Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Cysteine-Rich α-Conotoxin SII Displays Novel Interactions at the Muscle Nicotinic Acetylcholine Receptor.

α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 310-helix frequently employed by framework I α-conotoxins; α-SII acted at the muscle nAChR with half-maximal inhibitory concentrations (IC50) of 120 nM (adult) and 370 nM (fetal) though weakly at neuronal nAChRs. Truncation of α-SII to a two disulfide bond amidated peptide with framework I disulfide connectivity led to similar activity. Surprisingly, the more constrained α-SII was less stable under mild reducing conditions and displayed a unique docking mode at the nAChR.

Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider.

The King Baboon spider, Pelinobius muticus, is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus, but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including NaV1.8, KV2.1, and tetrodotoxin-sensitive NaV channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms.

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, Doratifera vulnerans.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.

Mutational analysis of ProTx-I and the novel venom peptide Pe1b provide insight into residues responsible for selective inhibition of the analgesic drug target NaV1.7.

Management of chronic pain presents a major challenge, since many currently available treatments lack efficacy and have problems such as addiction and tolerance. Loss of function mutations in the SCN9A gene lead to a congenital inability to feel pain, with no other sensory deficits aside from anosmia. SCN9A encodes the voltage-gated sodium (NaV) channel 1.7 (NaV1.7), which has been identified as a primary pain target. However, in developing NaV1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other NaV subtypes that are critical for survival. Since spider venoms are an excellent source of NaV channel modulators, we screened a panel of spider venoms to identify selective NaV1.7 inhibitors. This led to identification of two novel NaV modulating venom peptides (ß/µ-theraphotoxin-Pe1a and ß/µ-theraphotoxin-Pe1b (Pe1b) from the arboreal tarantula Phormingochilus everetti. A third peptide isolated from the tarantula Bumba pulcherrimaklaasi was identical to the well-known ProTx-I (ß/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the NaV1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and NaV channel subtype selectivity. Several analogues had improved potency against NaV1.7, and altered specificity against other NaV channels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of NaV1.7.

A tetrapeptide class of biased analgesics from an Australian fungus targets the µ-opioid receptor.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.

Antiallodynic effects of the selective NaV1.7 inhibitor Pn3a in a mouse model of acute postsurgical pain: evidence for analgesic synergy with opioids and baclofen.

Pain is the leading cause of disability in the developed world but remains a poorly treated condition. Specifically, postsurgical pain continues to be a frequent and undermanaged condition. Here, we investigate the analgesic potential of pharmacological NaV1.7 inhibition in a mouse model of acute postsurgical pain, based on incision of the plantar skin and underlying muscle of the hind paw. We demonstrate that local and systemic treatment with the selective NaV1.7 inhibitor µ-theraphotoxin-Pn3a is effectively antiallodynic in this model and completely reverses mechanical hypersensitivity in the absence of motor adverse effects. In addition, the selective NaV1.7 inhibitors ProTx-II and PF-04856264 as well as the clinical candidate CNV1014802 also reduced mechanical allodynia. Interestingly, co-administration of the opioid receptor antagonist naloxone completely reversed analgesic effects of Pn3a, indicating an involvement of endogenous opioids in the analgesic activity of Pn3a. In addition, we found superadditive antinociceptive effects of subtherapeutic Pn3a doses not only with the opioid oxycodone but also with the GABAB receptor agonist baclofen. Transcriptomic analysis of gene expression changes in dorsal root ganglia of mice after surgery did not reveal any changes in mRNA expression of endogenous opioids or opioid receptors; however, several genes involved in pain, including Runx1 (Runt related transcription factor 1), Cacna1a (CaV2.1), and Cacna1b (CaV2.2), were downregulated. In summary, these findings suggest that pain after surgery can be successfully treated with NaV1.7 inhibitors alone or in combination with baclofen or opioids, which may present a novel and safe treatment strategy for this frequent and poorly managed condition.

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold.

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (KV) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric ß-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type KV channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.

Novel venom-derived inhibitors of the human EAG channel, a putative antiepileptic drug target.

Recently, we and other groups revealed that gain-of-function mutations in the human ether à go-go voltage-gated potassium channel hEAG1 (Kv10.1) lead to developmental disorders with associated infantile-onset epilepsy. However, the physiological role of hEAG1 in the central nervous system remains elusive. Potent and selective antagonists of hEAG1 are therefore much sought after, both as pharmacological tools for studying the (patho)physiological functions of this enigmatic channel and as potential leads for development of anti-epileptic drugs. Since animal venoms are a rich source of potent ion channel modifiers that have been finely tuned by millions of year of evolution, we screened 108 arachnid venoms for hEAG1 inhibitors using electrophysiology. Two hit peptides (Aa1a and Ap1a) were isolated, sequenced, and chemically synthesised for structure-function studies. Both of these hEAG1 inhibitors are C-terminally amidated peptides containing an inhibitor cystine knot motif, which provides them with exceptional stability in both plasma and cerebrospinal fluid. Aa1a and Ap1a are the most potent peptidic inhibitors of hEAG1 reported to date, and they present a novel mode of action by targeting both the activation and inactivation gating of the channel. These peptides should be useful pharmacological tools for probing hEAG1 function as well as informative leads for the development of novel anti-epileptic drugs.

Conotoxin Φ-MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti-Apoptotic Activity.

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two ß-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC50 17.85 µm) while inhibiting apoptosis (EC50 2.2 µm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors.

Δ-Myrtoxin-Mp1a is a Helical Heterodimer from the Venom of the Jack Jumper Ant that has Antimicrobial, Membrane-Disrupting, and Nociceptive Activities.

Δ-Myrtoxin-Mp1a (Mp1a), a 49-residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26-mer A chain and a 23-mer B chain connected by two disulfide bonds in an antiparallel arrangement. Combination of the individual synthetic chains through aerial oxidation remarkably resulted in the self-assembly of Mp1a as a homogenous product without the need for directed disulfide-bond formation. NMR analysis revealed a well-defined, unique structure containing an antiparallel α-helix pair. Dual polarization interferometry (DPI) analysis showed strong interaction with supported lipid bilayers and insertion within the bilayers. Mp1a caused non-specific Ca2+ influx in SH-SY5Y cells with a half maximal effective concentration (EC50 ) of 4.3 µm. Mp1a also displayed broad-spectrum antimicrobial activity, with the highest potency against Gram-negative Acinetobacter baumannii (MIC 25 nm). Intraplantar injection (10 µm) in mice elicited spontaneous pain and mechanical allodynia. Single- and two-chain mimetics of Mp1a revealed functional selectivity.

Modulatory features of the novel spider toxin µ-TRTX-Df1a isolated from the venom of the spider Davus fasciatus.

BACKGROUND AND PURPOSE: Naturally occurring dysfunction of voltage-gated sodium (NaV ) channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel NaV modulators, we investigated spider venoms for new inhibitors of NaV channels. EXPERIMENTAL APPROACH: We used high-throughput screens to identify a NaV modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modelling and a rodent pain model. KEY RESULTS: We identified a potent NaV inhibitor named µ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by NaV 1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited voltage-gated calcium (CaV 3) currents but had no activity against the voltage-gated potassium (KV 2) channel. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the NaV channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all NaV subtypes tested (hNaV 1.1-1.7). Df1a also slowed fast inactivation of NaV 1.1, NaV 1.3 and NaV 1.5 and modified the voltage-dependence of activation and inactivation of most of the NaV subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which probably explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviours induced by the NaV activator OD1. CONCLUSION AND IMPLICATIONS: µ-TRTX-Df1a shows potential as a new molecule for the development of drugs to treat pain disorders mediated by voltage-gated ion channels.

Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain.

Loss-of-function mutations of Na(V)1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na(V)1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na(V)1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na(V)1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na(V)1.7 inhibitors and significantly reduced in Na(V)1.7(-/-) mice. To validate the use of the model for profiling Na(V)1.7 inhibitors, we determined the Na(V) selectivity and tested the efficacy of the reported Na(V)1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na(V)1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na(V)1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na(V) channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na(V)1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na(V)1.7 inhibitors.

Identification and Characterization of ProTx-III [µ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens.

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (NaV) channels, we screened spider venoms for inhibitors of human NaV1.7 (hNaV1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel NaV1.7 inhibitor, µ-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNaV1.7 > hNaV1.6 > hNaV1.2 > hNaV1.1 > hNaV1.3 channels in fluorescent assays. NaV1.7 inhibition was diminished (IC50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 µM. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNaV1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNaV1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNaV1.7 inhibitors for treatment of chronic pain.

Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl.

A safety-catch cysteine protecting group, S-4,4'-dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high-resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide-bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4 nM (non-native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells.

A tarantula-venom peptide antagonizes the TRPA1 nociceptor ion channel by binding to the S1-S4 gating domain.

BACKGROUND: The venoms of predators have been an excellent source of diverse highly specific peptides targeting ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1). RESULTS: We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by coexpression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. We constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site. CONCLUSIONS: These studies establish our approach, which we term "toxineering," as a generally applicable method for isolation of novel ion channel modifiers and design of ion channel modifiers with altered specificity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing biophysical mechanisms of TRPA1 gating and the physiology of TRPA1 function in nociceptors, as well as for potential clinical application in the context of pain and inflammation.

Isolation, characterization and total regioselective synthesis of the novel µO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels.

The µO-conotoxins are notable for their unique selectivity for Na(v)1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel µO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to µO-conotoxin MrVIB. To overcome the synthetic challenges posed by µO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of µO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Na(v)1.8 (IC50 95.9±74.3 nM) and Na(v)1.4 (IC50 81±16 nM), with significantly lower affinity for other Na(v) subtypes (IC50 431-6203 nM; Na(v)1.5>1.6∼1.7∼1.3∼1.1∼1.2). This improved approach to µO-conotoxin synthesis will facilitate the optimization of µO-conotoxins as novel analgesic molecules to improve pain management.

α-Conotoxin ImI incorporating stable cystathionine bridges maintains full potency and identical three-dimensional structure.

The two disulfide bonds of α-conotoxin ImI, a peptide antagonist of the α7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a γ-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to α-conotoxin ImI by (1)H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC(50) = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC(50) = 6.41 ± 0.09), whereas those of 2 (pIC(50) = 5.96 ± 0.09) and 4 (pIC(50) = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S═O) to a 3-fold increase (3S═O(B)) in potencies.

Modulating oxytocin activity and plasma stability by disulfide bond engineering.

Disulfide bond engineering is an important approach to improve the metabolic half-life of cysteine-containing peptides. Eleven analogues of oxytocin were synthesized including disulfide bond replacements by thioether, selenylsulfide, diselenide, and ditelluride bridges, and their stabilities in human plasma and activity at the human oxytocin receptor were assessed. The cystathionine (K(i) = 1.5 nM, and EC50 = 32 nM), selenylsulfide (K(i) = 0.29/0.72 nM, and EC50 = 2.6/154 nM), diselenide (K(i) = 11.8 nM, and EC50 = 18 nM), and ditelluride analogues (K(i) = 7.6 nM, and EC50 = 27.3 nM) retained considerable affinity and functional potency as compared to oxytocin (K(i) = 0.79 nM, and EC50 = 15 nM), while shortening the disulfide bridge abolished binding and functional activity. The mimetics showed a 1.5-3-fold enhancement of plasma stability as compared to oxytocin (t(½) = 12 h). By contrast, the all-D-oxytocin and head to tail cyclic oxytocin analogues, while significantly more stable with half-lives greater than 48 h, had little or no detectable binding or functional activity.