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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 (Na V ) channel 1.7 (Na V 1.7), which has been identified as a primary pain target. However, in developing Na V 1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other Na V subtypes that are critical for survival. Since spider venoms are an excellent source of Na V channel modulators, we screened a panel of spider venoms to identify selective Na V 1.7 inhibitors. This led to identification of two novel Na V modulating venom peptides (β/μ-theraphotoxin-Pe1a and β/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 (β/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the Na V 1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and Na V channel subtype selectivity. Several analogues had improved potency against Na V 1.7, and altered specificity against other Na V channels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of Na V 1.7., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Conopeptides are neurotoxic peptides in the venom of marine cone snails and have broad therapeutic potential for managing pain and other conditions. Here, we identified the single-disulfide peptides Czon1107 and Cca1669 from the venoms of Conus zonatus and Conus caracteristicus , respectively. We observed that Czon1107 strongly inhibits the human α3β4 (IC 50 15.7 ± 3.0 μm) and α7 (IC 50 77.1 ± 0.05 μm) nicotinic acetylcholine receptor (nAChR) subtypes, but the activity of Cca1669 remains to be identified. Czon1107 acted at a site distinct from the orthosteric receptor site. Solution NMR experiments revealed that Czon1107 exists in equilibrium between conformational states that are the result of a key Ser 4 -Pro 5 cis-trans isomerization. Moreover, we found that the X-Pro amide bonds in the inter-cysteine loop are rigidly constrained to cis conformations. Structure-activity experiments of Czon1107 and its variants at positions P5 and P7 revealed that the conformation around the X-Pro bonds ( cis-trans ) plays an important role in receptor subtype selectivity. The cis conformation at the Cys 6 -Pro 7 species for similar, short bioactive peptides that allosterically modulate ligand-gated receptor function.Conus species for similar, short bioactive peptides that allosterically modulate ligand-gated receptor function., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Mohan et al.)

Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus , the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that "messy" processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.

Venoms from snakes are rich sources of highly active proteins with potent affinity towards a variety of enzymes and receptors. Of the many distinct toxicities caused by envenomation, neurotoxicity plays an important role in the paralysis of prey by snakes as well as by venomous sea snails and insects. In order to improve the analytical discovery component of venom toxicity profiling, this paper describes the implementation of microfluidic high-resolution screening (HRS) to obtain neurotoxicity fingerprints from venoms that facilitates identification of the neurotoxic components of envenomation. To demonstrate this workflow, 47 snake venoms were profiled using the acetylcholine binding protein (AChBP) to mimic the target of neurotoxic proteins, in particular nicotinic acetylcholine receptors (nAChRs). In the microfluidic HRS system, nanoliquid chromatographic (nanoLC) separations were on-line connected to both AChBP profiling and parallel mass spectrometry (MS). For virtually all neurotoxic elapid snake venoms tested, we obtained bioactivity fingerprints showing major and minor bioactive zones containing masses consistent with three-finger toxins (3FTxs), whereas, viperid and colubrid venoms showed little or no detectable bioactivity. Our findings demonstrate that venom interactions with AChBP correlate with the severity of neurotoxicity observed following human envenoming by different snake species. We further, as proof of principle, characterized bioactive venom peptides from a viperid (Daboia russelli) and an elapid (Aspidelaps scutatus scutatus) snake by nanoLC-MS/MS, revealing that different toxin classes interact with the AChBP, and that this binding correlates with the inhibition of α7-nAChR in calcium-flux cell-based assays. The on-line post-column binding assay and subsequent toxin characterization methodologies described here provide a new in vitro analytic platform for rapidly investigating neurotoxic snake venom proteins., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cone snail venoms are considered a treasure trove of bioactive peptides. Despite over 800 species of cone snails being known, each producing over 1000 venom peptides, only about 150 unique venom peptides are structurally and functionally characterized. To overcome the limitations of the traditional low-throughput bio-discovery approaches, multi-omics systems approaches have been introduced to accelerate venom peptide discovery and characterisation. This "venomic" approach is starting to unravel the full complexity of cone snail venoms and to provide new insights into their biology and evolution. The main challenge for venomics is the effective integration of transcriptomics, proteomics, and pharmacological data and the efficient analysis of big datasets. Novel database search tools and visualisation techniques are now being introduced that facilitate data exploration, with ongoing advances in related omics fields being expected to further enhance venomics studies. Despite these challenges and future opportunities, cone snail venomics has already exponentially expanded the number of novel venom peptide sequences identified from the species investigated, although most novel conotoxins remain to be pharmacologically characterised. Therefore, efficient high-throughput peptide production systems and/or banks of miniaturized discovery assays are required to overcome this bottleneck and thus enhance cone snail venom bioprospecting and accelerate the identification of novel drug leads., Competing Interests: The authors declare no conflict of interest.

Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.

The adsorption mechanism for three peptides was studied under overloaded conditions through adsorption isotherm measurements in the presence of an ion-pairing reagent, trifluoroacetic acid (TFA), on an end-capped C 18 -bonded stationary phase. The overall aim of the study was to obtain a better understanding of how the acetonitrile and the TFA fractions in the eluent affected the overloaded elution profiles and the selectivity between peptides using mechanistic modelling and multivariate design of experiments. When studying the effect of TFA, direct evidence for ion pair formation between a peptide and TFA in acetonitrile-water solutions was provided by fluorine-proton nuclear Overhauser NMR enhancement experiments and the adsorption of TFA on the stationary phase was measured by frontal analysis. The adsorption isotherms for each peptide were then determined by the inverse method at eight TFA concentrations ranging from 2.6mM to 37.3mM (0.02-0.29vol-%) in isocratic elution. The equilibrium between the peptide ion and the peptide-TFA complex was modelled by coupling the mass-balance to reaction kinetics and determining separate adsorption isotherms for the two species. We found that a Langmuir isotherm described the elution profile of peptide-TFA complex well while the peptide ion was described by a bi-Langmuir adsorption isotherm since it exhibited strong secondary interactions. The elution profiles had an unfavorable shape at low TFA concentrations consisting of a spike in their front and a long tailing rear due to the secondary interactions for the peptide ion having very low saturation capacity. The acetonitrile dependence on the adsorption isotherms was studied by determination of adsorption isotherms directly from elution profiles obtained in gradient elution which enabled a broad acetonitrile interval to be studied. Here, it was found that the column saturation capacity was quickly reached at very low acetonitrile fractions and that there were significant variations in adsorption with the molecular weight. Finally, practical implications for method development are discussed based on an experimental design where gradient slope and TFA concentrations are used as factors., (Copyright © 2017. Published by Elsevier B.V.)

Voltage-gated sodium (Na V ) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal Na V channels inhibiting peak current of hNa V 1.1, rNa V 1.2, hNa V 1.6, and hNa V 1.7 while concurrently inhibiting fast inactivation of hNa V 1.1 and rNa V 1.3. The DII and DIV S3-S4 loops of Na V channel voltage sensors are important for the interaction of Pre1a with Na V channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between Na V channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a's potent and selective inhibitory effect on the fast inactivation process of Na V 1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of Na V channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.

Crotalphine is a structural analogue to a novel analgesic peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. Although crotalphine's analgesic effect is well established, its direct mechanism of action remains unresolved. The aim of the present study was to investigate the effect of crotalphine on ion channels in peripheral pain pathways. We found that picomolar concentrations of crotalphine selectively activate heterologously expressed and native TRPA1 ion channels. TRPA1 activation by crotalphine required intact N-terminal cysteine residues and was followed by strong and long-lasting desensitization of the channel. Homologous desensitization of recombinant TRPA1 and heterologous desensitization in cultured dorsal root ganglia neurons was observed. Likewise, crotalphine acted on peptidergic TRPA1-expressing nerve endings ex vivo as demonstrated by suppression of calcitonin gene-related peptide release from the trachea and in vivo by inhibition of chemically induced and inflammatory hypersensitivity in mice. The crotalphine-mediated desensitizing effect was abolished by the TRPA1 blocker HC030031 and absent in TRPA1-deficient mice. Taken together, these results suggest that crotalphine is the first peptide to mediate antinociception selectively and at subnanomolar concentrations by targeting TRPA1 ion channels.

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.

Selective activation of peripheral κ opioid receptors (KORs) may overcome the dose-limiting adverse effects of conventional opioid analgesics. We recently developed a vicinal disulfide-stabilized class of peptides with subnanomolar potency at the KOR. The aim of this study was to assess the analgesic effects of one of these peptides, named conorphin-1, in comparison with the prototypical KOR-selective small molecule agonist U-50488, in several rodent pain models. Surprisingly, neither conorphin-1 nor U-50488 were analgesic when delivered peripherally by intraplantar injection at local concentrations expected to fully activate the KOR at cutaneous nerve endings. While U-50488 was analgesic when delivered at high local concentrations, this effect could not be reversed by coadministration with the selective KOR antagonist ML190 or the nonselective opioid antagonist naloxone. Instead, U-50488 likely mediated its peripheral analgesic effect through nonselective inhibition of voltage-gated sodium channels, including peripheral sensory neuron isoforms NaV1.8 and NaV1.7. Our study suggests that targeting the KOR in peripheral sensory nerve endings innervating the skin is not an alternative analgesic approach.

Chemical synthesis of peptides can allow the option of sequential formation of multiple cysteines through exploitation of judiciously chosen regioselective thiol-protecting groups. We report the use of 2-nitroveratryl (oNv) as a new orthogonal group that can be cleaved by photolysis under ambient conditions. In combination with complementary S-pyridinesulfenyl activation, disulfide bonds are formed rapidly in situ. The preparation of Fmoc-Cys(oNv)-OH is described together with its use for the solid-phase synthesis of complex cystine-rich peptides, such as insulin., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ca(v)2.2 is a calcium channel subtype localized at nerve terminals, including nociceptive fibers, where it initiates neurotransmitter release. Ca(v)2.2 is an important contributor to synaptic transmission in ascending pain pathways, and is up-regulated in the spinal cord in chronic pain states along with the auxiliary α2δ1 subunit. It is therefore not surprising that toxins that inhibit Ca(v)2.2 are analgesic. Venomous animals, such as cone snails, spiders, snakes, assassin bugs, centipedes and scorpions are rich sources of remarkably potent and selective Ca(v)2.2 inhibitors. However, side effects in humans currently limit their clinical use. Here we review Ca(v)2.2 inhibitors from venoms and their potential as drug leads.

Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Conopeptides and conotoxins are small peptides produced by cone snails as a part of their predatory/defense strategies that target key ion channels and receptors in the nervous system. Some of these peptides also potently target mammalian ion channels involved in pain pathways. As a result, these venoms are a source of valuable pharmacological and therapeutic agents. The traditional approach towards conopeptide discovery relied on activity-guided fractionation, which is time consuming and resource-intensive. In this review, we discuss the advances in the fields of transcriptomics, proteomics and bioinformatics that now allow researchers to integrate these three platforms towards a more efficient discovery strategy. In this review, we also highlight the challenges associated with the wealth of data generated with this integrated approach and briefly discuss the impact these methods could have on the field of toxinology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

μ-Conotoxins are peptide blockers of voltage-gated sodium channels (sodium channels), inhibiting tetrodotoxin-sensitive neuronal (Na(v) 1.2) and skeletal (Na(v) 1.4) subtypes with highest affinity. Structure-activity relationship studies of μ-conotoxins SIIIA, TIIIA, and KIIIA have shown that it is mainly the C-terminal part of the three-loop peptide that is involved in binding to the sodium channel. In this study, we characterize the effect of N- and C-terminal extensions of μ-conotoxins SIIIA, SIIIB, and TIIIA on their potency and selectivity for neuronal versus muscle sodium channels. Interestingly, extending the N- or C-terminal of the peptide by introducing neutral, positive, and/or negatively charged residues, the selectivity of the native peptide can be altered from neuronal to skeletal and the other way around. The results from this study provide further insight into the binding profile of μ-conotoxins at voltage-gated sodium channels, revealing that binding interactions outside the cysteine-stablilized loops can contribute to μ-conotoxin affinity and sodium channel selectivity., (Copyright © 2012 Wiley Periodicals, Inc.)

Cone snails have evolved many 1000s of small, structurally stable venom peptides (conopeptides) for prey capture and defense. Whilst < 0.1% have been pharmacologically characterised, those with known function typically target membrane proteins of therapeutic importance, including ion channels, transporters and GPCRs. Several conopeptides reduce pain in animals models, with one in clinical development (χ-conopeptide analogue Xen2174) and one marketed (ω- conotoxin MVIIA or Prialt) for the treatment of severe pain. In addition to their therapeutic potential, conopeptides have been valuable probes for studying the role of a number of key membrane proteins in normal and disease physiology.

Venoms of snakes, scorpions, spiders, insects, sea anemones, and cone snails are complex mixtures of mostly peptides and small proteins that have evolved for prey capture and/or defense. These deadly animals have long fascinated scientists and the public. Early studies isolated lethal components in the search for cures and understanding of their mechanisms of action. Ion channels have emerged as targets for many venom peptides, providing researchers highly selective and potent molecular probes that have proved invaluable in unraveling ion channel structure and function. This minireview highlights molecular details of their toxin-receptor interactions and opportunities for development of peptide therapeutics.

Norepinephrine (NE) amplifies the strength of descending pain inhibition, giving inhibitors of spinal NET clinical utility in the management of pain. chi-MrIA isolated from the venom of a predatory marine snail noncompetitively inhibits NET and reverses allodynia in rat models of neuropathic pain. An analogue of chi-MrIA has been found to be a suitable drug candidate. On the basis of the NMR solution structure of this related peptide, Xen2174 (3), and structure-activity relationships of analogues, a pharmacophore model for the allosteric binding of 3 to NET is proposed. It is shown that 3 interacts with NET predominantly through amino acids in the first loop, forming a tight inverse turn presenting amino acids Tyr7, Lys8, and Leu9 in an orientation allowing for high affinity interaction with NET. The second loop interacts with a large hydrophobic pocket within the transporter. Analogues based on the pharmacophore demonstrated activities that support the proposed model. On the basis of improved chemical stability and a wide therapeutic index, 3 was selected for further development and is currently in phase II clinical trials.

The plant Momordica cochinchinensis has traditionally been used in Chinese medicine to treat a variety of illnesses. A range of bioactive molecules have been isolated from this plant, including peptides, which are the focus of this study. Here we report the isolation and characterization of two novel peptides, MCoCC-1 and MCoCC-2, containing 33 and 32 amino acids, respectively, which are toxic against three cancer cell lines. The two peptides are highly homologous to one another, but show no sequence similarity to known peptides. Elucidation of the three-dimensional structure of MCoCC-1 suggests the presence of a cystine knot motif, also found in a family of trypsin inhibitor peptides from this plant. However, unlike its structural counterparts, MCoCC-1 does not inhibit trypsin. MCoCC-1 has a well-defined structure, characterized mainly by a triple-stranded antiparallel beta-sheet, but unlike the majority of cystine knot proteins MCoCC-1 contains a disordered loop presumably as a result of flexibility in a localized region of the molecule. Of the cell lines tested, MCoCC-1 is the most toxic against a human melanoma cell line (MM96L) and is nonhemolytic to human erythrocytes. The role of these peptides within the plant remains to be determined.

Venom peptides offer enormous opportunity for the discovery of peptide drug leads. This review focusses on the potential of cone snails that have developed arrays of small peptides as part of highly evolved venoms used for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of these conopeptides reduce pain in animals models and several are now in preclinical and clinical development for the treatment of severe pain often associated with diseases such as cancer.

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V 1a receptor.

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a d-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture.

Xen2174 is a structural analogue of Mr1A, a chi-conopeptide recently isolated from the venom of the marine cone snail, Conus marmoreus. Although both chi-conopeptides are highly selective inhibitors of the norepinephrine transporter (NET), Xen2174 has superior chemical stability relative to Mr1A. It is well-known that tricyclic antidepressants (TCAs) are also potent NET inhibitors, but their poor selectivity relative to other monoamine transporters and various G-protein-coupled receptors, results in dose-limiting side-effects in vivo. As TCAs and the alpha(2)-adrenoceptor agonist, clonidine, have established efficacy for the relief of neuropathic pain, this study examined whether intrathecal (i.t.) Xen2174 alleviated mechanical allodynia in rats with either a chronic constriction injury of the sciatic nerve (CCI-rats) or an L5/L6 spinal-nerve injury. The anti-allodynic responses of i.t. Mr1A and i.t. morphine were also investigated in CCI-rats. Paw withdrawal thresholds were assessed using calibrated von Frey filaments. Bolus doses of i.t. Xen2174 produced dose-dependent relief of mechanical allodynia in CCI-rats and in spinal nerve-ligated rats. Dose-dependent anti-allodynic effects were also produced by i.t. bolus doses of Mr1A and morphine in CCI-rats, but a pronounced 'ceiling' effect was observed for i.t. morphine. The side-effect profiles were mild for both chi-conopeptides with an absence of sedation. Confirming the noradrenergic mechanism of action, i.t. co-administration of yohimbine (100 nmol) with Xen2174 (10 nmol) abolished Xen2174s anti-allodynic actions. Xen2174 appears to be a promising candidate for development as a novel therapeutic for i.t. administration to patients with persistent neuropathic pain.

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.; Schroder, C. I.; Kaye, D. M.; Adams, D. J.; Alewood, P. F.; Lewis, R. J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency., (Copyright 2005 Wiley Periodicals, Inc.)

The defensive use of cone snail venom is hypothesised to have first arisen in ancestral worm-hunting snails and later repurposed in a compartmentalised venom duct to facilitate the dietary shift to molluscivory and piscivory. Consistent with its placement in a basal lineage, we demonstrate that the C. distans venom gland lacked distinct compartmentalisation. Transcriptomics revealed C. distans expressed a wide range of structural classes, with inhibitory cysteine knot (ICK)-containing peptides dominating. To better understand the evolution of the venom gland compartmentalisation, we compared C. distans to C. planorbis, the earliest diverging species from which a defence-evoked venom has been obtained, and fish-hunting C. geographus from the Gastridium subgenus that injects distinct defensive and predatory venoms. These comparisons support the hypothesis that venom gland compartmentalisation arose in worm-hunting species and enabled repurposing of venom peptides to facilitate the dietary shift from vermivory to molluscivory and piscivory in more recently diverged cone snail lineages. [ABSTRACT FROM AUTHOR]

Given the important role of voltage-gated sodium (NaV) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant NaV channels, we screened the venom from Australian theraphosid species against the human pain target hNaV1.7. Using assay-guided fractionation, we isolated a 33-residue inhibitor cystine knot (ICK) peptide (Ssp1a) belonging to the NaSpTx1 family. Recombinant Ssp1a (rSsp1a) inhibited neuronal hNaV subtypes with a rank order of potency hNaV1.7 > 1.6 > 1.2 > 1.3 > 1.1. rSsp1a inhibited hNaV1.7, hNaV1.2 and hNaV1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNaV1.7 and rSsp1a-bound hNaV1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNaV1.7 to trap the channel in its resting state. Nuclear magnetic resonance spectroscopy revealed key structural features of Ssp1a, including an amphipathic surface with hydrophobic and charged patches shown by docking studies to comprise the interacting surface. This study provides the basis for future structure-function studies to guide the development of subtype selective inhibitors. [ABSTRACT FROM AUTHOR]

Spider venom-derived cysteine knot peptides are a mega-diverse class of molecules that exhibit unique pharmacological properties to modulate key membrane protein targets. Voltage-gated sodium channels (NaV) are often targeted by these peptides to allosterically promote opening or closing of the channel by binding to structural domains outside the channel pore. These effects can result in modified pain responses, muscle paralysis, cardiac arrest, priapism, and numbness. Although such effects are often deleterious, subtype selective spider venom peptides are showing potential to treat a range of neurological disorders, including chronic pain and epilepsy. This review examines the structure–activity relationships of cysteine knot peptides from spider venoms that modulate NaV and discusses their potential as leads to novel therapies for neurological disorders. [ABSTRACT FROM AUTHOR]

Venom-derived peptides have attracted much attention as potential lead molecules for pharmaceutical development. A well-known example is Huwentoxin-IV (HwTx-IV), a peptide toxin isolated from the venom of the Chinese bird-eating spider Haplopelma schmitdi. HwTx-IV was identified as a potent blocker of a human voltage-gated sodium channel (hNaV1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at NaV1.7 (IC50 ~26 nM) and selectivity over the cardiac NaV subtype (NaV1.5). Mutagenesis studies aimed at optimising the potency of the peptide resulted in the development of a triple-mutant of HwTx-IV (E1G, E4G, Y33W, m3-HwTx-IV) with significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. The activity of m3-HwTx-IV against other NaV subtypes was, however, not investigated. Similarly, the structure of the mutant peptide was not characterised, limiting the interpretation of the observed increase in potency. In this study we produced isotope-labelled recombinant m3-HwTx-IV in E. coli, which enabled us to characterise the atomic-resolution structure and dynamics of the peptide by NMR spectroscopy. The results show that the structure of the peptide is not perturbed by the mutations, whilst the relaxation studies reveal that residues in the active site of the peptide undergo conformational exchange. Additionally, the NaV subtype selectivity of the recombinant peptide was characterised, revealing potent inhibition of neuronal NaV subtypes 1.1, 1.2, 1.3, 1.6 and 1.7. In parallel to the in vitro studies, we investigated NaV1.7 target engagement of the peptide in vivo using a rodent pain model, where m3-HwTx-IV dose-dependently suppressed spontaneous pain induced by the NaV1.7 activator OD1. Thus, our results provide further insight into the structure and dynamics of this class of peptides that may prove useful in guiding the development of inhibitors with improved selectivity for analgesic NaV subtypes. [ABSTRACT FROM AUTHOR]

Transcriptome sequencing is now widely adopted as an efficient means to study the chemical diversity of venoms. To improve the efficiency of analysis of these large datasets, we have optimised an analysis pipeline for cone snail venom gland transcriptomes. The pipeline combines ConoSorter with sequence architecture-based elimination and similarity searching using BLAST to improve the accuracy of sequence identification and classification, while reducing requirements for manual intervention. As a proof-of-concept, we used this approach reanalysed three previously published cone snail transcriptomes from diverse dietary groups. Our pipeline method generated similar results to the published studies with significantly less manual intervention. We additionally found undiscovered sequences in the piscovorous Conus geographus and vermivorous Conus miles and identified sequences in incorrect superfamilies in the molluscivorus Conus marmoreus and C. geographus transcriptomes. Our results indicate that this method can improve toxin detection without extending analysis time. While this method was evaluated on cone snail transcriptomes it can be easily optimised to retrieve toxins from other venomous animals. [ABSTRACT FROM AUTHOR]

Background and Purpose Chronic pain is a serious worldwide health issue, with current analgesics having limited efficacy and dose-limiting side effects. Humans with loss-of-function mutations in the voltage-gated sodium channel NaV1.7 (h NaV1.7) are indifferent to pain, making h NaV1.7 a promising target for analgesic development. Since spider venoms are replete with NaV channel modulators, we examined their potential as a source of h NaV1.7 inhibitors. Experimental Approach We developed a high-throughput fluorescent-based assay to screen spider venoms against h NaV1.7 and isolate 'hit' peptides. To examine the binding site of these peptides, we constructed a panel of chimeric channels in which the S3b-S4 paddle motif from each voltage sensor domain of h NaV1.7 was transplanted into the homotetrameric KV2.1 channel. Key Results We screened 205 spider venoms and found that 40% contain at least one inhibitor of h NaV1.7. By deconvoluting 'hit' venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide μ-TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited h NaV1.7 with a high level of selectivity over all other subtypes, except h NaV1.1. We showed that Hd1a is a gating modifier that inhibits h NaV1.7 by interacting with the S3b- S4 paddle motif in channel domain II. The structure of Hd1a, determined using heteronuclear NMR, contains an inhibitor cystine knot motif that is likely to confer high levels of chemical, thermal and biological stability. Conclusion and Implications Our data indicate that spider venoms are a rich natural source of h NaV1.7 inhibitors that might be useful leads for the development of novel analgesics. [ABSTRACT FROM AUTHOR]

Chemical synthesis of peptides can allow the option of sequential formation of multiple cysteines through exploitation of judiciously chosen regioselective thiol-protecting groups. We report the use of 2-nitroveratryl (oNv) as a new orthogonal group that can be cleaved by photolysis under ambient conditions. In combination with complementary S-pyridinesulfenyl activation, disulfide bonds are formed rapidly in situ. The preparation of Fmoc-Cys(oNv)-OH is described together with its use for the solid-phase synthesis of complex cystine-rich peptides, such as insulin. [ABSTRACT FROM AUTHOR]

During selenate respiration by Thauera selenatis, the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed nO cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor A (SefA). and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichla coil, and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coil when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication. [ABSTRACT FROM AUTHOR]

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets α1-adrenoceptors (ρ-TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (χ-MrIA and χ-MrIB from the mollusk-hunting C. marmoreus). ρ-TIA and χ-MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs. [ABSTRACT FROM AUTHOR]

A novel conotoxin belonging to the ‘four-loop’ structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transl-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the ω-, δ- and κ-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins. [ABSTRACT FROM AUTHOR]

The interactions of χ-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [³H]norepinephrine (K[sub i] 1.89 µM) than [³H]dopamine (K[sub i] 4.33 µM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA (to 7 µM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [³H]norepinephrine uptake for three mutations (in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrlA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine. [ABSTRACT FROM AUTHOR]

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 1H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC50 = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC50 = 6.41 ± 0.09), whereas those of 2 (pIC50 = 5.96 ± 0.09) and 4 (pIC50 = 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═OB) in potencies. [ABSTRACT FROM AUTHOR]

Abstract: Conotoxins are highly constrained peptide toxins that exhibit pharmaceutically relevant biological activities. We herein report the extent of absorption and profile of distribution of a native α-conotoxin, MII and a lipophilic analogue of MII (N-LaaMII) after intravenous (iv) and oral administration to male Sprague–Dawley rats. N-LaaMII is formed by coupling 2-amino-d,l-dodecanoic acid (Laa) to the N-terminus of MII and has previously been shown to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII while maintaining the potency in inhibition of nAChRs of the parent peptide. Both peptides crossed the GI tract after oral administration (∼6% after 30m). While Laa conjugation did not significantly improve absorption, it did greatly increase the accumulation of the compound in the liver after iv administration. Neither peptide crossed the blood–brain barrier to any significant extent. This is the first study of the in vivo biodistribution of an α-conotoxin after oral administration. [Copyright &y& Elsevier]

Monoamine neurotransmitter transporters for norepinephrine (NE), dopamine and serotonin are important targets for antidepressants and analgesics. The conopeptide χ-MrIA is a noncompetitive and highly selective inhibitor of the NE transporter (NET) and is being developed as a novel intrathecal analgesic. We used site-directed mutagenesis to generate a suite of mutated transporters to identify two amino acids (Leu469 and Glu382) that affected the affinity of χ-MrIA to inhibit [³H]NE uptake through human NET. Residues that increased the Kd of a tricyclic antidepressant (nisoxetine) were also identified (Phe207, Ser225, His296, Thr381, and Asp473), Phe207, Ser225, His296, and Thr381 also affected the rate of NE transport without affecting NE Km. In a new model of NET constructed from the bLeuT crystal structure, χ-MrIA-interacting residues were located at the mouth of the transporter near residues affecting the binding of small molecule inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Non-peptide mimetics based on an anthranilamide ‘scaffold’ possessing fragments that mimic Lys2, Tyr13 and Arg17 in ω-conotoxin GVIA have been prepared. Compounds were assayed for binding to the voltage-gated calcium channels Cav2.2 (‘N-type’) and Cav2.1 (‘P/Q-type’) in rat brain. The primary synthetic target, 2-(6-amino-hexanoylamino)-5-(3-guanidino-propoxy)-N-[4-(4-hydroxyphenoxy)-phenyl]-benzamide (2a), exhibited low μM binding to Cav2.2 and was more than 30-fold selective for Cav2.2 over Cav2.1. [Copyright &y& Elsevier]

A benzothiazole-derived compound (4a) designed to mimic the Cα–Cβ bond vectors and terminal functionalities of Lys2, Tyr13 and Arg17 in ω-conotoxin GVIA was synthesised, together with analogues (4b–d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of ω-conotoxin. The fully functionalised mimetic (4a) showed low μM binding affinity to N-type VGCCs (IC50=1.9 μM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC50= 4.1 μM) showed a greater than 25-fold selectivity for the N-type channel. [Copyright &y& Elsevier]

χ-Conopeptide MrIA (χ-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the notepinephrine transporter (NET). We investigated whether χ-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 µM) on the activity of the dopamine transporter and the seretonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [³H]nisoxetine and [³H]mazindol, to the expressed rat and human NET was inhibited by χ-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by χ-MrIA was competitive in nature. It has previously been demonstrated that χ-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for χ-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of χ-MrIA was found to be dependent on Na[sup +] with the conopeptide becoming a less effective blocker of [³H]norepinephrine by the NET under the conditions of reduced extracellular Na[sup +]. In this respect, χ-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of χ-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of χ-MrIA and a His in loop 2 played a dominant role in the interaction between χ-MrIA and the NET. Hα chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of χ-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET. [ABSTRACT FROM AUTHOR]

A peptide contained in the venom of the predatory marine snail Conus tulipa, ρ-TIA, has previously been shown to possess α[sub 1]-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, ρ-TIA inhibited α[sub 1]-adrenoreceptor-mediated increases in cytosolic Ca[sup 2+] concentration that were triggered by norepinephrine, but did not affect presynaptic α[sub 2]-adrenoreceptor-mediated responses. In radio-ligand binding assays using [[sup 125]I]HEAT, ρ-TIA displayed slightly greater potency at the α[sub 1B] than at the α[sub 1A] or α[sub 1D] subtypes. Moreover, although it did not affect the rate of association for [³H]prazosin binding to the α[sub 1B]adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by ρ-TIA. N-terminally truncated analogs of ρ-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of ρ-TIA (Arg[sup 4]). An alanine walk of ρ-TIA confirmed the importance of Arg[sup 4] for activity and revealed a number of other residues clustered around Arg[sup 4] that contribute to the potency of ρ-TIA. The unique allosteric antagonism of ρ-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive α[sub 1]-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective. [ABSTRACT FROM AUTHOR]