L11.1 - Venoms and Toxins - Jackson.pdf

Venom and toxins part 1: evolutionary context of venom Dr Timothy Jackson ([email protected]) Australian Venom Research Unit (AVRU) Australian Venom Research Unit Overview 1. 2. 3. 4. 5. Evolutionary context of venom Toxins as tools Evolution as a guide to biodiscovery How we study...

Venom and toxins part 1: evolutionary context of venom Dr Timothy Jackson ([email protected]) Australian Venom Research Unit (AVRU) Australian Venom Research Unit Overview 1. 2. 3. 4. 5. Evolutionary context of venom Toxins as tools Evolution as a guide to biodiscovery How we study venom Learning outcomes Poison versus venom • Classic meme of unknown (to me) provenance: “If you bite it and you die, it’s poisonous; if it bites you and you die, it’s venomous.” • Pedant’s peril – “poison” is a general term; “venom” is a specialised type of poison. • Only in biological (evolutionary) contexts is the poison/venom distinction meaningful. • Yes, there are poisonous snakes (in both senses of the word)! What is venom? • A secretory “cocktail” • Produced in a specialised tissue (e.g. a venom gland, cnidocyte, etc.) • Actively delivered (e.g. via a bite or sting) • Contains molecules that disrupt target organism’s physiology • Evolved to facilitate feeding, defence, or competitor deterrence Ø Venom is a functional trait Picture by Ryan Ellis, used with permission What is a toxin? (toxinology versus toxicology) • • • • Toxins (in the biological context) are the active constituents of venoms and poisons Venom toxins are typically proteinaceous (peptides and enzymes) Huge range of activities Toxinology is the study of toxins of biological origin (i.e. from animals, plants, microbes); toxicology is the study of “poisons” and poisoning more generally Evolutionary context of venom • Venom is a functional trait • Designed (by evolution) for specific purposes, typically feeding and/or defence o Snake venom’s primary function is prey subjugation (not killing), but it is also used defensively and defensive specialistions occur (e.g. spitting cobras) o Some venomous organisms (e.g. cone snails) produce two distinct venoms – one for offence and one for defence • Coevolution – venomous organisms are in a “chemical arms race” with their prey/predators Co-variation of venom composition and ecology: the example of Pseudonaja textilis • The eastern brown snake (P. textilis) undergoes a major shift in feeding ecology as it grows from a juvenile to an adult – this is called an ontogenetic shift. • Over the same developmental as the dietary shift, the venom changes dramatically in both composition and activity (which are two sides of the same coin). Photo by David Kirshner, used with permission Photo by Matt Summerville, used with permission Australian Venom Research Unit Molecular evolution of toxin genes • The same gene families have been recruited as toxins many times independently • These gene families are functionally diverse, but share numerous characteristics: o Appropriate function o Disulfide bonding o High apparent rate of duplication • Characteristics that facilitate the accumulation of variation on which selection can act – toxin genes are highly evolvable. Evolution of resistance to venom • Evidence of arms races – sometimes the prey/predators of venomous organisms evolve a degree of resistance. • Several pathways: o Toxin inhibitors (deployed against enzymatic toxins) – circulate in the blood o “Molecular cage” (hedgehog anti-protease) o Altered targets – point mutations in receptor/target prevent toxin binding but allow endogenous ligand to bind o “Repurposed toxin” – change in affinity causes toxin to bind alternate receptor, with corresponding shift in effect (e.g. grasshopper mouse turning scorpion stings from painful to pleasurable) • How does venom stay ahead, given the life/dinner principle? o Hyper-evolvability of venom Wikimedia commons Toxin sequestration • Some animals are not only resistant to toxins, they steal them for their own ends • Is this any different to humans finding “drugs” in nature? • More common with (biological) poisons (e.g. poison arrow frogs; keelback snakes; butterflies; many tetrodotoxin-sequestering animals etc.) • Venomous examples include o Blue-ringed octopus (Hapalochlaena sp.) – sequestration of tetrodotoxin from endosymbiotic bacteria o Nudibranch (Aeolidiidae) “kleptocnidae” – deployment of stinging cells from hydrozoan prey Australian Venom Research Unit Wikimedia commons Venom and toxins part 2: Toxins as tools What is a tool? • “A device or implement used to carry out a particular function” • Humans used to be thought of as “the tool-making species” • Human exceptionalism is a preevolutionary fairy tale • Tool use is widespread amongst animals • Primates and birds not only use tools, but actually “fashion” them Exaptation – a crucial concept for the tool kit of the evolutionary thinker • Property (effect) versus function – all functions are properties, but not all properties are functions (see Jackson and Fry 2016) • An “adaptation” is a trait/character that has been shaped by selection for its current use - a function is the usage/purpose of that trait. • Two forms of exaptation (Gould and Vrba 1982): • When a property with no selective history (i.e. an “epiphenomenon”) is “co-opted” for a new function (i.e. a property becomes a function) • When a trait that has been selected for a specific function is co-opted for a new function • i.e., when a new function arises from an existing property • Concept originates with Darwin – 6th edition of “On the origin…” • Exaptation is crucial for the evolution of novelty – ex nihilo nihil fit – you can’t get something from nothing. Tool use and exaptation • Rocks were not “designed” for anything • Turning something we find in nature into a tool is exaptation • What is biodiscovery? • Is toxin sequestration tool use? “Biodiscovery” evolved from “ethnopharmacology” • Pharmacology is an ancient discipline – today we call all non-western pharmacological practices “ethnopharmacology” • Culture evolves oHumans are not distinct from “nature” oWhat we call “biodiscovery” today is really just a fancy version of what humans have been doing for millennia •“Nature” is always the starting point in the search for drugs owe do not come up with novel molecules from scratch oThere are more possible combinations of 20 amino acids than there are atoms in the universe •As Salvador Dali said - “I do not take drugs, I am drugs.” oHumans, like all biological systems, are intrinsically pharmacological…..and this is why pharmacology students should study evolution! Venom and drugs – overdoses and off-target effects • “Pharmakon” – ancient Greek word that can be translated as either “drug”, “poison” or “remedy”…an ambiguity preserved in English by colloquial usage of the word “drug” • “Solo dosis facit venenum” – the dose makes the poison o “All things are poison, and nothing is without poison, the dosage alone makes it so a thing is not a poison.” – Paracelsus (physicist, alchemist, astrologer and quintessential “Renaissance Man”) • The most basic principle of toxicity – overdose. • Better yet – rate increase (Orgel’s First Rule – “Whenever a spontaneous process is too slow or too inefficient a protein will evolve to speed it up or make it more efficient.”) o Specific deletions in regulatory components of snake venom factor Xa dramatically increase its rate of activity relative to endophysiological homologue • ”Intentional” off-target effects – take a regulatory protein/peptide and change its affinity (i.e. shift targeting) for instant toxic effect Typical functions of venom toxins • Haemotoxicity o Procoagulant activity – inducing clot formation, typically by activating or mimicking thrombin. Can lead to death by stroke (via formation of numerous microthrombi) or venom-induced consumption coagulopathy (VICC) o Anticoagulant activity – achieved via multiple pathways including fibrinogen-destruction, prevention of platelet aggregation and platelet destruction (thrombocytopenia) • Haemorrhagic activity – disruption of microvessel integrity causing bleeding, including systemic haemorrhage exacerbated by haemotoxins • Neurotoxicity o numerous pathways including pre- and post-synaptic; receptor antagonists & agonists; channel activators and blockers, etc. o May be reversible or irreversible (including physical destruction of synapses) o May result in flaccid or excitatory paralysis • Myotoxicity – destruction of muscle cells • Cytotoxicity and necrosis • Cardiovascular disturbances, including induction of either hypo- or hypertension • Lethality is typically not a function of toxins/venom, it is a side-effect. Haemotoxicity Neurotoxicity Part 3: Evolution as a guide to biodiscovery Dr Timothy N. W. Jackson Toxins are a model system for studying effector/receptor interactions • Toxins have evolved over many millions of years to engender specific physiological consequences: toxins are “drugs” designed by evolution • A venomous organism produces a purpose-built pharmacopeia: they are nature’s apothecaries, offering a complex suite of pharmacologically active compounds • Toxins “know” (i.e. possess information about) their targets: we can use them to understand these effector/receptor interactions, which are all-important in modern medicine o Investigational ligands o Lead compounds for drug development • Orgel’s Second Rule – “Evolution is cleverer than you are.” o Note however the complementarity of the evolutionary and mechanistic perspectives: we can learn a great deal about mechanism by studying the activity of molecules in context • Note parallels with your practical “using drugs to understand autonomic mechanisms…” Evolution as a guide to biodiscovery Evolution as a guide to biodiscovery Venomous taxa occur across the animal kingdom and there are many thousands of candidate toxins There is a great deal of convergence in both toxin recruitment and targeting, however…. ….there is a great deal of divergence, even amongst closely related taxa How can we make rational choices about where to concentrate our efforts? o The principle of phylogenetic distance – focus on distantly related taxa o The principle of ecological distance – examine taxa that deploy their venom against different target organisms o The principle of evolutionary rate – focus on toxin gene families that are known to evolve rapidly and readily acquire new functions • Use high-throughput screening technologies (both –omics based and functional) to minimise selection biases • Understand the sources of variation in venom composition and activity • • • • Australian Venom Research Unit Three-finger toxins (3FTx) – a case study in neofunctionalisation Three-finger toxins (3FTx) – a case study in neofunctionalisation Picture by Stewart Macdonald, used with permission. Picture by David Williams, AVRU Three-finger toxins (3FTx) – a case study in neofunctionalisation • • • • • • • • • Typically antagonists of post-synaptic nAChR (⍺-neurotoxins) o Most commonly ⍺1 neuromuscular nAChR, the most “plesiotypic” forms have approximately 100-fold greater affinity for reptilian (including avian) ⍺1 than mammalian o Type II ⍺-neurotoxins also bind ⍺7 (neuronal) nAChR, and κ-neurotoxins bind ⍺3 Aminergic toxins o Antagonists and agonists of muscarinic and adrenergic receptors Anti-cholinesterases o Bind acetylcholinesterase (AChE) and prevent it from hydrolysing acetylcholine Calcium channel blockers o Antagonise L-type calcium channels, inhibiting transmission of action potential Acid-sensing ion channel (ASIC) blockers o Reversibly bind/antagonise ASIC, including those in nociceptors (thus potential as an analgesic) Platelet-aggregation inhibitors o Anticoagulant Cytotoxins o Bind cell membranes via hydrophobic interaction, thus disturbing their integrity (can lead to horrific tissue damage in bite victims) Synergistic toxins o Individually non-toxic, but potentiate the activity of other 3FTx Note parallels with your practical “using drugs as tools to understand autonomic mechanisms” o E.g., interference with function of acetylcholine at multiple levels (interaction with nAChR; inhibition of cholinesterases, etc) o What therapeutic benefits or adverse affects might a drug derived from 3FTx possess/cause? Toxins as tools – investigational ligands and clinical diagnostics • • • • α-bungarotoxin was used in the initial characterisation of “cholinergic receptor proteins” AKA nicotinic acetylcholine receptors (nAChR) o Irreversible antagonist of α1 nAChR – prevents ACh binding. o Still being used in the characterisation of nAChR structure-function relationships and as a pharmacological probe Tetrodotoxin (TTX) has been used to investigate structure-function relationships of sodium (Na+) channels and has helped identify different subtypes of these channels in humans Ecarin and hirudin o Ecarin is a procoagulant (cleaves prothrombin to form thrombin) snake venom metalloprotease (SVMP) from the venom of the saw-scaled viper (Echis carinatus) o Hirudin is a thrombin-inhibiting peptide from leech venom, which is used as an anticoagulant treatment o Ecarin is used to quantify the effect of the thrombin-inhibition, in an assay known as “Ecarin clotting time” Toxins have helped us understand biochemical mechanisms in ways that contribute to drug design and diagnosis Australian Venom Research Unit Echis pyramidum – photograph by David Williams (AVRU) Venom to drugs - examples • Captopril – angiotensin-converting enzyme (ACE) inhibitor o Based on bradykinin-potentiating peptides from the venom of Bothrops jararaca o Orally active vasodilator o Used in treatment of hypertension, congestive heart disease and diabetic nephropathy • Exenatide – glucagon-like peptide-1 agonist o Synthetic exendin-4, a peptide from gila monster (Heloderma suspectum) venom o Used to treat diabetes due to numerous useful activities including increased insulin secretion; decreased glucagon release; decrease of gastric emptying; suppression of appetite; & reduction of liver fat • Eptifibatide & tirofiban o Based on peptides from the venom of a pygmy rattlesnake (Sistrurus miliaris barbouri) and sawscaled viper (Echis carinatus), respectively o Delay clot formation (anticoagulant activity via prevention of platelet aggregation) • Ziconotide o Synthetic version of peptide from cone snail (Conus magus) venom o Potent analgesic • Note however the distinct possibility of adverse affects when developing a toxin into a drug…. Part 4: How we study venom and toxins Dr Timothy N. W. Jackson How we study venom – “Venomics” • • • Venomics is the application of “-omics” technology to study of venom (predominantly at the descriptive level). Combines proteomics…. o Decomplexification of the venom proteome with high-performance liquid chromatography (HP-LC) o Further purification by gel electrophoresis (SDS-PAGE) o Identification of proteins eluted and digested from gel spots with tandem mass spectrometry (MS2) …with transcriptomics o Next-generation sequencing (e.g. Illumina hiseq) of toxin-encoding mRNA extracted from secretory tissue of venom system (e.g. venom glands) o Used to create databases against which MS2 data can be matched, and for bioinformatics studies Australian Venom Research Unit How we study venom - antivenomics • Addition of affinity chromatography to the venomics pipeline o Antibodies from an antivenom are immobilised in a matrix and then incubated with venom to assess immunorecognition o Bound and unbound fractions are eluted separately and compared with HP-LC, SDSPAGE and MS2 • Provides an indication of the potential effectiveness of an antivenom in treating bites from a specific species of snake • In vivo neutralisation of lethality remains the gold standard for demonstration of antivenom effectiveness How we study venom - bioinformatics • mRNA sequences must first be assembled and curated o Extraction and sequencing of RNA o Sequences identified by similarity-based search techniques such as BLAST (Basic Local Alignment Search Tool) o Toxin-encoding genes identified • Resultant data may be analysed in numerous ways o Phylogenetics – assessing the evolutionary relationships of toxin genes o Evolutionary rate estimations – assessing the relative influence of negative and positive selection, as well as neutral evolution, and determining the overall rate of change o Structural modelling via homology with experimentally determined protein structures Australian Venom Research Unit How we study venom - function • Either crude (whole) venom, toxin fractions, or purified toxins may be analysed in a wide range of functional assays, which largely overlap with those utilised in classical pharmacology. • Organ bath tissue assays: o o o o • Enzymology: Chick biventer cervicis Rat phrenic nerve Rat/rabbit ileum (e.g. prac 2) Rat aortic ring Gel-based zymography Plate-based absorbance assays o o o o o Myotoxicity Haemorrhage Dermonecrosis Edema Cytotoxicity • In vitro assessments of: • Cell-based assays • Electrophysiology (patch-clamping and voltageclamping) • Haematology: o o o o o Plasma clotting assays Platelet aggregation assays Fibrinogen assays • In vivo: o o Australian Venom Research Unit Cardiovascular disturbances Lethality The burden of snakebite • • • • • Not a major public health issue in Australia o Under 500 snakebite hospitalisations annually o 1-2 deaths on average A different situation elsewhere, particularly in the tropical developing world o Up to 2.7 million cases annually o Up to 400,000 cases of permanent disablement o Up to 125,000 deaths Snakebite envenoming was recently recognised by the World Health Organisation (WHO) as a Class A Neglected Tropical Disease (NTD) Predominantly affects the rural poor A “multifactorial crisis” o Lack of effective antivenoms o Inability to access antivenoms o Poor hospital care o High density human/snake populations o Inadequate housing (not snakeproof) o Inadequate footwear o http://biomedicalsciences.unimelb.edu.au/departments/pharmacology/engage/avru/discover/snakebiteNTD Australian Venom Research Unit (AVRU) • • • • • Founded in 1994 by Struan Sutherland, formerly of Commonwealth Serum Laboratories (CSL) A multidisciplinary research group within the University of Melbourne’s Department of Pharmacology & Therapeutics Working closely with the WHO to mitigate the burden of snakebite worldwide Ongoing projects in Papua New Guinea and India Here in Melbourne: o Knowledge-hub for venom-related community engagement and education o Australian Venomous Injury Project (epidemiology and geospatial modelling of venomous bites and stings in Australia) o Venom/toxin pharmacology (in collaboration with the Cardiovascular Therapeutics Unit) o Venomics (composition of Australian and international snake venoms) o Antivenomics (auditing Australian and international antivenom efficacy) o Evolution of venom – the “systems perspective” (from molecular evolution of toxin genes to venom composition, venomous snake ecology, and venom system anatomy) Australian Venom Research Unit Learning outcomes • After studying this topic, you should be able to: o Give examples of venom toxins that have been used in drug design or as investigational ligands o Describe some common functions of venom toxins o Understand the complementarity of evolutionary and mechanistic investigations o Describe some of the basic methods used in the study of venoms and toxins and link these to those used in pharmacology more broadly o Give examples of neofunctionalisation within a toxin class and consider potential therapeutic benefits or adverse effects of drugs derived from toxins o Appreciate the wonders of the natural world! Australian Venom Research Unit