1. Gives an overview of the development of drugs from peptides and proteins present in venom.
2. Six drugs derived from venoms have already been approved, and there are many more in development.
3. Medicinal chemists, pharmacologists, poisons experts, toxicologists
4. Issues in Toxicology Series, Toxicology Research, MedChemComm
5. The pharmaceutical industry has become increasingly interested in biologics from animal venoms as a potential source for therapeutic agents in recent years, the different aspects of benefit and risk are discussed in this book.
About the Author
Professor Glenn King has been working on animal venoms since 1996.He has extensive experience in the discovery, production, and structural and functional characterization of venom proteins, and is intimately aware of the issues surrounding their development as drugs and insecticides. In 2006,Professor King founded an agricultural biotechnology company, Vestaron Corporation, that is developing spider-venom peptides discovered in the lab as bioinsecticides.His current research is largely focussed on the development of venom peptides as analgesics for the treatment of chronic pain.
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Venoms to Drugs
Venom as a Source for the Development of Human Therapeutics
By Glenn F. King
The Royal Society of ChemistryCopyright © 2015 The Royal Society of Chemistry
All rights reserved.
Seeing the Woods for the Trees: Understanding Venom Evolution as a Guide for Biodiscovery
BRYAN G. FRY, IVAN KOLUDAROV, TIMOTHY N. W. JACKSON, MANDË HOLFORD, YVES TERRAT, NICHOLAS R. CASEWELL, EIVIND A. B. UNDHEIM, IRINA VETTER, SYED A. ALI, DOLYCE H. W. LOW, AND KARTIK SUNAGAR
1.1 The Fundamental Problems
The majority of commercial drugs being used today in both developed and developing countries are based on natural products. Most of these products are based upon plants, but research into animal venoms holds great potential for the discovery of novel medicinally useful natural products. Knowledge of the evolutionary origins of venom proteins/peptides and the forces shaping the biodiversity seen today is crucial for efficient biodiscovery. In addition, efficient utilisation of venom toxins in drug design and development cannot be achieved without recognition of the true biochemical, ecological, morphological, and pharmacological diversity of venoms and associated venom systems. A major limitation of the use of venom proteins thus far has been the very narrow taxonomical range studied. Entire groups of venomous animals remain virtually ignored. Those that have been examined have apparently been selected due to their medical significance or ease of collection, rather than as a result of their ecological or evolutionary uniqueness.
Venom is defined as "a secretion, produced in a specialised gland in one animal and delivered to a target animal through the infliction of a wound (regardless of how tiny it may be), which contains molecules that disrupt normal physiological or biochemical processes in the victim so as to facilitate feeding or defence by the producing animal". This definition encompasses creatures normally considered venomous (e.g., scorpions, snakes, and spiders) as well as animals that have not been traditionally recognised as such (e.g., leeches, ticks, and vampire bats). Acknowledgement of the evolutionary analogy of the recruitment and use of toxins in all these animals increases the number of known independent occasions in which venom has evolved independently. In addition, this acknowledgement improves our understanding of the factors underlying the evolution of venoms and their associated proteins while also drawing attention to the vast pool of unstudied toxins. Venom has been a key innovation in the evolutionary history of an incredibly diverse range of animals. Even using the traditional definition of venom, venom systems are believed to have evolved independently on at least 20 occasions in extant lineages (Figure 1.1). Intriguing fossil evidence has also led to speculation about the possibility of extinct venomous lineages represented by the theropod dinosaur Sinornithosaurus and the extinct pantolestid mammal Bisonalveus browni. If lineages such as ticks, leeches, vampire bats, etc. are rightfully recognised as venomous, the number of independent evolutionary events in which venom has arisen increases to over 30.
The evolutionary selection pressure upon defensive venoms (e.g., those of fish and bees) is largely directed at the development of streamlined venom that has the primary action of immediate, intense localised pain. In contrast, predatory venoms are shaped by a classic co-evolutionary arms race, where evolving venom resistance in prey and the evolution of novel venom composition exerts reciprocal selective pressures on one another in a situation that conforms to the Red Queen hypothesis of Van Valen. Powerful purifying selection pressures acting on predatory venoms for millions of years have resulted in highly complex modern venom arsenals that consist of potent compounds with exquisite target specificity. Variation in venom composition is not only observed between different lineages, but also between the closely related species within a clade. Intraclade differences in venom composition often arise as a result of the evolution of prey-specific toxins in species with specialised diets. Significant variation in venom profile has even been demonstrated within individual species with widespread geographical distributions. Venom can also vary intraspecifically as the result of numerous other factors, including sibling differences and ontogenic changes in prey preference or behaviour. In Sydney funnel-web spiders (Atrax robustus), juvenile male spiders and female spiders of all ages have similar insecticidal predatory venoms, whilst sexually mature males (who stop feeding and leave the burrows in search of females) have a vertebrate-specific defensive venom. It is this adaptive complexity and innovation that makes predatory venoms ideal candidates for the discovery of therapeutic lead compounds.
The majority of venom components have evolved to target physiological systems reachable by the bloodstream. In particular, the neurological and haemostatic systems have been convergently targeted via a myriad of innovative pathways (Figure 1.2). A consistent feature of venom proteins is a stable molecular scaffold of cross-linked cysteines (see Chapter 2 for further details of disulfide-rich toxin scaffolds); this characteristic appears to facilitate modification of non-structural residues, which in turn facilitates protein neo-/sub-functionalisation. A remarkable degree of convergence exists not only in terms of toxin molecular scaffolding, but also in target specificity and bioactivity. The superimposition of sequences from functionally convergent toxins reveals tremendously useful information regarding structure–function relationships. An example of this is the platelet-aggregation inhibiting RGD tripeptide motif. This motif has been independently derived on numerous occasions within a myriad of distinct protein scaffolds, ranging from snakes (two different occasions: disintegrins and three-finger toxins) to a wide variety of invertebrate species, including ticks (e.g., Ixodes spp., Argas spp., Rhipicephalus spp., Amblyomma spp.), tabanid flies (e.g., Tabanus spp.), true bugs (e.g., Triatoma spp., Rhodnius prolixus), mosquitoes (e.g., Anopheles spp., Aedes spp., Culex spp.), sand flies (e.g., Lutzomyia spp., Phlebotomus spp.), leeches (e.g., Macrobdella spp., Placobdella spp.), and worms (e.g., Ancylostoma spp.). This reinforces the fact that biological targets within prey animals are the primary drivers of the evolution of toxin structures.
Snakes, spiders, scorpions, marine cone snails, and sea anemones represent the majority of venomous organisms that have been studied, with other venomous lineages remaining neglected. Moreover, even within these well-studied lineages, there has been a significant taxonomical bias. Partly as a result of the limited taxonomic range studied, the majority of known venom components remain poorly understood, and it is likely that many more venom components await discovery. The complex nature of venom makes it energetically expensive to produce. Hence, most venomous organisms have evolved a highly sophisticated cocktail that can efficiently aid in predation and/or defence, even when secreted in very small quantities. The small amount of venom produced by many venomous organisms was a major obstacle that impeded venom exploration in the past. Even in snakes, which may produce copious amounts of venom, particularly large amounts (multi-gram) were necessary for the discovery novel venom components that are secreted in miniscule amounts. For smaller animals such as spiders, the tiny amounts secreted made many species impossible to investigate using protein-based approaches.
Another impediment to venom exploration has been the difficulty of extracting venom from species that do not store secreted venom in readiness for delivery, or that have venom delivery systems that are difficult to access or stimulate. For instance, the venom delivery apparatus of non-front-fanged snakes is located at the back of the mouth and, unlike those of many front-fanged snakes, the venom glands do not contain an appreciable lumen for venom storage, instead only secreting venom as required. Hence, it was very difficult and time-consuming to obtain sufficient quantities of venom from such snakes for the "proteome-only" oriented venom research of the past. Even chemical stimulation of venom secretion (e.g., injection of pilocarpine into the venom gland) has been unsuccessful in overcoming some of the aforementioned complications, impeding venom research in a large group of other organisms (e.g., coleoids, centipedes, non-front-fanged snakes, spiders, and vampire bats). In some cases, these difficulties have been overcome through the application of considerable amounts of time and effort. For example, it took over 200 pilocarpine-stimulated milkings (venom extraction) of Coelognathus radiatus to obtain 110 mg of crude venom, which yielded 10 mg of pure α-colubritoxin in the first study of a three-finger toxin (3FTx) from a non-elapid snake. In other cases, however, the difficulties described above have proven impossible to surmount.
With the advent of next-generation RNA sequencing, venom exploration has become more efficient, as researchers can now rapidly construct transcriptome libraries of entire venom glands, without depending on proteinaceous venomous secretions directly. While transcriptomics will rapidly yield full-length precursor sequences, the prediction of propeptide cleavage sites, other than those that are conventionally dibasic, may be impossible. Moreover, transcriptomics alone cannot unravel post-translational modifications (PTMs), which are often crucial for the biological activities of venom components. For example, a sulfo-tyrosine PTM is required for the bioactivity of lizard venom cholecystotoxin; synthetic analogues lacking this PTM are completely inactive. For these reasons, a combined proteomic–transcriptomic approach is essential for the most effective venom exploration (see Chapter 3).
Genome information remains scarce for species other than those routinely used as model organisms in genetics research. To date, genomes are available for only a few venomous animals. Knowledge of the location and organisation of venom-encoding genes can greatly increase our understanding of their molecular evolutionary history. Availability of genomic information will facilitate easier amplification of specific venom-encoding genes. Moreover, this will enable researchers to use small amounts of tissue or other non-destructive samples for sequencing of venom-encoding genes, not only making venom exploration in rare venomous organisms easier and more sustainable, but also overcoming the difficulties of obtaining permits for destructive sampling for research.
Another major problem that has affected venom research is the difficulty of obtaining the venomous animals themselves. Most well-studied venomous organisms have been those that are locally common in the regions in which the research was performed. Researchers often restrict their venom collection to species represented in local serpentariums or that are available from other venom suppliers. Samples acquired from third parties in this manner are often associated with uncertainties regarding geographical origin and sometimes even basic taxonomy. For example, the Sigma pharmaceutical catalogue entry for Oxyuranus scutellatus venom (see http://www.sigmaaldrich.com/catalog/product/SIGMA/V3129) states "This venom may be from subspecies O. s canni (Papuan taipan) or O. s scutellatus (Australian taipan) or a mixture from both"; with a note that "Physical characteristics are almost identical". This is despite the existence of abundant research showing that venoms may vary appreciably across a relatively short continuous geographical range, let alone the sort of variance that may occur between completely disjunct localities. Considerable differences in toxicity and antivenom coverage have recently been demonstrated for O. s. canni and O. s. scutellatus, which highlights the fact that disregarding the geographical origin of samples is unacceptable in venom research.
The taxonomical bias in toxinology is starkly evident when sequenced toxins are mapped against organismal diversity. For example, in elapid snake venom research, two genera (Bungarus and Naja) account for almost 40% of all published sequences (Table 1.1). Moreover, almost 40% of all 3FTxs have been sequenced from Naja alone. Despite the diversity of toxin forms present, some toxin types are known from transcriptomic studies only. Similarly, of the 3FTxs known from the non-front fanged snake lineages, the majority are known from a single transcriptomic study. Only three studies have characterised the bioactivity of fully-sequenced 3FTx from non-front-fanged snake venoms. This bias is not unique to snakes, as the other venomous lineages that have received toxinological attention have suffered similar levels of taxonomical bias. For example, although scorpion venoms have received more research attention than the venoms of any other lineage, only 50 or so of the approximately 1700 species of scorpion recognised today have been examined. The major focus has been on basal families such as Buthidae, which account for more than 50% of all known scorpion toxin sequences. These basal families are known to have separated from all other scorpion families about 350 million years ago, suggesting that there is likely to be a plethora of novel venom components that remain undiscovered in the other families. Similarly, despite spiders being the most speciose group of venomous animals, represented by ~45 000 recognised species, venom exploration remains primarily restricted to large mygalomorph species. For example, tarantulas account for more than one quarter of all spider toxins isolated to date, although they represent only ~2% of the taxonomic diversity of spiders. Furthermore, it is suggested that the currently recognised species constitute only ~25% of existing species. Spiders evolved from the stem arachnid ancestor about 300 million years ago during the Carboniferous period. Spider venoms contain a range of low molecular weight peptides and proteins that are neurotoxic, haemotoxic or cytotoxic in activity. It is likely that the highly complex nature of the venom is responsible for the tremendous success and diversification of the spiders as a group. Continuing this theme, the venom has not been thoroughly characterised from a single species of centipede amongst the 3300 species of centipedes known today. The forcipules, or poison claws, which are modified front legs used for delivering venom into the prey, have been identified in centipede fossils dating back to the early Devonian period, 400 million years ago. This suggests that centipedes, along with scorpions, possess one of the most ancient venom delivery apparatuses. Despite this, centipede venom research is very much in its infancy.
1.2 The Solutions
The most efficient venom exploration approach is multidisciplinary and encompasses various fields and techniques, including:
Organismal selection based upon phylogenetic position and ecological niche occupied
Transcriptomics and in silico studies
Molecular evolution and phylogenetics of toxins
Excerpted from Venoms to Drugs by Glenn F. King. Copyright © 2015 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents
Venomous animals: evolution and ecology; Chemistry and structural biology of animal venoms; Venoms-based drug discovery: proteomic and transcriptomic approaches; Venoms-based drug discovery: bioassays, high-throughput screens, and target identification; Reptile venoms as a platform for drug development; Cone snail venoms as a platform for drug development; Scorpion venoms as a platform for drug development; Spider-venom peptides as a platform for drug development; Case study 1: development of the analgesic drugs Prialt® and Xen2174; Case study 2: development of exenatide for treatment of type 2 diabetes; Case study 3: development of ShK for the treatment of autoimmune diseases; Development of venom natriuretic peptides for treating congestive heart failure; Engineering venom peptides to improve their stability and bioavailability; Manufacturing of venom-derived therapeutic peptides; Venoms to drugs: prospects and pitfalls