Publications by authors named "Baldomero M Olivera"

220 Publications

Neuroactive Type-A γ-Aminobutyric Acid Receptor Allosteric Modulator Steroids from the Hypobranchial Gland of Marine Mollusk, .

J Med Chem 2021 May 5. Epub 2021 May 5.

Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.

In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A-E (-), from the hypobranchial gland of the mollusk . Compounds - were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue was used for a detailed pharmacological study. Compound significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABARs). In a patch-clamp experiment, out of the six subunit combinations tested, exhibited subtype selectivity, most strongly antagonizing αβγ and αβγ receptors (IC 1.5 and 1.0 μM, respectively). Although the structures of - differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABARs, expanding the known chemical space of neuroactive steroids.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00562DOI Listing
May 2021

Cannabinoid receptor agonists from Conus venoms alleviate pain-related behavior in rats.

Pharmacol Biochem Behav 2021 Mar 25;205:173182. Epub 2021 Mar 25.

University of Miami, Miller School of Medicine, Miami Project, 1095 NW 14(th) terrace, Miami, FL 33136, USA.

Cannabinoid (CB) receptor agonists show robust antinociceptive effects in various pain models. However, most of the clinically potent CB1 receptor-active drugs derived from cannabis are considered concerning due to psychotomimetic side effects. Selective CB receptor ligands that do not induce CNS side effects are of clinical interest. The venoms of marine snail Conus are a natural source of various potent analgesic peptides, some of which are already FDA approved. In this study we evaluated the ability of several Conus venom extracts to interact with CB1 receptor. HEK293 cells expressing CB1 receptors were treated with venom extracts and CB1 receptor internalization was analyzed by immunofluorescence. Results showed C. textile (C. Tex) and C. miles (C. Mil) samples as the most potent. These were serially subfractionated by HPLC for subsequent analysis by internalization assays and for analgesic potency evaluated in the formalin test and after peripheral nerve injury. Intrathecal injection of C. Tex and C. Mil subfractions reduced flinching/licking behavior during the second phase of formalin test and attenuated thermal and mechanical allodynia in nerve injury model. Treatment with proteolytic enzymes reduced CB1 internalization of subfractions, indicating the peptidergic nature of CB1 active component. Further HPLC purification revealed two potent antinociceptive subfractions within C. Tex with CB1 and possible CB2 activity, with mild to no side effects in the CB tetrad assessment. CB conopeptides can be isolated from these active Conus venom-derived samples and further developed as novel analgesic agents for the treatment of chronic pain using cell based or gene therapy approaches.
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http://dx.doi.org/10.1016/j.pbb.2021.173182DOI Listing
March 2021

Discovery of a Potent Conorfamide from Using a Novel Zebrafish Larvae Assay.

J Nat Prod 2021 Apr 25;84(4):1232-1243. Epub 2021 Mar 25.

School of Biological Sciences, University of Utah, 257 S 1400 E, Salt Lake City, Utah 84112, United States.

Natural products such as conotoxins have tremendous potential as tools for biomedical research and for the treatment of different human diseases. Conotoxins are peptides present in the venoms of predatory cone snails that have a rich diversity of pharmacological functions. One of the major bottlenecks in natural products research is the rapid identification and evaluation of bioactive molecules. To overcome this limitation, we designed a set of light-induced behavioral assays in zebrafish larvae to screen for bioactive conotoxins. We used this screening approach to test several unique conotoxins derived from different cone snail clades and discovered that a conorfamide from , CNF-Ep1, had the most dramatic alterations in the locomotor behavior of zebrafish larvae. Interestingly, CNF-Ep1 is also bioactive in several mouse assay systems when tested and . Our novel screening platform can thus accelerate the identification of bioactive marine natural products, and the first compound discovered using this assay has intriguing properties that may uncover novel neuronal circuitry.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01297DOI Listing
April 2021

Small-molecule mimicry hunting strategy in the imperial cone snail, .

Sci Adv 2021 Mar 12;7(11). Epub 2021 Mar 12.

Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT 84112, USA.

Venomous animals hunt using bioactive peptides, but relatively little is known about venom small molecules and the resulting complex hunting behaviors. Here, we explored the specialized metabolites from the venom of the worm-hunting cone snail, Using the model polychaete worm , we demonstrate that venom contains small molecules that mimic natural polychaete mating pheromones, evoking the mating phenotype in worms. The specialized metabolites from different cone snails are species-specific and structurally diverse, suggesting that the cones may adopt many different prey-hunting strategies enabled by small molecules. Predators sometimes attract prey using the prey's own pheromones, in a strategy known as aggressive mimicry. Instead, uses metabolically stable mimics of those pheromones, indicating that, in biological mimicry, even the molecules themselves may be disguised, providing a twist on fake news in chemical ecology.
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http://dx.doi.org/10.1126/sciadv.abf2704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954447PMC
March 2021

A Serendipitous Path to Pharmacology.

Annu Rev Pharmacol Toxicol 2021 01;61:9-23

School of Biological Sciences, University of Utah, Salt Lake City, Utah 84112, USA; email:

My path to research in neuropharmacology has been a coalescing of my training as a molecular biologist and my intense interest in an esoteric group of animals, the fish-hunting cone snails. Attempting to bridge these two disparate worlds has led me to an idiosyncratic career as a pharmacologist.
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http://dx.doi.org/10.1146/annurev-pharmtox-030320-113510DOI Listing
January 2021

Chronicling changes in the somatosensory neurons after peripheral nerve injury.

Proc Natl Acad Sci U S A 2020 10 5;117(42):26414-26421. Epub 2020 Oct 5.

School of Biological Sciences, University of Utah, Salt Lake City, UT 84112;

Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain.
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http://dx.doi.org/10.1073/pnas.1922618117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584894PMC
October 2020

Purification and Characterization of the Pink-Floyd Drillipeptide, a Bioactive Venom Peptide from (Gastropoda: Conoidea: Drilliidae).

Toxins (Basel) 2020 08 7;12(8). Epub 2020 Aug 7.

The Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines.

The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, >20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.
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http://dx.doi.org/10.3390/toxins12080508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472735PMC
August 2020

Integration of the pSLT Plasmid into the Chromosome Results in a Temperature-Sensitive Growth Defect Due to Aberrant DNA Replication.

J Bacteriol 2020 09 23;202(20). Epub 2020 Sep 23.

School of Biological Sciences, University of Utah, Salt Lake City, Utah, USA

A mutant of serovar Typhimurium was isolated that simultaneously affected two metabolic pathways as follows: NAD metabolism and DNA repair. The mutant was isolated as resistant to a nicotinamide analog and as temperature-sensitive for growth on minimal glucose medium. In this mutant, 's 94-kb virulence plasmid pSLT had recombined into the chromosome upstream of the NAD salvage pathway gene This insertion blocked most transcription of , which reduced uptake of the nicotinamide analog. The pSLT insertion mutant also exhibited phenotypes associated with induction of the SOS DNA repair system, including an increase in filamentous cells, higher exonuclease III and catalase activities, and derepression of SOS gene expression. Genome sequencing revealed increased read coverage extending out from the site of pSLT insertion. The two pSLT replication origins are likely initiating replication of the chromosome near the normal replication terminus. Too much replication initiation at the wrong site is probably causing the observed growth defects. Accordingly, deletion of both pSLT replication origins restored growth at higher temperatures. In studies that insert a second replication origin into the chromosome, both origins are typically active at the same time. In contrast, the integrated pSLT plasmid initiated replication in stationary phase after normal chromosomal replication had finished. The gradient in read coverage extending out from a single site could be a simple but powerful tool for studying replication and detecting chromosomal rearrangements. This technique may be of particular value when a genome has been sequenced for the first time to verify correct assembly.
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http://dx.doi.org/10.1128/JB.00380-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515242PMC
September 2020

Curses or Cures: A Review of the Numerous Benefits Versus the Biosecurity Concerns of Conotoxin Research.

Biomedicines 2020 Jul 22;8(8). Epub 2020 Jul 22.

Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.

Conotoxins form a diverse group of peptide toxins found in the venom of predatory marine cone snails. Decades of conotoxin research have provided numerous measurable scientific and societal benefits. These include their use as a drug, diagnostic agent, drug leads, and research tools in neuroscience, pharmacology, biochemistry, structural biology, and molecular evolution. Human envenomations by cone snails are rare but can be fatal. Death by envenomation is likely caused by a small set of toxins that induce muscle paralysis of the diaphragm, resulting in respiratory arrest. The potency of these toxins led to concerns regarding the potential development and use of conotoxins as biological weapons. To address this, various regulatory measures have been introduced that limit the use and access of conotoxins within the research community. Some of these regulations apply to all of the ≈200,000 conotoxins predicted to exist in nature of which less than 0.05% are estimated to have any significant toxicity in humans. In this review we provide an overview of the many benefits of conotoxin research, and contrast these to the perceived biosecurity concerns of conotoxins and research thereof.
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http://dx.doi.org/10.3390/biomedicines8080235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460000PMC
July 2020

Transcriptomic Profiling Reveals Extraordinary Diversity of Venom Peptides in Unexplored Predatory Gastropods of the Genus Clavus.

Genome Biol Evol 2020 05;12(5):684-700

A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Science, Moscow, Russia.

Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration.
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http://dx.doi.org/10.1093/gbe/evaa083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259678PMC
May 2020

αM-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish.

Toxins (Basel) 2020 03 21;12(3). Epub 2020 Mar 21.

Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail . Injections of αM-MIIIJ induced paralysis in goldfish () but not mice. Intracellular recording from skeletal muscles of fish () and frog () revealed that αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC of ~0.1 μM. With comparable potency, αM-MIIIJ reversibly blocked ACh-gated currents (I) of voltage-clamped oocytes exogenously expressing nAChRs cloned from zebrafish () muscle. αM-MIIIJ also protected against slowly-reversible block of I by α-bungarotoxin (α-BgTX, a snake neurotoxin) and α-conotoxin EI (α-EI, from another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that αM-MIIIJ inhibited the binding of fluorescently-tagged α-BgTX at neuromuscular junctions of , and . (Note, we observed that αM-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in oocytes, but with ICs ~100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that αM-MIIIJ inhibits muscle nAChRs and furthermore apparently does so by interfering with the binding of ACh to its receptor. Comparative alignments with homologous sequences identified in other fish hunters revealed that αM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.
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http://dx.doi.org/10.3390/toxins12030197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150935PMC
March 2020

Boholamide A, an APD-Class, Hypoxia-Selective Cyclodepsipeptide.

J Nat Prod 2020 04 18;83(4):1249-1257. Epub 2020 Mar 18.

Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.

Calcium homeostasis is implicated in some cancers, leading to the possibility that selective control of calcium might lead to new cancer drugs. On the basis of this idea, we designed an assay using a glioblastoma cell line and screened a collection of 1000 unique bacterial extracts. Isolation of the active compound from a hit extract led to the identification of boholamide A (), a 4-amido-2,4-pentadieneoate (APD)-class peptide. Boholamide A () applied in the nanomolar range induces an immediate influx of Ca in glioblastoma and neuronal cells. APD-class natural products are hypoxia-selective cytotoxins that primarily target mitochondria. Like other APD-containing compounds, is hypoxia selective. Since APD natural products have received significant interest as potential chemotherapeutic agents, provides a novel APD scaffold for the development of new anticancer compounds.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00038DOI Listing
April 2020

An integrative approach to the facile functional classification of dorsal root ganglion neuronal subclasses.

Proc Natl Acad Sci U S A 2020 03 20;117(10):5494-5501. Epub 2020 Feb 20.

School of Biological Sciences, University of Utah, Salt Lake City, UT 841120;

Somatosensory neurons have historically been classified by a variety of approaches, including structural, anatomical, and genetic markers; electrophysiological properties; pharmacological sensitivities; and more recently, transcriptional profile differentiation. These methodologies, used separately, have yielded inconsistent classification schemes. Here, we describe phenotypic differences in response to pharmacological agents as measured by changes in cytosolic calcium concentration for the rapid classification of neurons in vitro; further analysis with genetic markers, whole-cell recordings, and single-cell transcriptomics validated these findings in a functional context. Using this general approach, which we refer to as tripartite constellation analysis (TCA), we focused on large-diameter dorsal-root ganglion (L-DRG) neurons with myelinated axons. Divergent responses to the K-channel antagonist, κM-conopeptide RIIIJ (RIIIJ), reliably identified six discrete functional cell classes. In two neuronal subclasses (L1 and L2), block with RIIIJ led to an increase in [Ca] Simultaneous electrophysiology and calcium imaging showed that the RIIIJ-elicited increase in [Ca] corresponded to different patterns of action potentials (APs), a train of APs in L1 neurons, and sporadic firing in L2 neurons. Genetically labeled mice established that L1 neurons are proprioceptors. The single-cell transcriptomes of L1 and L2 neurons showed that L2 neurons are Aδ-low-threshold mechanoreceptors. RIIIJ effects were replicated by application of the K1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional K1.1/K1.2 heteromeric channels. Using this approach on other neuronal subclasses should ultimately accelerate the comprehensive classification and characterization of individual somatosensory neuronal subclasses within a mixed population.
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http://dx.doi.org/10.1073/pnas.1911382117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071849PMC
March 2020

Characterization of the First Conotoxin from , a Vermivorous Cone Snail from the Cabo Verde Archipelago.

Mar Drugs 2019 Jul 24;17(8). Epub 2019 Jul 24.

Department of Biology, University of Utah, 257 S 1400 E, Salt Lake City, UT 84112, USA.

is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting . In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.
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http://dx.doi.org/10.3390/md17080432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723684PMC
July 2019

α-Conotoxin VnIB from Conus ventricosus is a potent and selective antagonist of α6β4* nicotinic acetylcholine receptors.

Neuropharmacology 2019 10 28;157:107691. Epub 2019 Jun 28.

Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA; Department of Psychiatry, University of Utah, Salt Lake City, UT, 84108, USA; George E. Wahlen Veterans Affair Medical Center, Salt Lake City, UT, 84148, USA. Electronic address:

α6-containing (α6*) nicotinic acetylcholine receptors (nAChRs) are expressed throughout the periphery and the central nervous system and constitute putative therapeutic targets in pain, addiction and movement disorders. The α6β2* nAChRs are relatively well studied, in part due to the availability of target specific α-conotoxins (α-Ctxs). In contrast, all native α-Ctxs identified that potently block α6β4 nAChRs exhibit higher potencies for the closely related α6β2β3 and/or α3β4 subtypes. In this study, we have identified a novel peptide from Conus ventricosus with pronounced selectivity for the α6β4 nAChR. The peptide-encoding gene was cloned from genomic DNA and the predicted mature peptide, α-Ctx VnIB, was synthesized. The functional properties of VnIB were characterized at rat and human nAChRs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. VnIB potently inhibited ACh-evoked currents at rα6β4 and rα6/α3β4 nAChRs, displayed ∼20-fold and ∼250-fold lower potencies at rα3β4 and rα6/α3β2β3 receptors, respectively, and exhibited negligible effects at eight other nAChR subtypes. Interestingly, even higher degrees of selectivity were observed for hα6/α3β4 over hα6/α3β2β3 and hα3β4 receptors. Finally, VnIB displayed fast binding kinetics at rα6/α3β4 (on-rate t = 0.87 min, off-rate t = 2.7 min). The overall preference of VnIB for β4* over β2* nAChRs is similar to the selectivity profiles of other 4/6 α-Ctxs. However, in contrast to previously identified native α-Ctxs targeting α6* nAChRs, VnIB displays pronounced selectivity for α6β4 nAChRs over both α3β4 and α6β2β3 receptors. VnIB thus represents a novel molecular probe for elucidating the physiological role and therapeutic properties of α6β4* nAChRs.
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http://dx.doi.org/10.1016/j.neuropharm.2019.107691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693646PMC
October 2019

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework.

J Biol Chem 2019 05 11;294(22):8745-8759. Epub 2019 Apr 11.

From the Department of Biology, Linderstrøm-Lang Centre for Protein Science, University of Copenhagen, 2200 Copenhagen N., Denmark,

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from , a peptide with a VI/VII cysteine framework. This framework has Cys-Cys/Cys-Cys/Cys-Cys connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked β-hairpins with opposing β-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.
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http://dx.doi.org/10.1074/jbc.RA119.007491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552430PMC
May 2019

-Anethole of Fennel Oil is a Selective and Nonelectrophilic Agonist of the TRPA1 Ion Channel.

Mol Pharmacol 2019 04 24;95(4):433-441. Epub 2019 Jan 24.

Departments of Biology (T.M., B.M.O., R.W.T.), Pharmacology and Toxicology (T.M., C.A.R.), and Ophthalmology and Visual Sciences (O.Y., D.K.), University of Utah, Salt Lake City, Utah.

Transient receptor potential (TRP) cation channels are molecular targets of various natural products. TRPA1, a member of TRP channel family, is specifically activated by natural products such as allyl isothiocyanate (mustard oil), cinnamaldehyde (cinnamon), and allicin (garlic). In this study, we demonstrated that TRPA1 is also a target of -anethole in fennel oil (FO) and fennel seed extract. Similar to FO, -anethole selectively elicited calcium influx in TRPA1-expressing mouse sensory neurons of the dorsal root and trigeminal ganglia. These FO- and anethole-induced calcium responses were blocked by a selective TRPA1 channel antagonist, HC-030031. Moreover, both FO and -anethole induced calcium influx and transmembrane currents in HEK293 cells stably overexpressing human TRPA1 channels, but not in regular HEK293 cells. Mutation of the amino acids S873 and T874 binding site of human TRPA1 significantly attenuated channel activation by -anethole, whereas pretreating with glutathione, a nucleophile, did not. Conversely, activation of TRPA1 by the electrophile allyl isothiocyanate was abolished by glutathione, but was ostensibly unaffected by mutation of the ST binding site. Finally, it was found that -anethole was capable of desensitizing TRPA1, and unlike allyl isothiocyanate, it failed to induce nocifensive behaviors in mice. We conclude that -anethole is a selective, nonelectrophilic, and seemingly less-irritating agonist of TRPA1.
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http://dx.doi.org/10.1124/mol.118.114561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408737PMC
April 2019

Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric K1 channels.

Proc Natl Acad Sci U S A 2019 01 28;116(3):1059-1064. Epub 2018 Dec 28.

Institute of Physiology, Christian-Albrechts-University Kiel, 24118 Kiel, Germany;

The vast complexity of native heteromeric K channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from precisely targets "asymmetric" K channels composed of three K1.2 subunits and one K1.1 or K1.6 subunit with 100-fold higher apparent affinity compared with homomeric K1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional K1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative K1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by () covalent linkage of members of the mammalian Shaker-related K1 family to K1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K channel-mediated currents in heterologous expression systems; () molecular dynamics simulations of asymmetric K1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and () evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric K channel complexes that operate in native tissues.
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http://dx.doi.org/10.1073/pnas.1813161116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338859PMC
January 2019

Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins.

Int J Mol Sci 2018 Oct 31;19(11). Epub 2018 Oct 31.

Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 ( Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.
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http://dx.doi.org/10.3390/ijms19113418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275033PMC
October 2018

Conopeptides promote itch through human itch receptor hMgprX1.

Toxicon 2018 Nov 21;154:28-34. Epub 2018 Sep 21.

Department of Anesthesiology and Center for the Study of Itch, Washington University School of Medicine, St. Louis MO 63110, USA. Electronic address:

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 7-14 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.
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http://dx.doi.org/10.1016/j.toxicon.2018.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299835PMC
November 2018

Pain therapeutics from cone snail venoms: From Ziconotide to novel non-opioid pathways.

J Proteomics 2019 01 16;190:12-20. Epub 2018 May 16.

Departments of Biology, University of Utah, Salt Lake City, UT, United States.

There have been numerous attempts to develop non-opioid drugs for severe pain, but the vast majority of these efforts have failed. A notable exception is Ziconotide (Prialt®), approved by the FDA in 2004. In this review, we summarize the present status of Ziconotide as a therapeutic drug and introduce a wider framework: the potential of venom peptides from cone snails as a resource providing a continuous pipeline for the discovery of non-opioid pain therapeutics. An auxiliary theme that we hope to develop is that these venoms, already a validated starting point for non-opioid drug leads, should also provide an opportunity for identifying novel molecular targets for future pain drugs. This review comprises several sections: the first focuses on Ziconotide as a therapeutic (including a historical retrospective and a clinical perspective); followed by sections on other promising Conus venom peptides that are either in clinical or pre-clinical development. We conclude with a discussion on why the outlook for discovery appears exceptionally promising. The combination of new technologies in diverse fields, including the development of novel high-content assays and revolutionary advancements in transcriptomics and proteomics, puts us at the cusp of providing a continuous pipeline of non-opioid drug innovations for pain. SIGNIFICANCE: The current opioid epidemic is the deadliest drug crisis in American history. Thus, this review on the discovery of non-opioid pain therapeutics and pathways from cone snail venoms is significant and timely.
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http://dx.doi.org/10.1016/j.jprot.2018.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214764PMC
January 2019

Structure and Biological Activity of a Turripeptide from Unedogemmula bisaya Venom.

Biochemistry 2017 11 1;56(45):6051-6060. Epub 2017 Nov 1.

Marine Science Institute, University of the Philippines , P. Velasquez Street, Diliman, Quezon City 1101, Philippines.

The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC-C-C-CC) in the M-superfamily of conopeptides. The three-dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C-C C-C, C-C. The peptide inhibited the activity of the α9α10 nicotinic acetylcholine receptor with relatively low affinity (IC, 10.2 μM). Initial Constellation Pharmacology data revealed an excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons.
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http://dx.doi.org/10.1021/acs.biochem.7b00485DOI Listing
November 2017

Stenotrophomonas-Like Bacteria Are Widespread Symbionts in Cone Snail Venom Ducts.

Appl Environ Microbiol 2017 Dec 16;83(23). Epub 2017 Nov 16.

Department of Medicinal Chemistry, L.S. Skaggs Pharmacy Institute, University of Utah, Salt Lake City, Utah, USA

Cone snails are biomedically important sources of peptide drugs, but it is not known whether snail-associated bacteria affect venom chemistry. To begin to answer this question, we performed 16S rRNA gene amplicon sequencing of eight cone snail species, comparing their microbiomes with each other and with those from a variety of other marine invertebrates. We show that the cone snail microbiome is distinct from those in other marine invertebrates and conserved in specimens from around the world, including the Philippines, Guam, California, and Florida. We found that all venom ducts examined contain diverse 16S rRNA gene sequences bearing closest similarity to bacteria. These sequences represent specific symbionts that live in the lumen of the venom duct, where bioactive venom peptides are synthesized. In animals, symbiotic bacteria contribute critically to metabolism. Cone snails are renowned for the production of venoms that are used as medicines and as probes for biological study. In principle, symbiotic bacterial metabolism could either degrade or synthesize active venom components, and previous publications show that bacteria do indeed contribute small molecules to some venoms. Therefore, understanding symbiosis in cone snails will contribute to further drug discovery efforts. Here, we describe an unexpected, specific symbiosis between bacteria and cone snails from around the world.
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http://dx.doi.org/10.1128/AEM.01418-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691409PMC
December 2017

Divergence of the Venom Exogene Repertoire in Two Sister Species of Turriconus.

Genome Biol Evol 2017 09;9(9):2211-2225

Department of Biology, University of Utah.

The genus Conus comprises approximately 700 species of venomous marine cone snails that are highly efficient predators of worms, snails, and fish. In evolutionary terms, cone snails are relatively young with the earliest fossil records occurring in the Lower Eocene, 55 Ma. The rapid radiation of cone snail species has been accompanied by remarkably high rates of toxin diversification. To shed light on the molecular mechanisms that accompany speciation, we investigated the toxin repertoire of two sister species, Conus andremenezi and Conus praecellens, that were until recently considered a single variable species. A total of 196 and 250 toxin sequences were identified in the venom gland transcriptomes of C. andremenezi and C. praecellens belonging to 25 and 29 putative toxin gene superfamilies, respectively. Comparative analysis with closely (Conus tribblei and Conus lenavati) and more distantly related species (Conus geographus) suggests that speciation is associated with significant diversification of individual toxin genes (exogenes) whereas the expression pattern of toxin gene superfamilies within lineages remains largely conserved. Thus, changes within individual toxin sequences can serve as a sensitive indicator for recent speciation whereas changes in the expression pattern of gene superfamilies are likely to reflect more dramatic differences in a species' interaction with its prey, predators, and competitors.
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http://dx.doi.org/10.1093/gbe/evx157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604253PMC
September 2017

Modulating the Serotonin Receptor Spectrum of Pulicatin Natural Products.

J Nat Prod 2017 08 26;80(8):2360-2370. Epub 2017 Jul 26.

Marine Science Institute, University of the Philippines, Diliman , Quezon City 1101, Philippines.

Serotonin (5-HT) receptors are important in health and disease, but the existence of 14 subtypes necessitates selective ligands. Previously, the pulicatins were identified as ligands that specifically bound to the subtype 5-HT in the 500 nM to 10 μM range and that exhibited in vitro effects on cultured mouse neurons. Here, we examined the structure-activity relationship of 30 synthetic and natural pulicatin derivatives using binding, receptor functionality, and in vivo assays. The results reveal the 2-arylthiazoline scaffold as a tunable serotonin receptor-targeting pharmacophore. Tests in mice show potential antiseizure and antinociceptive activities at high doses without motor impairment.
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http://dx.doi.org/10.1021/acs.jnatprod.7b00317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025773PMC
August 2017

Linking neuroethology to the chemical biology of natural products: interactions between cone snails and their fish prey, a case study.

J Comp Physiol A Neuroethol Sens Neural Behav Physiol 2017 Sep 27;203(9):717-735. Epub 2017 May 27.

Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.

From a biological perspective, a natural product can be defined as a compound evolved by an organism for chemical interactions with another organism including prey, predator, competitor, pathogen, symbiont or host. Natural products hold tremendous potential as drug leads and have been extensively studied by chemists and biochemists in the pharmaceutical industry. However, the biological purpose for which a natural product evolved is rarely addressed. By focusing on a well-studied group of natural products-venom components from predatory marine cone snails-this review provides a rationale for why a better understanding of the evolution, biology and biochemistry of natural products will facilitate both neuroscience and the potential for drug leads. The larger goal is to establish a new sub-discipline in the broader field of neuroethology that we refer to as "Chemical Neuroethology", linking the substantial work carried out by chemists on natural products with accelerating advances in neuroethology.
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http://dx.doi.org/10.1007/s00359-017-1183-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656512PMC
September 2017

The Venom Repertoire of Conus gloriamaris (Chemnitz, 1777), the Glory of the Sea.

Mar Drugs 2017 May 20;15(5). Epub 2017 May 20.

Department of Biology, University of Utah, Salt Lake City 84112, UT, USA.

The marine cone snail Conus gloriamaris is an iconic species. For over two centuries, its shell was one of the most prized and valuable natural history objects in the world. Today, cone snails have attracted attention for their remarkable venom components. Many conotoxins are proving valuable as research tools, drug leads, and drugs. In this article, we present the venom gland transcriptome of C. gloriamaris, revealing this species' conotoxin repertoire. More than 100 conotoxin sequences were identified, representing a valuable resource for future drug discovery efforts.
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http://dx.doi.org/10.3390/md15050145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450551PMC
May 2017

TRPA1 expression levels and excitability brake by K channels influence cold sensitivity of TRPA1-expressing neurons.

Neuroscience 2017 06 10;353:76-86. Epub 2017 Apr 10.

Department of Biology, University of Utah, 257 S. 1400 E., Salt Lake City, UT 84112, United States. Electronic address:

The molecular sensor of innocuous (painless) cold sensation is well-established to be transient receptor potential cation channel, subfamily M, member 8 (TRPM8). However, the role of transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in noxious (painful) cold sensation has been controversial. We find that TRPA1 channels contribute to the noxious cold sensitivity of mouse somatosensory neurons, independent of TRPM8 channels, and that TRPA1-expressing neurons are largely non-overlapping with TRPM8-expressing neurons in mouse dorsal-root ganglia (DRG). However, relatively few TRPA1-expressing neurons (e.g., responsive to allyl isothiocyanate or AITC, a selective TRPA1 agonist) respond overtly to cold temperature in vitro, unlike TRPM8-expressing neurons, which almost all respond to cold. Using somatosensory neurons from TRPM8-/- mice and subtype-selective blockers of TRPM8 and TRPA1 channels, we demonstrate that responses to cold temperatures from TRPA1-expressing neurons are mediated by TRPA1 channels. We also identify two factors that affect the cold-sensitivity of TRPA1-expressing neurons: (1) cold-sensitive AITC-sensitive neurons express relatively more TRPA1 transcripts than cold-insensitive AITC-sensitive neurons and (2) voltage-gated potassium (K) channels attenuate the cold-sensitivity of some TRPA1-expressing neurons. The combination of these two factors, combined with the relatively weak agonist-like activity of cold temperature on TRPA1 channels, partially explains why few TRPA1-expressing neurons respond to cold. Blocking K channels also reveals another subclass of noxious cold-sensitive DRG neurons that do not express TRPM8 or TRPA1 channels. Altogether, the results of this study provide novel insights into the cold-sensitivity of different subclasses of somatosensory neurons.
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http://dx.doi.org/10.1016/j.neuroscience.2017.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477667PMC
June 2017

Structure and activity of contryphan-Vc2: Importance of the d-amino acid residue.

Toxicon 2017 Apr 17;129:113-122. Epub 2017 Feb 17.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia. Electronic address:

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.
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http://dx.doi.org/10.1016/j.toxicon.2017.02.012DOI Listing
April 2017

A minimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin.

Nat Struct Mol Biol 2016 Oct 12;23(10):916-920. Epub 2016 Sep 12.

Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Insulins in the venom of certain fish-hunting cone snails facilitate prey capture by rapidly inducing hypoglycemic shock. One such insulin, Conus geographus G1 (Con-Ins G1), is the smallest known insulin found in nature and lacks the C-terminal segment of the B chain that, in human insulin, mediates engagement of the insulin receptor and assembly of the hormone's hexameric storage form. Removal of this segment (residues B23-B30) in human insulin results in substantial loss of receptor affinity. Here, we found that Con-Ins G1 is monomeric, strongly binds the human insulin receptor and activates receptor signaling. Con-Ins G1 thus is a naturally occurring B-chain-minimized mimetic of human insulin. Our crystal structure of Con-Ins G1 reveals a tertiary structure highly similar to that of human insulin and indicates how Con-Ins G1's lack of an equivalent to the key receptor-engaging residue Phe is mitigated. These findings may facilitate efforts to design ultrarapid-acting therapeutic insulins.
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http://dx.doi.org/10.1038/nsmb.3292DOI Listing
October 2016