Critical residue properties for potency and selectivity of α-Conotoxin RgIA towards α9α10 nicotinic acetylcholine receptors

The α9α10 nicotinic acetylcholine receptor (nAChR) has been characterized as an effective anti-pain target that functions through a non-opioid mechanism. However, as a pentameric ion channel comprised of two different subunits, the specific targeting of α9α10 nAChRs has proven challenging. Previously the 13-amino-acid peptide, RgIA, was shown to block α9α10 nAChRs with high potency and specificity. This peptide, characterized from the venom of the carnivorous marine snail, Conus regius, produced analgesia in several rodent models of chronic pain. Despite promising pre-clinical data in behavioral assays, the number of specific α9α10 nAChR antagonists remains small and the physiological mechanisms of analgesia remain cryptic. In this study, we implement amino-acid substitutions to definitively characterize the chemical properties of RgIA that contribute to its activity against α9α10 nAChRs. Using this mutational approach, we determined the vital role of biochemical side-chain properties and amino acids in the second loop that are amenable to substitutions to further engineer next-generation analogs for the blockade of α9α10 nAChRs

Dual activity lysophosphatidic acid receptor pan-antagonist/autotaxin inhibitor reduces breast cancer cell migration in vitro and causes tumor regression in vivo

Signal transduction modifiers that modulate the lysophosphatidic acid (LPA) pathway have potential as anticancer agents. Herein, we describe metabolically stabilized LPA analogues that reduce cell migration and invasion and cause regression of orthotopic breast tumors in vivo. Two diastereoisomeric α-bromophosphonates (BrP-LPA) were synthesized, and the pharmacology was determined for five LPA G protein-coupled receptors (GPCRs). The syn and anti diastereomers of BrP-LPA are pan-LPA GPCR antagonists and are also nanomolar inhibitors of the lysophospholipase D activity of autotaxin, the dominant biosynthetic source of LPA. Computational models correctly predicted the diastereoselectivity of antagonism for three GPCR isoforms. The anti isomer of BrP-LPA was more effective than syn isomer in reducing migration of MDA-MB-231 cells, and the anti isomer was superior in reducing invasion of these cells. Finally, orthotopic breast cancer xenografts were established in nude mice by injection of MB-231 cells in an in situ cross-linkable extracellular matrix. After 2 weeks, mice were treated with the BrP-LPA alone (10 mg/kg), Taxol alone (10 mg/kg), or Taxol followed by BrP-LPA. All treatments significantly reduced tumor burden, and BrP-LPA was superior to Taxol in reducing blood vessel density in tumors. Moreover, both the anti- and syn-BrP-LPA significantly reduced tumors at 3 mg/kg. ©2009 American Association for Cancer Research

Conopeptides promote itch through human itch receptor hMgprX1

Members of Mas related G-protein coupled receptors (Mrgpr) are known to mediate itch. To date, several compounds have been shown to activate these receptors, including chloroquine, a common antimalarial drug, and peptides of the RF-amide family. However, specific ligands for these receptors are still lacking and there is a need for novel compounds that can be used to modulate the receptors in order to understand the cellular and molecular mechanism in which they mediate itch. Some cone snail venoms were previously shown to induce itch in mice. Here, we show that the venom of Conus textile induces itch through activation of itch-sensing sensory neurons, marked by their sensitivity to chloroquine. Two RF-amide peptides, CNF-Tx1 and CNF-Tx2, were identified in a C. textile venom gland transcriptome. These belong to the conorfamide family of peptides which includes previously described peptides from the venoms of Conus victoriae (CNF-Vc1) and Conus spurius (CNF-Sr1 and CNF-Sr2). We show that CNF-Vc1 and CNF-Sr1 activate MrgprC11 whereas CNF-Vc1 and CNF-Tx2 activate the human MrgprX1 (hMrgprX1). The peptides CNF-Tx1 and CNF-Sr2 do not activate MrgprC11 or hMrgprX1. Intradermal injection of CNF-Vc1 and CNF-Tx2 into the cheek of a transgenic mouse expressing hMrgprX1 instead of endogenous mouse Mrgprs resulted in itch-related scratching thus demonstrating the in vivo activity of these peptides. Using truncated analogues of CNF-Vc1, we identified amino acids at positions 7-18 as important for activity against hMrgprX1. The conopeptides reported here are tools that can be used to advance our understanding of the cellular and molecular mechanism of itch mediated by Mrgprs

Probing the Redox States of Sodium Channel Cysteines at the Binding Site of μO§-Conotoxin GVIIJ

μO§-Conotoxin GVIIJ is a 35-amino acid peptide that readily blocks six of eight tested Na_V1 subunit isoforms of voltage-gated sodium channels. μO§-GVIIJ is unusual in having an S-cysteinylated cysteine (at residue 24). A proposed reaction scheme involves the peptide–channel complex stabilized by a disulfide bond formed via thiol–disulfide exchange between Cys24 of the peptide and a Cys residue at neurotoxin receptor site 8 in the pore module of the channel (specifically, Cys910 of rat Na_V1.2). To examine this model, we synthesized seven derivatives of μO§-GVIIJ in which Cys24 was disulfide-bonded to various thiols (or SR groups) and tested them on voltage-clamped <i>Xenopus laevis</i> oocytes expressing Na_V1.2. In the proposed model, the SR moiety is a leaving group that is no longer present in the final peptide–channel complex; thus, the same <i>k</i>_off value should be obtained regardless of the SR group. We observed that all seven derivatives, whose <i>k</i>_on values varied over a 30-fold range, had the same <i>k</i>_off value. Concordant results were observed with Na_V1.6, for which the <i>k</i>_off was 17-fold larger. Additionally, we tested two μO§-GVIIJ derivatives (where SR was glutathione or a free thiol) on two Na_V1.2 Cys replacement mutants (Na_V1.2­[C912A] and Na_V1.2­[C918A]) without and with reduction of channel disulfides by dithiothreitol. The results indicate that Cys910 in wild-type Na_V1.2 has a free thiol and conversely suggest that in Na_V1.2­[C912A] and Na_V1.2­[C918A], Cys910 is disulfide-bonded to Cys918 and Cys912, respectively. Redox states of extracellular cysteines of sodium channels have hitherto received scant attention, and further experiments with GVIIJ may help fill this void

Hormone-like peptides in the venoms of marine cone snails

The venoms of cone snails (genus Conus) are remarkably complex, consisting of hundreds of typically short, disulfide-rich peptides termed conotoxins. These peptides have diverse pharmacological targets, with injection of venom eliciting a range of physiological responses, including sedation, paralysis and sensory overload. Most conotoxins target the prey's nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules in the venoms of cone snails we systematically mined the venom gland transcriptomes of several cone snail species and examined secreted venom peptides in dissected and injected venom of the Australian cone snail Conus victoriae. Using this approach we identified several novel hormone/neuropeptide-like toxins, including peptides similar to the bee brain hormone prohormone-4, the mollusc ganglia neuropeptide elevenin, and thyrostimulin, a member of the glycoprotein hormone family, and confirmed the presence of insulin. We confirmed that at least two of these peptides are not only expressed in the venom gland but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers an exciting new avenue for the discovery of novel pharmacological tools and drug candidates, complementary to conotoxins