show effects of site-specific glycosylation on the in vitro and in vivo pharmacological properties of this neuropeptide. NT analogs containing O-linked disaccharides (β-melibiose and α-TF antigen) or β-lactose unit linked via a PEG3-spacer were designed and chemically synthesized using Fmoc chemistry. For the latter analog, Fmoc-Glu-(β-Lac-PEG3-amide) was prepared. Our results indicate that the addition of the disaccharides did not negatively affect the subnanomolar affinity nor the low nanomolar agonist potency for the neurotensin receptor subtype 1 (NTS1). Interestingly, three glycosylated analogs exhibited subpicomolar potency in the 6 Hz limbic seizure mouse model of pharmacoresistant epilepsy following intracerebroventricular administration. Our results suggest for the first time that chemically-modified NT analogs may lead to novel antiepileptic therapies.
neurotensin; anticonvulsant; glycosylation; binding; agonist
The endogenous neuropeptide galanin has anticonvulsant and analgesic properties mediated by galanin receptors expressed in the central and peripheral nervous systems. Our previous work showed that combination of truncation of the galanin peptide along with N-and C-terminal modifications afforded analogs that suppressed seizures or pain following intraperitoneal administration. To generate orally-active galanin analogs, the previously reported lead compound Gal-B2 (NAX 5055) was redesigned by (1) central truncation, (2) introduction of D-amino acids, (3) and addition of backbone spacers. Analog D-Gal(7-Ahp)-B2, containing 7-amino heptanoic acid as a backbone spacer and oligo-D-lysine motif at the C-terminus, exhibited anticonvulsant and analgesic activity post intraperitoneal administration. Oral administration of D-Gal(7-Ahp)-B2 demonstrated analgesic activity with reduction in both acute and inflammatory pain in the mouse formalin model of pain at doses as low as 8 mg/kg.
galanin; analgesic peptides; orally-active; backbone prosthesis; neuropeptide
The µ-conotoxin KIIIA is a three disulfide-bridged blocker of voltage-gated sodium channels (VGSCs). The Lys7 residue in KIIIA is an attractive target for manipulating the selectivity and efficacy of this peptide. Here, we report the design and chemical synthesis of µ-conopeptoid analogues (peptomers) in which we replaced Lys7 with peptoid monomers of increasing side-chain size: N-methylglycine, N-butylglycine and N-octylglycine. In the first series of analogues, the peptide core contained all three disulfide bridges; whereas in the second series, a disulfide-depleted selenoconopeptide core was used to simplify oxidative folding. The analogues were tested for functional activity in blocking the Nav1.2 subtype of mammalian VGSCs exogenously expressed in Xenopus oocytes. All six analogues were active, with the N-methylglycine analogue, [Sar7]KIIIA, the most potent in blocking the channels while favoring lower efficacy. Our findings demonstrate that the use of N-substituted Gly residues in conotoxins show promise as a tool to optimize their pharmacological properties as potential analgesic drug leads.
µ-conotoxins; KIIIA, peptomers; selenocysteine; diselenide; sodium channels; electrophysiology
µ-SIIIA, a novel µ-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-sensitive sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remained unknown. We have determined the structure of µ-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, µ-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11–16, while residues 3–5 in the N-terminal region form a helix-like turn resembling 310 helix. The Trp12 and His16 side chains are in close proximity, as in the related conotoxin µ-SmIIIA, but Asn2 is further away. Dynamics measurements show that the N-terminus and Ser9 have larger magnitude motions on the sub-ns timescale, while the C-terminus is more rigid. Cys4, Trp12 and Cys13 undergo significant conformational exchange on µs - ms timescales. µ-SIIIA is a potent, nearly irreversible blocker of NaV1.2, but also blocks NaV1.4 and NaV1.6 with submicromolar potency. The selectivity profile of µ-SIIIA, including poor activity against the cardiac sodium channel, NaV1.5, is similar to that of the closely related µ-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal NaV1.2. The structural and functional characterization described in this paper of an analgesic µ-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.
Among the μ-conotoxins that block vertebrate voltage-gated sodium channels (VGSCs), some have been shown to be potent analgesics following systemic administration in mice. We have determined the solution structure of a new representative of this family, μ-BuIIIB, and established its disulfide connectivities by direct mass spectrometric collision induced dissociation fragmentation of the peptide with disulfides intact. The major oxidative folding product adopts a 1-4/2-5/3-6 pattern with the following disulfide bridges: Cys5-Cys17, Cys6-Cys23 and Cys13-Cys24. The solution structure reveals that the unique N-terminal extension in μ-BuIIIB, which is also present in μ-BuIIIA and μ-BuIIIC but absent in other μ-conotoxins, forms part of a short α-helix encompassing Glu3 to Asn8. This helix is packed against the rest of the toxin and stabilized by the Cys5-Cys17 and Cys6-Cys23 disulfide bonds. As such, the side chain of Val1 is located close to the aromatic rings of Trp16 and His20, which are located on the canonical helix that displays several residues found to be essential for VGSC blockade in related μ-conotoxins. Mutations of residues 2 and 3 in the N-terminal extension enhanced the potency of μ-BuIIIB for NaV1.3. One analog, [d-Ala2]BuIIIB, showed a 40-fold increase, making it the most potent peptide blocker of this channel characterized to date and thus a useful new tool with which to characterize this channel. Based on previous results for related μ-conotoxins, the dramatic effects of mutations at the N-terminus were unanticipated, and suggest that further gains in potency might be achieved by additional modifications of this region.
The μ-conotoxin μ-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12 and Arg14) all reside on an α-helix with the exception of Arg14. To further probe structure-activity relationships of this toxin against VGSC subtypes, we have characterised the analogue μ-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in μ-KIIIA were replaced with Ala. Its structure is quite similar to that of μ-KIIIA, indicating that the Cys1-Cys9 disulfide bond could be removed without any significant distortion of the α-helix bearing the key residues. Consistent with this, μ-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of μ-KIIIA, namely, NaV1.2 > NaV1.4 > NaV1.7 ≥ NaV1.1 > NaV1.3 > NaV1.5. Kinetics of block were obtained for NaV1.2, NaV1.4 and NaV1.7, and in each case both kon and koff values of μ-KIIIA[C1A,C9A] were larger than those of μ-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an α-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on- and off-rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.
Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-Methyl D-Asparate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity to the most studied NR2B-selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogs for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings from Tyr5 substitutions in other conantokins, conRl-B [L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis and pharmacology for discovering new bioactive natural products.
Conus peptides; conantokin; NMDA antagonist; NR2B subunits; epilepsy; anticonvulsant
A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl-A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl-A is a 24-residue peptide containing three gamma-carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl-A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl-A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca++. The order of potency for blocking the four NMDA receptor subtypes by ConRl-A was NR2B>NR2D>NR2A>NR2C. This peptide has a greater discrimination between NR2B and NR2C then any other ligand reported so far. In summary, ConRl-A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from genus Conus.
Conantokin; Molecular phylogeny; Conformational interconversion; Helical peptide; Electrophysiology
In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely-related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric CID fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on NMR data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as for μ-KIIIA, and both blocked NaV1.2 (Kd 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA/μ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means to further expand the snail's repertoire of active peptides.
Mobile medical applications (mHealth), music, and video games are being developed and tested for their ability to improve pharmacotherapy outcomes and medication adherence. Pleiotropic mechanism of music and gamification engages an intrinsic motivation and the brain reward system, supporting therapies in patients with neurological disorders, including neuropathic pain, depression, anxiety, or neurodegenerative disorders. Based on accumulating results from clinical trials, an innovative combination treatment of epilepsy seizures, comorbidities, and the medication non-adherence can be designed, consisting of antiepileptic drugs and disease self-management software delivering clinically beneficial music. Since creative elements and art expressed in games, music, and software are copyrighted, therefore clinical and regulatory challenges in developing copyrighted, drug–device therapies may be offset by a value proposition of the exclusivity due to the patent–independent protection, which can last for over 70 years. Taken together, development of copyrighted non-pharmacological treatments (e-therapies), and their combinations with pharmacotherapies, offer incentives to chronically ill patients and outcome-driven health care industries.
chronic disease; psychiatric disorders; dopamine; Mozart; epilepsy; depression; fatigue
Disulfide bridges, which stabilize the native conformation of conotoxins impose a challenge in the synthesis of smaller analogs. In this work, we describe the synthesis of a minimized analog of the analgesic μ-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal NaV1.2 and skeletal muscle NaV1.4. Three disulfide-deficient analogs of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1-C9, C2-C15 or C4-C16 was removed. Deletion of the first bridge only slightly affected the peptide’s bioactivity. To further minimize this analog, the N-terminal residue was removed and two non-essential Ser residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting “polytide” analog retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogs containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics.
conopeptide; conotoxin; sodium channels; backbone spacers; disulfide bridges
This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
Conantokins are venom peptides from marine cone snails that are NMDA receptor antagonists. Here, we report the characterization of a 24 AA conantokin from Conus brettinghami (1), conantokin-Br (con-Br), the first conantokin that does not have the conserved glutamate residue at position 2. Molecular modeling studies suggest that con-Br has a helical structure between residues 2–13. In contrast to other characterized conantokins, con-Br has a high potency for NMDA receptors with NR2D subunits. To identify determinants for NR2D potency, we synthesized chimeras of con-Br and conantokin-R (con-R), the latter has a ~30-fold lower potency for the NR2D subtype. The characterization of two reciprocal chimeras (con-Br/R and con-R/Br), comprising the first 9–10 N-terminal AAs of each conantokin followed by the corresponding C-terminal AAs of the other conantokin demonstrates that determinants for NR2D selectivity are at the N-terminal region. Additional analogs comprising 1–3 amino acid substitutions from each peptide into the homologous region of the other led to the identification of a key determinant; a Tyr residue in position 5 increases potency for NR2D, while Val at this locus causes a decrease. The systematic definition of key determinants in the conantokin peptides for NMDA receptor subtype selectivity is an essential component in the development of conantokin peptides that are highly selective for each specific NMDA receptor subtype.
Conotoxin genes are among the most rapidly evolving genes currently known; however, despite the well-established hypervariability of the intercysteine loops, the cysteines demonstrate significant conservation, with a site-specific codon bias for each cysteine in a family of conotoxins. Herein we present a novel rationale behind the codon-level conservation of the cysteines that comprise the disulfide scaffold. We analyze cysteine codon conservation using an internal reference and phylogenetic tools; our results suggest that the established codon conservation can be explained as the result of selective pressures linked to the production efficiency and folding of conotoxins, driving the conservation of cysteine at the amino-acid level. The preservation of cysteine has resulted in maintenance of the ancestral codon in most of the daughter lineages, despite the hypervariability of adjacent residues. We propose that the selective pressures acting on the venom components of cone snails involve an interplay of biosynthetic efficiency, activity at the target receptor and the importance of that activity to effective prey immobilization. Functional redundancy in the venom can thus serve as a buffer for the energy expenditure of venom production.
In cysteine-rich peptides, diselenides can be used as a proxy for disulfide bridges, since the energetic preference for diselenide bonding over mixed selenium-sulfur bonds simplifies folding. Herein we report that an intramolecular diselenide bond efficiently catalyzes the oxidative folding of selenopeptide analogs of conotoxins, and serves as a reagentless method to substantially accelerate formation of various native disulfide bridging patterns.
Diselenide; oxidative folding; kinetics; conotoxin; selenopeptide
Conotoxins comprise a large group of peptidic neurotoxins that employ diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of non-critical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot (ICK) motif family and blocks N-type calcium channels. Removal of a non-critical Cys1–Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally non-critical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionary preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.
disulfide bridges; conotoxins; structure-function; oxidative folding; calcium channels
μ-Conotoxin KIIIA (μ-KIIIA) blocks mammalian voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. Previous structure-activity studies of μ-KIIIA identified a helical pharmacophore for VGSC blockade. This suggested a route for designing truncated analogues of μ-KIIIA by incorporating the key residues into an α-helical scaffold. As (i, i+4) lactam bridges constitute a proven approach for stabilizing α-helices, we designed and synthesized six truncated analogues of μ-KIIIA containing single lactam bridges at various locations. The helicity of these lactam analogues was analysed by NMR spectroscopy, and their activities were tested against mammalian VGSC subtypes NaV1.1 through 1.7. Two of the analogues, Ac-cyclo9/13[Asp9,Lys13]KIIIA7–14 and Ac-cyclo9/13[Lys9,Asp13]KIIIA7–14, displayed µM activity against VGSC subtypes NaV1.2 and NaV1.6; importantly, the subtype selectivity profile for these peptides matched that of μ-KIIIA. Our study highlights structure-activity relationships within these helical mimetics and provides a basis for the design of additional truncated peptides as potential analgesics.
The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA.
Manipulation of the flagellar secretion system bypasses the problems of inclusion body formation and cellular degradation that occur during conventional recombinant protein expression. This work serves as a proof of principle for the use of engineered bacterial cells for rapid purification of recombinant neuroactive peptides and proteins by exploiting secretion via the well-characterized flagellar type III secretion system.
Anticonvulsant neuropeptides play an important role in controlling neuronal excitability that leads to pain or seizures. Based on overlapping inhibitory mechanisms, many anticonvulsant compounds have been found to exhibit both analgesic and antiepileptic activities. An analgesic neuropeptide W (NPW) targets recently deorphanized G-protein coupled receptors. Here, we tested the hypothesis that the analgesic activity of NPW may lead to the discovery of its antiepileptic properties. Indeed, direct administration of NPW into the brain potently reduced seizures in mice. To confirm this discovery, we rationally designed, synthesized, and characterized NPW analogues that exhibited anticonvulsant activities following systemic administration. Our results suggest that the combination of neuropeptide repositioning and engineering NPW analogues that penetrate the blood-brain barrier could provide new drug leads, not only for the treatment of epilepsy and pain but also for studying effects of this peptide on regulating feeding and energy metabolism coupled to leptin levels in the brain.
Neuropeptide W; neuropeptide repositioning; lipidization-cationization; anticonvulsant; metabolic stability; systemic bioavailability
A protease from ribosomal peptide biosynthesis macrocyclizes diverse substrates, including those resembling nonribosomal peptide and hybrid polyketide-peptide products. The proposed mechanism is analogous to thioesterase-catalyzed chemistry, but the substrates are amide bonds rather than thioesters.
Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged μ-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of another disulfide bridge with a diselenide bridge) dramatically simplified its oxidative folding while preserving the peptide’s ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogues containing a single disulfide bridge allowed rapid positional scanning at Lys7 of μ-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide’s selectivity in blocking a neuronal (Nav1.2) over a muscle (Nav1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high-throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.
Conotoxins; diselenide bridges; selenocysteines; oxidative folding; disulfide-rich peptides
The possibility of independently manipulating the affinity and efficacy of pore-blocking ligands of sodium channels is of interest for the development of new drugs for the treatment of pain. The analgesic µ-conotoxin KIIIA, a 16-residue peptide with three disulfide bridges, is a pore-blocker of voltage-gated sodium channels, including the neuronal subtype NaV1.2 (Kd of 5 nM). At saturating concentrations, µ-KIIIA incompletely blocks the sodium current of NaV1.2, leaving a 5% residual current (rINa). Lys7 is an important residue: the mutation K7A decreases both the efficacy (i.e., increases rINa to 23%) and the affinity of the peptide (Kd, 115 nM). In this report, various replacements of residue 7 were examined to determine whether affinity and efficacy were inexorably linked. Because of their facile chemical synthesis, KIIIA analogs were used that had as a core structure the disulfide-depleted KIIIA[C1A,C2U,C9A,C5U] (where U is selenocysteine) or ddKIIIA. The analogs ddKIIIA and ddKIIIA[K7X], where X represents one of nine different amino acids, were tested on voltage-clamped Xenopus oocytes expressing rat NaV1.2 or NaV1.4. Their affinities ranged from 0.01 to 36 µM and rINa's from 2 to 42%, and these two variables appeared uncorrelated. Instead, rINa varied inversely with side chain size, and remarkably charge and hydrophobicity appeared inconsequential. The ability to manipulate a µ-conopeptide's affinity and efficacy, as well as its capacity to interfere with subsequent tetrodotoxin-binding, greatly expands its scope as a reagent to probe sodium channel structure and function, and may also lead to the development of µ-conotoxins as safe analgesics.
Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged μ-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of a disulfide- with a diselenide-bridge) dramatically simplified its oxidative folding while preserving the peptide’s ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogs containing a single disulfide bridge allowed rapid positional scanning at Lys7 of μ-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide’s selectivity in blocking a neuronal (Nav1.2) over a muscle (Nav1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.
conotoxins; diselenide bridges; selenocysteines; oxidative folding; disulfide-rich peptides
Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern: C—C—CC—C—C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of ω-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogs were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analog, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogs. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide crosslinks. Both in vivo and in vitro activities of the analogs were assessed; block of N-type calcium channels was comparable among the analogs and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including α-, µ- and ω- conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.
Galanin modulates seizures in the brain through two galanin receptor subtypes, GalR1 and GalR2. To generate systemically-active galanin receptor ligands that discriminate between GalR1 and GalR2, the GalR1-preferring analogue, Gal-B2 (or NAX 5055), was rationally redesigned to yield GalR2-preferring analogues. Systematic truncations of the N-terminal backbone led to [N-Me, des-Sar]Gal-B2, containing N-methyl tryptophan: this analogue exhibited 18-fold preference in binding toward GalR2, maintained agonist activity, and exhibited potent anticonvulsant activity in mice following intraperitoneal administration.