VENOMOUS CONE SNAILS (FISH - HUNTING SPECIES) Some kill people: Conus geographus, 70% fatality rate. 3 F2 4 different clades of fish-hunting cone snails harpoon tooth proboscis tip Lightning-strike cabal -Conotoxin - INCREASES Na channel conductance k-Conotoxin - Blocks K channels Others - ? k-PVIIA CRIONQKCFQHLDDCCSRKCNRFNKCV -PVIA EACYAOGTFCGIKOGLCCSEFCLPGVCFG Prey Capture Excitotoxic Neuromuscular 1 Shock 2 Block Very rapid, fish stunned Irreversible paralysis Lightning-strike cabal Lightning strike constellation -Conotoxin - INCREASES Na channel conductance k-Conotoxin - Blocks K channels -Conotoxin - Activates Na Channels Con-ikot-ikot - Inhibits Glu receptor desensitization Motor cabal Motor constellation w-Conotoxin - Blocks Ca channels a-Conotoxin - Competitive nicotinic receptor inhibitor y-Conotoxin - Nicotinic receptor channel blocker? m-Conotoxin - BLOCKS Na channel conductance Conus geographus • The Deadliest Snail in the Ocean Net Strategy Sensory Deadening Neuromuscular Block (Nirvana Cabal) (Motor Cabal) Nirvana Cabal Sedated, quiescent state Motor Cabal Neuromuscular transmission block Nirvana cabal Targeted to sensory circuitry: s-Conotoxin - 5HT3 receptor blocker * Conantokin - NMDA receptor blocker * “Sluggish” peptide “Sleeper” peptides “Weaponized” insulin Mature venom insulin is post-translationally modified Con-Ins G1 Highly expressed in venom gland Highly abundant in C. geographus venom Helena Hemami-Safavi Activity testing Adam Douglass SafaviSantosh-Hemami Karanth et al. 2015, Amnon PNAS Schlegel Venom insulin: proposed mechanism of action Adminstration of insulin causes glucose uptake from the blood into liver and muscle tissue Insulin overdose: rapid depletion of blood glucose leads to insufficient glucose supply for the brain: dizziness, nausea, coma and death Insulin shock, hypoglycemic shock Insulin as a murder weapon, the Sunny von Bülow case: American heiress and socialite. Her husband, Claus von Bülow, was convicted of attempting her murder by insulin overdose C. geographus insulin: the Sunny von Bülow peptide Smallest insulin described from a natural source Why so small? A chain B chain Structural Features of Human/Fish Insulin Insulin Hexamer Insulin Dimer A chain GIVEQCCTSICSLYQLENYCN B chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT hexamerization dimerization http://watcut.uwaterloo.ca/webnotes/Metabolism/diabetesHbA1c.html Oligomerization leads to slow (IV) absorption rates Insulin receptor Diabetes is treated by using a combination of basal (hexameric) and fast-acting (monomeric) insulins FISH-HUNTING CONE SNAILS HAVE EVOLVED A MONOMERIC INSULIN Michael Lawrance, WEHI A chain B chain ActiveMonomeractive Loss of activity Insulin Signaling Activation 1.5 human insulin l e ConG1_Cys v e l DOI 1.0 Insulin Signat ling Activation k ConG1_desPTM 1.5 A p human insulin e v l i t e 0.5 ConG1_Cys a v l e e l DOI 1.0 r t k ConG1_desPTM A p e -2 0 2 4 v i t 0.5 a l log [insulin] (nM) e r -2 0 2 4 log [insulin] (nM) Chemical total synthesis of DOI mutants 1. 25% mercaptoethanol/DMF, r.t., 2 h. 2. DTNP, DCM, r.t., 1 h SBu-t Mmt t-BuO2C O 3. 1% TFA, 5% TIS, DCM S S 2 mins, 6 times. GIVEQCCTSICSLYQLENYC N N OH H H GIVEQCCTSICSLYQLENYCN Trt 4. DCM, r.t., 5 h. Acm Acm 5. 5% TIS, 2.5% H2O, 2 1 92.5% TFA, r.t., 2.5 h. 2.5% TIS, 2.5% H2O, S 95% TFA, DTDP, r.t., N 2-chlorotrityl Resin 2.5 h. FVNQHLCGSHLVEAYYLVCGER FVNQHLCGSHLVEAYYLVCGER OH Acm Acm S S GIVEQCCTSICSLYQLENYCN OH S 1. 6 M urea, 0.2 M NH4HCO3, pH 8 S 2 + 4 S S 2. I2, AcOH/H2O FVNQHLCGSHLVEAYYLVCGER OH 5 B15 Tyr DOI analog Xiaochun Xiong, Danny Chou Current Progress • By utilizing the structural insights from Con-Ins-G1, our current lead DOI analogue has a comparable bioactivity as con-Ins-G1. • By introducing additional mutations learned from Con- Ins-G1, our goal is to identify monomericMaria human Disotuar DOI, Danny Chou analog with full native insulin potency. Summary • Human insulin is hexameric, which leads to delayed action after subcutaneous injections. • Monomeric human insulin (DOI) has 1,000 fold weaker bioactivity due to the lack of B24 Phe. • Venom insulin, Con-Ins-G1, has reasonable bioactivity toward human insulin receptor although it lacks B24 Phe. • Crystal structure of Con-Ins-G1 suggests the importance of B15 & B20 Tyr in compensating the role of B24 Phe. • Through chemical protein synthesis, the importance of B15 & B20 Tyr is confirmed. • Further protein engineering efforts are currently underway to develop a monomeric human insulin with full bioactivity. Nirvana Cabal peptides that reached clinical trials Sluggish * Contulakin-G <ESEEGGSNATKKPYIL Neurotensin <ELYENKPRRPYIL (Intractable Pain) Molecular target: Neurotensin Receptor Nirvana Cabal peptides that reached clinical trials Sleeper (Subtype-specific NMDA receptor antagonist) Conantokin Gly Glu Gla Gla Leu Gln Gla Asn Gln Gla Glu Ile Arg Gla Lys Ser Asn NH2 COO OOC COO CO2 CH2 CH CH CH 2 Vitamin K 2 carboxylase C C Glu Gla (Epilepsy - Subtype-specific NMDA receptor antagonist) Molecular target: NMDA Receptor (NR2B subunit) ω-MVIIA CKGKGAKCSRLMYDCCTGSCRSGKC# ω-GVIA CKSOGSSCSOTSYNCCRSCNOYTKRCY# Conus geographus venom components, Biomedical Applications Con Ins GI Preclinical Diabetes (C. geographus insulin) Contulakin G Phase I Intractable Cancer Pain Conantokin G Phase I Epilepsy ω-Conotoxin GVIA Diagnostic Lambert-Eaton Syndrome ω-Conotoxin MVIIA FDA Approved Neuropathic Pain Drug MORE PEPTIDE THERAPEUTICS SHOULD BE DISCOVERED FROM STUDYING CHEMICAL INTERACTIONS BETWEEN MARINE ANIMALS Biomedical applications from studying one single chemical interaction: (between Conus geographus and its fish prey) 1 FDA Approved drug: (Neuropathic pain; target: Cav 2.2 Ca Channel) 2 Compounds in human clinical trials: Contulakin G (Cancer pain; Target: Neurotensin Receptor) Conantokin G (Epilepsy; Target: NMDA Receptor) 1 Diagnostic agent: Radiolabeled ω-GVIA (Lambert-Eaton Syndrome) 1 Preclinical stage drug development: Con Ins-GI (Diabetes) ~800 Species of Cone Snails (family Conidae) Arguably the largest genus of marine invertebrates The venom of each different Conus species has a different set of peptides. With ~800 Conus species that each have ~100 peptides, there are over 100,000 pharmacologically active peptides Toxins in Conus venoms are used for more than prey capture: Defense against predators Deterrence of competitors Why so many different peptides in one venom? Venom peptides are used for more than prey capture: Defense against predators Deterrence of competitors EACH SPECIES HAS MULTIPLE BIOTIC INTERACTIONS Why does each species have different venom components? EACH SPECIES HAS ITS OWN PARTICULAR ECOLOGICAL NICHE Each Conus species has: Different prey spectrum, different predators, different competitors Conus sulcatus The Slowest Fish-Hunting Cone Biodiversity Chemical Biology Neuroscience Colubraria “The vampires of the Tropical Seas” Acknowledgments Early Characterization Venom Insulins Of Conotoxins HELENA HEMAMI-SAFAVI Luly Cruz (Philippines) Joanna Gajzwiak Bill Gray (Utah) Sam Robinson Doju Yoshikami (Utah) Aiping Lyu (Shanghai) Anthony Craig, Jean Rivier (La Jolla) Mark Yandell (Utah Medical School) Grey Bulaj (Utah) Danny Chou (Utah Medical School) Beatrix Uberheide (New York) Turris Venoms Jean Rivier, Judit Erchegyi (La Jolla) Gisela Concepcion (Philippines) Michael Lawrence, Ray Norton, Julia Imperial Briony Forbes (Australia) Estuardo Lopez-Vera (Mexico) Manuel Aguilar (Mexico) Constellation Pharmacology Utah Former Bea Ramiro (Philippines) Russ Teichert Undergraduates April Cabang Cheenu Raghurahman Craig Clark Maren Watkins Tosifa Memon Michael McIntosh Sam Espino David Griffin Financial Support Kevin Chase David Johnson National Institute of General Medical Na Channels, K Channels Aryan Azimi-Zonooz Science (GM48677) Heinz Terlau (Germany ) Chris Hopkins HHMI Professorship Grant Min-Min Zhang ICBG Grant (NIH Fogarty Center) Doju Yoshikami Neogastropoda Buccinoidea Muricoidea Olivoidea Conoidea Cancellarioidea Tonnoidea Conoidea (~15,000 species) CONIDAE (~800 SPECIES) CLAVATULIDAE DRILLIDAE* PSEUDOMELATOMIDAE* STRICTOSPIRIDAE RAPHITOMIDAE* CLATHURELLIDAE* MANGELIDAE* CONORBIDAE RURRIDAE TEREBRIDAE Cones Turrids Augers Cone Snails ~ 500 - 700 species (Conus, etc.) Auger Snails ~ 400 - 600 species (Terebra, etc.) Turrid Snails ~ 12,000 species (Turris, etc.) * Cone Snails * Auger Snails * * TURRIDS * * Small Molecules in Conus Venoms Conus genuanus Neves et al Org Lett 2015 How is Genuanine Made? • A novel nucleic acid – Derived from guanine • Methyl group modification known in some bacteria • Radical SAM enzyme • Hypothesis: Made by animal? • Only found in colored venom ducts – Radicals Endogenous insulins Highly conserved Purifying selection Venom insulins Hyperdiverse Positive selection Juxtaposition between a highly conserved (endogenous) and hypervariable (venom) insulin gene class Insulin Insulin is a peptide hormone of the pancreas, and is central to regulating carbohydrate and fat metabolism in the body. It causes cells in the liver, skeletal muscles, and fat tissue to absorb glucose from the blood. A chain B chain Within vertebrates, the amino acid sequence and peptide structure is strongly conserved (fish insulin is active in human cells) Insulins are found in many invertebrates where they exert similar effects Molluscan insulins have longer chains and an additional disulfide bond .