The Chemistry of Snake Venom and its Medicinal Potential PDF

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/361226075 The chemistry of snake venom and its medicinal potential Article in Nature Reviews Chemistry · June 2022 DOI: 10.1038/s41570-022-00393-7 CITATIONS...

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/361226075 The chemistry of snake venom and its medicinal potential Article in Nature Reviews Chemistry · June 2022 DOI: 10.1038/s41570-022-00393-7 CITATIONS READS 163 1,209 6 authors, including: Matilde Viegas Saulo Luís Da Silva LAQV@REQUIMTE. Faculdade de Ciências Universidade do Porto University of Porto 12 PUBLICATIONS 392 CITATIONS 119 PUBLICATIONS 3,280 CITATIONS SEE PROFILE SEE PROFILE Andreimar Soares Fiocruz Rondônia e Universidade Federal de Rondônia 383 PUBLICATIONS 11,323 CITATIONS SEE PROFILE All content following this page was uploaded by Saulo Luís Da Silva on 11 July 2022. The user has requested enhancement of the downloaded file.  REVIEWS The chemistry of snake venom and its medicinal potential Ana L. Oliveira 1,2, Matilde F. Viegas 1,2, Saulo L. da Silva 1,2 , Andreimar M. Soares , 3,4 Maria J. Ramos 1,2 and Pedro A. Fernandes 1,2 ✉ Abstract | The fascination and fear of snakes dates back to time immemorial, with the first scientific treatise on snakebite envenoming, the Brooklyn Medical Papyrus, dating from ancient Egypt. Owing to their lethality, snakes have often been associated with images of perfidy, treachery and death. However, snakes did not always have such negative connotations. The curative capacity of venom has been known since antiquity, also making the snake a symbol of pharmacy and medicine. Today, there is renewed interest in pursuing snake-​venom-​based therapies. This Review focuses on the chemistry of snake venom and the potential for venom to be exploited for medicinal purposes in the development of drugs. The mixture of toxins that constitute snake venom is examined, focusing on the molecular structure, chemical reactivity and target recognition of the most bioactive toxins, from which bioactive drugs might be developed. The design and working mechanisms of snake-​venom-​derived drugs are illustrated, and the strategies by which toxins are transformed into therapeutics are analysed. Finally, the challenges in realizing the immense curative potential of snake venom are discussed, and chemical strategies by which a plethora of new drugs could be derived from snake venom are proposed. Neurotoxicity More than 220,000 species, or approximately 15% of species8–15. Other factors, such as environmental condi- The ability of a substance to all animal diversity on earth, are venomous1. Venom tions, age, sex or type of prey available, can also affect negatively affect the structure endows predators with a chemical weapon far more venom composition10. or function of the central or potent than physical force. Animal venoms are com- This diversity is a double-​edged sword. The only peripheral nervous system. plex and sophisticated bioactive cocktails, the main efficient treatment for a snakebite is the administration components of which are proteins and peptides2. The of the specific antivenom, but the variability in venom best characterized animal venoms are probably those composition limits the availability and the upscaling derived from cone snails, spiders, scorpions and snakes. of the production of antivenoms16–18. There are an esti- 1 Department of Chemistry and Biochemistry, Faculty of The composition of the venoms of the first three is mated 2.7 million envenomings each year, which result Sciences, University of Porto, dominated by short (3–9-​kDa) disulfide-​rich peptides in >100,000 deaths and leave >400,000 victims with Porto, Portugal. that contain the inhibitor cysteine knot (ICK) motif, severe and permanent sequelae16–19. However, the com- 2 LAQV/Requimte, University although heavier proteins, including enzymes, are also positional diversity is a rich playground for medicinal of Porto, Porto, Portugal. present. ICK peptides are structurally very stable and chemists, providing a collection of highly specific and 3 Biotechnology Laboratory mainly target the nervous system, acting primarily bioactive compounds that offer many paths towards for Proteins and Bioactive on membrane channels or neuronal receptors3–5. The developing new therapeutic drugs1,4,20–22. Compounds from the Western Amazon, Oswaldo venom of a spider or cone snail might contain thousands In this Review, we first examine the chemical com- Cruz Foundation, National of different peptides, whereas a scorpion’s venom might position of snake venom, analysing the venoms of Institute of Epidemiology contain several hundred3,6. The large number of spider >200 snake species. We then discuss the mechanis- in the Western Amazon species (possibly >100,000) further increases venom tic details of the chemistry of the principal enzymatic (INCT-​EpiAmO), Porto Velho, diversity. venom toxins. Next, we review the development, struc- Brazil. Snake venoms typically consist of a mixture of 20 to ture and mode of action of approved snake-​venom-​based 4 Sao Lucas Universitary Center (UniSL), Porto Velho, >100 components, of which the majority (>90%) are drugs as well as those of compounds in clinical and pre- Brazil. peptides and proteins7, with the dominant bioactivities clinical testing. Finally, we conclude with an analysis of ✉e-​mail: [email protected] including neurotoxicity, haemotoxicity and cytotoxicity, the vast therapeutic potential of snake venom, pointing https://doi.org/10.1038/ depending on the snake species. Venom composition out chemical strategies for the transformation of venom s41570-022-00393-7 varies widely between species and even within the same into a repertoire of new drugs. NATuRe RevIewS | ChEMISTRy volume 6 | July 2022 | 451 0123456789();: Reviews Elapidae Viperidae 5% 3% 6% 4% 33% 7% 51% 9% 27% 15% 21% Bungarus Aspidelaps Bothops Crotalus 3% 5% 9% 5% 6% 7% 37% 6% 7% 8% 33% 45% 19% 8% 9% 10% 80% 16% 19% 29% 16% Micrurus Naja Echis Vipera 5% 4% 5% 7% 6% 7% 6% 8% 32% 14% 50% 54% 19% 13% 31% 64% 16% 14% 21% Hydrophis Lachesis Botriechis Bitis 4% 4% 6% 27% 6% 24% 10% 27% 10% 30% 12% 10% 66% 19% 25% 13% 26% 22% 21% 16% PLA2 SVSP SVMP LAAO CRiSP 3FTx KSPI CTL/SNACLEC DIS NP DEF Other The chemical composition of snake venom to be turned into medicines. Most snake venom toxins Haemotoxicity From the middle of the twentieth century, researchers belong to one of ~30 families23,24, although the venom The ability of a substance to negatively affect the observed the richness in the constituents of venom and of a given snake species can contain hundreds of bio- cardiovascular system or began to isolate and analyse the structures and activ- active compounds15,25. In the snake venoms of known disrupt haemostasis. ities of its toxins, as many of them have the potential composition, some protein families have many hundreds 452 | July 2022 | volume 6 www.nature.com/natrevchem 0123456789();:  Reviews ◀ Fig. 1 | Composition of the venom of snakes from the Elapidae and Viperidae venom composition for each species and the respective families. The large charts show the averaged composition of the venom of snake bibliographic sources. species from the Elapidae (elapids) or Viperidae (viperids) families. Each entry in the Some toxins act synergistically, and the combination charts corresponds to a protein family, in which we group tens to hundreds of isoforms. and proportion of each toxin determine the pathophys- Only protein families with an average abundance of >1% of the total venom proteome iology of snakebite envenomation17,28. The difference in are represented, except for the SVSPs in elapids, which are included for comparison with the viperids, and defensins, which although seldom present, can be abundant in the chemical composition of venom from elapids and the venom of certain species. The distribution of the proportion of the most abundant vipers (Fig. 1) leads to different clinical manifestations. protein families is shown in Supplementary Fig. 1. Data are from the proteomic studies of Envenoming by elapids mostly induces neurotoxic, the past 15 years; 143 entries for 2007–2017 are from Isbister and Tasoulis’s database cytotoxic and cardiotoxic manifestations, whereas of snake venom proteomes7; we assembled the additional entries for 2017–2021 from envenoming by vipers typically induces myotoxicity and the literature. The Atractaspididae and Colubridae snake families are not included haemotoxicity17. in the study because most are non-​venomous or their venoms are weak, not medically Elapid venoms mainly comprise peptides and pro- important and poorly studied (for venomics studies on colubrids see ref.15). Each species teins from seven families; secreted phospholipases A2 contributes with the same weight to the average; subspecies or species from different (PLA2s)32–36 and three-​finger toxins (3FTxs)36–39 are locations were averaged within the entry for the species. The entry ‘Other’ corresponds often major constituents and have a dominant role to unidentified components or components with an average abundance of 300 venomous Viperidae (viperids, commonly referred to as vipers, toxins that are present in smaller proportions (4–7%) heavy-​body snakes with long, and further divided into the true viper and pit viper sub- are LAAOs34,44–47, C-​type lectins and C-​type lectin-​like retractable front fangs. families). Elapids and vipers include almost all medically proteins52–54, and natriuretic peptides55–57. Their venom is frequently haemotoxic and cytotoxic. important snakes, although there are also some examples This analysis shows only part of the complexity of This family includes Old World in the Colubridae (colubrids) family. Figure 1 depicts snake venom, as hundreds of additional proteins, enzymes vipers, rattlesnakes and the averaged venom composition of 76 species and sub- and peptides can be present in the venom of each species. lanceheads, among others. species of the ~400 known elapids and 117 of the ~400 Medically important snakes known vipers. Only protein families with an average Interspecies variation Snake species that cause abundance of >1% of the total venom proteome were Snake venom shows both considerable intraspecies notable morbidity and considered here. However, because evolutionary and (Box 1) and interspecies variation. The fraction of mortality. This classification ecological factors can lead to considerable interpopu­ PLA2s and 3FTx in the venom of each elapid species depends on the venom toxicity, lation and intrapopulation variation in the chemical varies widely (Supplementary Fig. 1), with the percent- the frequency of snake–human interactions, the aggressiveness composition of venom, with numerous exceptions and age of each ranging from almost 0% to nearly 100%. of the snake and the dichotomies adding to the molecular richness at all tax- Interestingly, in most species, a lower fraction of PLA2 health-​care facilities. onomic levels, we also analysed the venom composition is compensated by a higher fraction of 3FTx, and vice of individual snake genera (only the most well-​studied versa. Thus, together, they represent, on average, >80% Myotoxicity Cytotoxicity specifically were considered), which illustrates the venom diversity. of the total venom proteome in most elapid species. directed to myocytes (muscle Supplementary Table 1 further illustrates the chemi- Kunitz-​type peptides generally represent 1,000 ern green mamba (Dendroaspis angusticeps), which is the groups IA and IIA are present in elapid and viperid proteins, most of which also devoid of PLA2s and rich in Kunitz-​type peptides venoms33,35. The enzymes in these groups have a molecu- bind carbohydrates in a and 3FTxs, but with the opposite proportions (16% and lar mass of 13–19 kDa, contain 5–8 disulfide bridges and Ca2+-​dependent manner. 69%, respectively). These two mambas may represent the form dimers in aqueous solution63. In cell membranes, The proteins share a C-​type lectin-​like domain in their most outstanding examples of the chemical diversity of PLA2s dissociate and bind as monomers63,64, and show carbohydrate-​binding region. elapid venoms. an affinity for membrane regions in which at least 15% In snake venoms, they are The composition of viperid venom varies widely of the phospholipids are negatively charged65 (Fig. 2a). haemotoxic. across genera (Fig. 1) and species (Supplementary Fig. 1 PLA2s can also be divided into acidic and basic iso- C-​type lectin-​like proteins and Supplementary Table 1). The PLA2, SVSP and forms, according to the isoelectric point (pI), with the A protein family whose SVMP enzymatic families represent an average of ~70% basic isoforms having a higher membrane affinity and members feature a domain of the viperid proteome. The proportion of PLA2 ranges thus higher toxicity32. with the C-​type lectin fold, from almost zero to >90%, with the distribution peaking The catalytic mechanism of PLA2s is still unclear which lacks critical structural at ~10% (Supplementary Fig. 1). The SVMP ratio also at the atomic level. Nevertheless, it is a two-​stage pro- elements to recognize and bind sugars. In snake venoms, these has a broad distribution, with a peak at ~40%. SVSPs cess, with the first corresponding to the binding of the proteins are haemotoxic. generally constitute 37% of the viperid the reaction65,66. An essential aspartate residue (Asp49) myotoxins. proteome in specific species. coordinates the Ca2+ cofactor, the mutation of which Defensins are small proteins ubiquitous across life renders the enzyme inactive. As metadata analyses have Lysosomes Membrane-​bound organelle that function as host defence peptides and have anti- shown, it is unusual for Ca2+ cofactors to participate in the containing digestive, hydrolytic microbial and/or immune signalling activities. The catalytic cycle67,68. Other divalent metal ions that are larger enzymes whose function is defensins found in viperid venom act on the Na+ and or smaller, or harder or softer, than Ca2+ lead to a nota- primarily the degradation of K+ channels of plasma membranes, including that of ble drop in activity69 for a reason not yet understood. In macromolecules, old cell parts and microorganisms. muscle cells (the sarcolemma), and accumulate in the the most-​accepted mechanism65, the active site histidine Lysosomes represent the lysosomes, causing analgesic, neurotoxic, myotoxic and residue (His48) abstracts a proton from a water molecule waste disposal of a cell. cytotoxic effects60,61. The accumulation in the lysosomes bound to the Ca2+ ion, and the resulting hydroxy group 454 | July 2022 | volume 6 www.nature.com/natrevchem 0123456789();:  Reviews Table 1 | Characteristics of the main families of snake venom toxins Toxin Snake Enzymatic Principal Major Most promising Representative Further family family activity biological pathophysiological therapeutic applications toxin structures reading targets activities PLA2 Elapids and Yes Plasma Acute skeletal Antibacterial activity 5TFV (basic); 1JIA 32–36 viperids membrane muscle necrosis, against Staphylococcus (basic); 1Y4L (PLA2 of myocytes flaccid paralysis, local aureus, Escherichia coli, homologue) and various inflammatory reactions Pseudomonas aeruginosa receptors in (oedema, leukocyte and Enterobacter the axolemma influx into tissues and aerogenes192; anti-​parasite (undetermined pain) effects155; antiviral activity molecular against HIV193,194 and target) dengue195 SVMP Elapids Yes Wide variety Predominantly, Haemostasis; 2W15 (P-​I bound to 34,36,40–42,49 (P-​III of targets; haemorrhagic blood coagulation, a peptidomimetic); SVMPs) and most notable activity but can fibrinolysis and platelet 2M75 (P-​II, DIS viperids are collagen cause the proteolytic aggregation78,168,196 domain); 3DSL (P-​III) (P-​I, IV and blood degradation of P-​II and P-​III coagulation fibrinogen and fibrin, SVMPs) factors induce apoptosis and inhibit platelet aggregation SVSP Elapids and Yes Mostly blood Imbalances the Prevention of thrombus 1OP0 (glycosylated); 34,36,43,50,51 viperids coagulation haemostatic system formation through 3S9B (RVV-​V in open factors through action in the fibrinogen depletion; form); 3S9C (RVV-​V coagulation cascade anticoagulant197,198; bound to factor V) on the fibrinolytic and applied in neurosurgical199 kallikrein–kinin systems and vertebro-​spinal200 procedures as a fibrin sealant 3FTx Elapids No Nicotinic and Neurotoxic effects, Regulation of blood 1QKD (short-​chain 36–39 muscarinic which cause paralysis; pressure201; treatment of α-​neurotoxin); acetylcholine muscle fasciculations; coagulation disorders202–204; 1YI5 (long-​chain receptors, and cardiac arrest analgesic action146,151,154 α-​neurotoxin); acetylcho- through lysis of 1KBA (κ-​neurotoxin linesterase and cardiomyocytes dimer); 3PLC cardiomyocytes (β-​cardiotoxin); 1F8U (undetermined (receptor-​bound molecular fasciculin); 4DO8 target) (muscarinic toxin) LAAO Elapids and Yes l-​amino acids; Haemorrhagic or Antimicrobial against 2IID (with 34,44–47 viperids substrate varies anticoagulant effects, P. aeruginosa, Candida l-​phenylalanine and among species induction of apoptosis, albicans, S. aureus; its cofactor FAD, and oedema and platelet antiparasitic against glycosylated); 4E0V aggregation or Leishmania chagasi (with cofactor FAD); inhibition and Leishmania 5TS5 (glycosylated, amazonensis205,206; potential and with cofactor antiviral against HIV-1 FAD) (ref.207) CRiSP Elapids and No Ca2+ channels, Inhibits angiogenesis, Antiparasitic against 6IMF (bound to 210 viperids K+ channels, increases vascular Leishmania and inhibitor peptide); and signalling permeability and trypanosome strains208; 1WVR (Ca2+-​channel cascades promotes inflammatory antimicrobial against blocker triflin); 3MZ8 involved in cell responses (leukocyte Gram-​negative bacteria (zinc-​bound natrin) adhesion and neutrophil and filamentous fungus209 infiltration) CTL/ Viperids No Platelet Diverse effects, Use in anticoagulant 1JZN (CTL in 52–54 SNACLEC and cellular including therapies211–213 complex with receptors, haemagglutination, galactose); 1UOS as well as mitogenic activity, (CTL convulxin); coagulation platelet aggregation, 3UBU (SNALEC factors, such as oedema, elevated bound to platelet factor IX and vascular permeability, glycoprotein Ib) factor X renal effects, hypotension, cytotoxicity and modulation of Ca2+ release from skeletal muscle sarcoplasmic reticulum NATuRe RevIewS | ChEMISTRy volume 6 | July 2022 | 455 0123456789();: Reviews Table 1 (cont.) | Characteristics of the main families of snake venom toxins Toxin Snake Enzymatic Principal Major Most promising Representative Further family family activity biological pathophysiological therapeutic applications toxin structures reading targets activities DIS Viperids No Integrins Disrupts cell–cell Anti-​inflammatory 1J2L (trimestatin); 107,217–219 adhesion and cell– and antiangiogenic for 3C05 (acostatin); matrix adhesion, and chronic inflammatory 1RMR (schistatin) inhibits angiogenesis processes214; template in the development of the anti-​platelet marketed drugs eptifibatide and tirofiban to treat thrombosis215; treatment for neoplastic processes216 NPs Elapids and No NP receptors A, Potent hypotensive Cardiorenal diseases; heart 4AA2 (BPPb 55–57 viperids B and C effects (vascular failure144 bound to an ACE-​I relaxation and a homologue); 4APJ decrease in myocardial (BPPb bound to contractility), leading ACE-​I) to rapid loss of consciousness KSPI Elapids and No Proteases and Inhibition effects Reduction of cyst 3BYB (textilinin-1); 48 viperids K+ channels on a range of development in polycystic 3D65 (textilinin-1 serine proteases, kidney diseases through in complex with including plasmin inhibition of vasopressin trypsin); 5M4V and trypsin, leading type 2 receptor pathways220 (mambaquaretin-1) to anticoagulation, fibrinolysis, inflammation and ion-​channel blocking DEF Elapids and No Skeletal muscle Myotoxic damage Anti-​proliferative, 4GV5 (crotamine, 60,61 viperids Na+ and K+ through depolarization anti-​nociceptive, X-​ray); 1H5O channels, lipid of skeletal muscles, and anti-​inflammatory, (crotamine, NMR) membranes and analgesic activity antifungal against sarcolemma C. albicans, anti-​Plasmodium, anti-​Leishmanial and anthelmintic129,221–224 Only toxins from the elapid and viperid families are included. Within each family, there are tens or hundreds of different isoforms. Although there are numerous species-​specific and isoform-​specific exceptions, the most common biological target(s) and pathophysiologic manifestations are reported. Representative structures, when available, are taken from the Protein Data Bank. Additional details on each toxin can be found in the referenced articles. We also refer readers to ref.225, which is an excellent and detailed source of information on the structure, bioactivity, pathophysiology and therapeutic applications of snake venom toxins. 3FTx, three-​finger toxin; ACE-​I, angiotensin-1 converting enzyme; BPPb, bradykinin potentiating peptide b; CRiSP, cysteine-​rich secretory protein; CTL/SNACLEC, C-​type lectin and C-​type lectin-​like; DEF, defensin; DIS, disintegrin; KSPI, Kunitz-​type serine protease inhibitor; LAAO, l-​amino acid oxidase; NMR, nuclear magnetic resonance; NP, natriuretic peptide; PLA2, phospholipase A2; RVV-​V, factor-​V activating enzyme from Russell’s viper venom; SVSP, snake venom serine protease; SVMP, snake venom metalloproteinase. attacks the sn-2 ester bond. A chain of water molecules Some neurotoxic PLA2s have even more refined probably mediates the proton transfer to His48. molecular mechanisms of action that serve as lessons In a twist of evolution, viper PLA2s split between for drug delivery. An example is β-​bungarotoxin from enzymes and catalytically inactive proteins, known as the venom of the Taiwan banded krait (Bungarus multi­ PLA2 homologues. The latter lack the Ca2+ cofactor owing cinctus), which comprises a PLA2–Kunitz-​type peptide to substitution of the Asp49 residue by lysine or, less heterodimer. The toxin travels silently through the commonly, by serine, arginine, glutamine or asparag- victim’s body, avoiding off-​target membranes owing ine. The (Lys49) PLA2 homologues are highly myo- to the partially occluded PLA2 active site and its low toxic, despite having no enzymatic activity. A sequence membrane affinity. When β-​bungarotoxin reaches of ~12 positively charged and hydrophobic residues the pre-​synaptic region of the neuromuscular junction, the at the C-​terminal region, with positive ends and a Kunitz-​type peptide recognizes and binds a specific mixed positive and hydrophobic core (such as the K+ channel, trapping the toxin at this location. This KKYRYYLKPLCKK sequence in MT-​II from the venom event exerts a first neurotoxic action. The PLA2 mon- of terciopelo (Bothrops asper)), is believed to penetrate omer, once firmly anchored at the membrane, opens and destabilize the sarcolemma (Fig. 2b), and thus pro- the active site and initiates hydrolysis of the membrane mote an influx of Ca2+ ions, which starts a chain of phospholipids, near the K+ channel, further enhancing Neuromuscular junction harmful events that leads to myotoxicity32,70,71. Further the neurotoxic effect75–77 (Fig. 2c). A specialized synapse investigation is needed to achieve an atomic-​level under- In summary, PLA2 enzymes and their inactive established between a motor standing of this effect. This stretch of residues alone homologues share extensive sequence and structural neuron and a muscle fibre through which signals for often has similar bioactivity to that of the whole protein similarity, and both induce myotoxicity but through muscle contraction are and is intensively investigated as a model to construct surprisingly diverse molecular mechanisms. The great transmitted. antimicrobial peptides69,72–74. diversity of PLA2 isoforms translates into a surprising 456 | July 2022 | volume 6 www.nature.com/natrevchem 0123456789();:  Reviews Fibrinogen variety of biological activities, underlying molecular haemodynamic pressure41. Continuous hydrolysis of A protein complex in the machinery and recognition targets, and, consequently, fibrinogen in vivo leads to weak, inefficient fibrin clots plasma of vertebrates that is a variety of drug discovery opportunities73. and hypofibrinogenaemia, and the hydrolysis of blood enzymatically and sequentially coagulation factors deregulates blood clotting78,79. converted into fibrin and Snake venom metalloproteinases P-​II SVMPs have an additional disintegrin domain that a fibrin-​based blood clot. Fibrinogen is responsible for SVMPs are mostly haemorrhagic and are classified into inhibits platelet aggregation through specific binding to stopping bleeding from blood three groups (P-​I to P-​III) according to the number of the blood platelet αIIBβ3 integrin — a vital protein that trig- vessels. domains (1–3), with further division into subgroups40,42. gers fibrinogen binding and platelet aggregation80. This P-​III SVMPs are the largest, more ancient and most com- action reinforces the haemorrhagic effect of collagen IV plex enzymes, from which the P-​II and P-​I enzymes evolved hydrolysis. through domain loss. Elapid venoms contain only P-​III P-​ I II SVMPs (Fig. 3) have a catalytic domain, a SVMPs. By contrast, viperid venoms contain SVMPs from disintegrin-like domain with a collagen-​binding three-​ each of the three groups, with SVMPs being a prominent amino acid Glu–Cys–Asp (ECD) motif (instead of the toxin, and often the most abundant one (Fig. 1). typical P-​II Arg–Gly–Asp (RGD) motif) and a cysteine- P-​I SVMPs comprise the catalytic domain only, which rich domain. The prominent role of the latter is substrate catalyses the hydrolysis of a vast array of physiologically recognition and binding. Nevertheless, the catalytic relevant enzymes and structural proteins. This domain domain is also involved in substrate recognition through is common to the three SVMP groups. Its hydrolytic an interesting conformational selection mechanism targets include collagen IV, fibrinogen and coagulation (Box 2). In some isoforms, a C-​type lectin-​like domain factors, with extensive haemorrhagic consequences41,49. is also present40. Hydrolysis of collagen IV weakens capillary walls, The reaction mechanism of SVMPs is not fully which causes them to collapse under otherwise normal clarified despite a wealth of X-​r ay structures 81–83. a c MT-I Sarcolemma KUN PLA2 b MT-II Sarcolemma Membrane K+ channel Fig. 2 | The three main types of PLA2 bound to their targets. a | Myotoxin I (MT-​I), a strongly myotoxic phospholipase A2 (PLA2) from the venom of a terciopelo viper (Bothrops asper), attached to the sarcolemma. MT-​I (PDB ID: 5TFV)226 is shown with a phospholipid substrate bound to the active centre. In the phospholipid, oxygen is red, phosphorus is orange, nitrogen is blue, carbon is grey and hydrogen is white; the enzyme is shown in light red, and the Ca2+ ion is shown in light green. The residues that form the protein–membrane interface and the PLA2–membrane binding geometry were identified through mutagenesis, fluorescence and X-​ray crystallography studies64,65. b | The PLA2 homologue myotoxin II (MT-​II), also from terciopelo venom (PDB ID: 1Y4L)227, bound to the sarcolemma. The C-​terminal region destabilizes and permeabilizes the membrane70. The protein is shown in light green, and the C-​terminal KKYRYYLKPLCKK sequence is shown in pink. c | β-​Bungarotoxin (PDB ID: 1 BUN)75 from the Taiwan banded krait (Bungarus multicinctus) bound to a neuronal membrane. The toxin travels silently through the victim’s body until its Kunitz (KUN) domain (green) recognizes and binds a presynaptic voltage-​gated K+ channel (violet, PDB ID:6PBX) with high specificity, trapping the PLA2 domain (light blue) at the neuronal membrane, where its active site, otherwise occluded, opens and starts degrading the adjacent phospholipids (bound phospholipid coloured by element)75–77. NATuRe RevIewS | ChEMISTRy volume 6 | July 2022 | 457 0123456789();: Reviews a His149 His145 His155 b Glu146 MET Zn2+ DIS Collagen Active GM6001 polypeptide site Tropo- collagen Tropocollagen CR SNACLEC Collagen fibre Fig. 3 | Structures of SVMPs and their substrates. a | The structure of the factor X activating enzyme RVV-​X (PDB ID: 2E3X)81 from the eastern Russell’s viper (Daboia siamensis). RVV-​X is a P-​III snake venom metalloproteinase (SVMP) isoform that is ubiquitous in species from the Indian subcontinent228. RVV-​X activates blood coagulation factor X by hydrolysing the Arg194–Ile195 position with such high specificity that it is used as a diagnostic tool for haematologic disorders26,93,94,229. The catalytic domain (MET) is coloured yellow, the disintegrin (DIS) domain is coloured green and the cysteine-​rich domain (CR) is coloured pink. RVV-​X has an additional C-​type lectin and C-​type lectin-​like protein (CLT/SNACLEC) domain, which is shown in blue. The inset shows the Zn2+ cofactor with its coordination shell and the peptidomimetic inhibitor GM6001, whose two coordinated oxygen atoms mimic the positions of the water molecule and the carbonyl of the substrate (superimposed on top of GM6001 with a translucent ball and stick representation). b | Illustrative scheme of daborhagin230, a highly haemorrhagic SVMP from Russell’s viper venom, bound to collagen IV at the basement membrane of capillaries. The colour scheme of the enzyme domains is the same as that of RVV-​X in part a. A collagen IV fibre is shown in light green, with a tropocollagen unit emphasized in dark green and drawn in a cartoon and tube representation. The hydrolysis of collagen IV weakens the mechanical stability of the capillary wall, which breaks down under regular haemodynamic forces, leading to massive haemorrhage. Daborhagin was modelled with the active site facing collagen IV. The mechanism is proposed82 to begin with the coordina- SVSPs deregulate homeostasis43,85. SVSPs that share some tion of the scissile carbonyl of the substrate to the Zn2+ ion of the fibrinogenolytic activities of thrombin have been in the active site, such that the carbonyl group is held named thrombin-​like enzymes. at an attacking distance from a water molecule also SVSPs are monomeric glycoproteins with ~228–239 bound to the Zn2+ centre. Following deprotonation of residues and a molecular mass of 26–67 kDa (refs34,43). the water molecule by a conserved glutamate residue This wide range of molecular masses is due to differ- (Glu146), the resulting hydroxide ion attacks the car- ent patterns of N-​glycosylation and O-​glycosylation. bonyl carbon to form a Zn2+-​bound gem-​diolate. Finally, The enzymes share the typical trypsin fold and the the neutral Glu146 protonates the peptide amine, leading highly conserved Ser195–His57–Asp102 catalytic triad to cleavage of the peptide bond. (chymotrypsin numbering) (Fig. 4). Six disulfide bonds stabilize the structures. Snake venom serine proteases Most SVSPs share the classical reaction mechanism SVSPs are primarily haemotoxic and interfere with of serine proteases. However, >20 SVSPs with variations blood coagulation, blood fibrinogen levels, blood pres- in the canonical catalytic triad have been found in snake sure and platelet aggregation34,36,43,50,51 (Fig. 4a,b), although venom transcripts86. Among the few of these that have there is one known example of an SVSP with K+-​channel been characterized, the horned viper (Vipera ammodytes blocking activity84 (Fig. 4c). The resistance of SVSPs to ammodytes) serine protease VaSP1, which bears the rare endogenous serine protease inhibitors endows them Ser195–Lys57–Asp102 triad, was surprisingly found with their toxic effects. Many of the activities of SVSPs to be catalytically active87, illustrating an unexpected mimic those of the enzyme thrombin, which is a vital richness in SVSP chemistry. component of the blood coagulation cascade. Each SVSP In contrast to thrombin, which activates many dif- exhibits one or more of the activities of thrombin and ferent coagulation factors (factor V, factor VIII, fac- sometimes has bioactivities that thrombin does not. But tor XI and factor XIII, as well as fibrinogen), each SVSP no SVSP possesses all the bioactivities of thrombin85, is highly substrate-​specific50. However, as different which makes them toxic and, in contrast to thrombin, SVSPs are specific for different sets of targets, a group 458 | July 2022 | volume 6 www.nature.com/natrevchem 0123456789();:  Reviews Intrathecal administration of isoforms can induce diverse physiological manifes- drugs approved by the FDA and EMA, in part because Invasive drug administration by tations. This recognition diversity is striking given their they affect a system whose physiology is well known injection through the skull or extensive mutual sequence identity (50–85%), which and easier to manipulate. As cardiovascular diseases the spine, allowing the drug to is a phenomenon known as the identity–selectivity are the leading cause of death globally, the develop- reach the cerebrospinal fluid, and thus the brain, without paradox50: their specificity cannot be understood from ment of snake-​venom-​derived drugs that target the crossing the blood–brain the primary sequence. Instead, the specificity appears to cardiovascular system is appealing. barrier. depend on a combination of subtle structural epitopes, primary and secondary binding sites, enzyme flexibil- Captopril. The antihypertensive drug captopril was the ity, glycosylation and water organization50. The precise first drug based on a bioactive component from snake specificity and intense haemoactivity of SVSPs make venom that was approved in the US by the FDA in 1981 them potential diagnostic and therapeutic tools in the and in European countries from 1984 onwards. The cardiovascular area. realization that envenoming by the South American pit The chemistry of snake venoms is partially under- viper jararaca (Bothrops jararaca) caused notable hypo- stood for the major enzymes, but further understanding tension led to the discovery of the vasodilator peptide at the atomic level is required. Computer simulations are bradykinin in its venom97. Subsequent studies led Sérgio one of the best ways to answer remaining questions, par- Ferreira and colleagues to discover a set of nine pep- ticularly given the recent advances in quantum mechan- tides in the venom of jararaca that potentiated the effect ical and classical mechanics methods, which enable of bradykinin, named bradykinin potentiating factors the reliable prediction and determination of complex (BPFs)98–100. BPFs inhibit the angiotensin-​converting chemical reaction mechanisms88–91. enzyme (ACE)101, which otherwise degrades bradykinin. The therapeutic potential of BPFs led the pharmaceuti- Drugs from snake venom toxins cal company Squibb to develop a drug against hyperten- Snake venom finds three major therapeutic applica- sion using BPF peptides (BPP5a and BPP9a, in particular) tions: pharmaceutical drugs4,92, toxin-​based diagnostic as templates102 (Fig. 5a). The result was captopril, a small, methods92–94 and biological markers for understanding synthetic, orally bioavailable and potent bioactive mol- human physiology26. We focus here on pharmaceutical ecule with a structure and electrostatics that mimic the drugs based on snake venom. This section discusses BPP5a Pro–Ala–Trp recognition motif for ACE. the snake venom toxins and toxin-​inspired mole- Captopril was a milestone in many ways: it was the cules that are being used to develop new drugs, focus- first drug developed from animal venom; it was created ing on the drugs approved by the US Food and Drug by converting toxic action into therapeutic action; it was Administration (FDA) and the European Medicines one of the first examples and a paradigm of ligand-​based Agency (EMA) as well as drugs under development in drug discovery; and it was the first drug targeting ACE, preclinical and clinical trials. rapidly becoming a blockbuster and saving countless lives100. Approved drugs To overcome the side effects of captopril caused by Snake venoms are typically cytotoxic, neurotoxic and its thiol group, Merck developed enalapril102,103 (Fig. 5a). haemotoxic. The anticancer potential of cytotoxins has The thiol group in captopril was replaced by a car- long been recognized92. Neurotoxins are of interest for boxylate, leading to a loss of potency, which was com- the treatment of neurological diseases. However, no pensated with additional modifications. The resulting drug derived from a snake venom neurotoxin has yet compound (enalaprilat) lacked oral bioavailability, most reached the market. The complexity of the human neu- probably owing to the ionic carboxylate. Enalaprilat was rological system, our insufficient understanding of this converted into its ethyl ester to overcome the problem, system and the difficulty in delivering medications to giving rise to enalapril, a prodrug with very good oral the nervous system contribute to the slow progress of bioavailability21,104 and approved by the FDA and EMA. this line of drug discovery20. Nevertheless, the FDA and Enalapril became Merck’s first billion-​dollar-​selling drug EMA approved ziconotide, a ω-​conotoxin peptide from in 1988. the magic cone snail (Conus magus), as an analgesic for Many ACE-​inhibitor drugs based on the BPP5a bind- severe chronic pain4,21,95,96. The main limitation of this ing motif were subsequently developed and approved. drug is its intrathecal administration route. In contrast to Examples include lisinopril, quinapril, ramipril, tran- neurotoxins, haemotoxins have given rise to numerous dolapril and moexipril105,106, which, despite being fre- quently dismissed in the snake-​based drug discovery Box 2 | Substrate recognition by snake venom metalloproteinases world, deserve to be considered snake-​venom-​based Despite extensive sequence identity, only some snake venom metalloproteinases (SVMPs) drugs. These drugs are among the most prescribed glob- bind collagen IV and have haemorrhagic activity. Molecular dynamics simulations ally and showcase the immense therapeutic potential of explained this paradox by revealing that in haemorrhagic SVMPs, the first half of the venoms, which is yet to be fully realized. Ω-​loop (residues 156–165) is highly flexible, whereas in non-​haemorrhagic SVMPs, the second part of the Ω-​loop (residues 166–175) is flexible instead. On the basis of this Tirofiban. Tirofiban is an antiplatelet drug approved by observation, the authors proposed that the flexibility of this loop is crucial for collagen IV the FDA in 1998 and the EMA in 1999 for treating acute recognition238 and, more recently, this hypothesis has been experimentally confirmed239. coronary syndrome107–110. Its structure is derived from Thus, the lesson here is that target recognition might rely on protein dynamics and the toxin echistatin111, a 49-​residue disintegrin from saw-​ not only on the static X-​ray structure; this constitutes a formidable challenge for drug scaled viper (Echis carinatus) venom. Echistatin com- discovery. petes with fibrinogen for binding to the αIIBβ3 integrin, NATuRe RevIewS | ChEMISTRy volume 6 | July 2022 | 459 0123456789();: Reviews which inhibits the final step in platelet aggregation92. minimal sequence for αIIBβ3 recognition. It binds sev- Echistatin thus reinforces the haemorrhagic activity of eral integrins with sub-​nanomolar affinity, with selec- saw-​scaled viper SVMPs. tivity for αIIBβ3 over others107. In high concentrations, Echistatin shares the RGD motif of the disinteg- the isolated RGD tripeptide also inhibits platelet aggre- rin domains of many P-​II-​type SVMPs, which is the gation. Tirofiban was modelled to replicate the RGD a FV14 Ser195 FV14 His57 Asp102 RVV-V b c Batroxobin Collinein-1 Membrane Fibrinogen K+ channel Fig. 4 | Structure of SVSPs and their substrates. a | The factor V activating enzyme from Russell’s viper venom (RVV-​V; PDB ID: 3S9C), which is a snake venom serine protease (SVSP) with specificity for blood coagulation factor V. RVV-​V is depicted in a complex with the 14-​residue terminal fragment of factor Va (residues 1533–1546), called FV14. RVV-​V releases the last 61 residues of factor V by hydrolysing its Arg1545–Ser1546 bond, generating procoagulant factor Va and mimicking one of the physiological roles of thrombin231. The inset shows the active site and factor V hydrolysis product. RVV-​V recognizes factor V through a selective induced-​fit mechanism that opens an otherwise closed subpocket. The strict specificity of RVV-​V for factor V makes it a useful diagnostic tool for measuring factor V levels, lupus anticoagulant levels and resistance to activated protein C93,94. b | Illustrative representation of the thrombin-​like Brazilian lancehead pit viper (Bothrops moojeni) SVSP batroxobin (Defibrase)4,21,118,119,232 bound to fibrinogen. Thrombin cleaves the Aα and the Bβ chains of fibrinogen and converts factor XIII into factor XIIIa, which generates crosslinked fibrin, whereas most SVSPs cleave either the Aα or the Bβ chain only85. Batroxobin cleaves only the Aα chain118,232. SVSPs therefore form abnormal, easily degradable fibrin clots that lead to fibrinogen depletion and hypofibrinogenaemia. The clotting time in the presence of batroxobin (reptilase time) is used in the clinic to diagnose several diseases93,94. Batroxobin was modelled from the homologue saxthrombin (PDB ID: 3S69). c | Collinein-1 from the neotropical rattlesnake (Crotalus durissus collilineatus) is the first example of an SVSP with specific K+-​channel blocking activity84. Through a mechanism that is independent of its enzyme activity, collinein-1 selectively inhibits the oncogenic hEAG1 channel (PDB ID:6PBX) among 12 tested voltage-​gated K+-​channels, with obvious antitumour implications. As K+ channels are known targets for many animal neurotoxins, the discovery of collinein-1 makes it tempting to speculate that some yet unknown SVSP isoforms might have found a neurotoxic role. Collinein-1 was modelled from the homologue thrombin-​like enzyme AhV_TL-​I (PDB ID: 4E7N) and is illustratively bound to the oncogenic hEAG1 channel. 460 | July 2022 | volume 6 www.nature.com/natrevchem 0123456789();:  Reviews a Fig. 5 | Approved drugs derived from snake venoms. W A P Several drugs derived from snake venoms have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The chemical BPP5a structures of these drugs are shown, with the region that mimics the snake toxin highlighted in grey. a | Nine hypotensive bradykinin potentiating peptides (BPPs) were O OH O isolated from the venom of the jararaca viper; they inspired the design of the antihypertensive drugs captopril and N Captopril enalapril. These drugs mimic the Trp–Ala–Pro (WAP) HS motif by which BPP5a (top right) recognizes its target: O the angiotensin-​converting enzyme (ACE). ACE is shown H N on the left in a complex with BPP5b, another BPP (PDB ID: N 6QS1). The ACE Zn2+ cofactor is shown in orange. O O b | The drug tirofiban was inspired by a disintegrin called HO Enalapril O echistatin found in the venom of the saw-​scaled viper. Echistatin, shown on the left (PDB ID: 6LSQ), binds specifically to the αIIBβ3 integrin through its Arg–Gly–Asp b (RGD) motif (coloured spheres and top right), which prevents platelet aggregation. In tirofiban, the piperidine D G R moiety replicates arginine, the aliphatic linker replicates glycine, and the tyrosine carboxyl group replicates the aspartic acid carboxylate. The (S)-​NHSO2nC4H9 group increases the affinity of tirofiban for its αIIBβ3 target. c | Eptifibatide is an antiplatelet drug inspired by the disintegrin babourin purified from the venom of Barbour’s O pygmy rattlesnake. A homology model of babourin H N (template PDB ID: 1J2L) is shown on the left. Most S OH disintegrins recognize the αIIBβ3 integrin through the O O NH RGD motif, but babourin uses a Lys–Gly–Asp (KGD) motif (coloured spheres and top right). Eptifibatide achieves O maximum selectivity owing to the fusion of the two motifs Tirofiban into the unnatural homoRGD motif. Additional peripheral residues and cyclization endow further molecular recognition capabilities and resistance to proteolysis. Eptifibatide. Eptifibatide is another antiplatelet drug c G D approved by the FDA in 1998 and EMA in 1999 that was K developed from a disintegrin (barbourin) found in the venom of Barbour’s pygmy rattlesnake (Sistrurus miliar­ ius barbourin)107,108,114,115. Barbourin binds the αIIBβ3 inte- H grin through a Lys–Gly–Asp (KGD) motif, rather than O O O the more common but less specific RGD motif. The H H H O H KGD motif provides excellent specificity for the αIIBβ3 HN N N N H H H O N integrin over other integrins115. H HNH H N Residues adjacent to the KGD motif greatly affect NH O H the affinity of barbourin. Therefore, these neighbour- H H ing regions were also elucidated during the develop- H H O ment of eptifibatide115,116. The final form of the drug H N S consists of a heptapeptide cyclized through a disulfide S H N bridge. Cyclization provides superior resistance to H H H O Eptifibatide proteolysis21,116,117. Interestingly, the motif presenting the H N H O highest affinity and specificity for the αIIBβ3 integrin was neither RGD nor KGD, but a ‘hybrid’ of these, homoRGD (Fig. 5c). This surprising result indicates that there are lim- its to the structural versatility of protein toxins based on motif of echistatin within a small synthetic molecule92. a small number of genetically encoded amino acids. The The affinity of tirofiban for αIIBβ3 was enhanced by the optimal structural solutions for molecular recognition (S)-​NHSO2nC4H9 extension, which interacts with an might not be achievable through genetically encoded αIIBβ3 exosite with which echistatin does not interact112 amino acids only, and might instead require complex and (Fig. 5b). The affinity and specificity of tirofiban thus sur- metabolic post-​translational modifications that are too pass those of echistatin. Tirofiban is another example of expensive for a secretion that a snake frequently depletes the transformation of a venom toxin into a life-​saving and reproduces. The versatility of synthetic chemistry drug. It is also one of the first documented successful presents an advantage that can be exploited to achieve pharmacophore-​based drug discovery applications113. affinity and specificity beyond what is observed in nature. NATuRe RevIewS | ChEMISTRy volume 6 | July 2022 | 461 0123456789();: Reviews In addition to the drugs approved by the FDA and the occlusive diseases, and peripheral and microcirculation EMA, other snake venom toxins have been approved for dysfunctions. clinical use in other countries and are described below. Haemocoagulase. Haemocoagulase (Reptilase)21,120 is an Batroxobin. Batroxobin (Defibrase) is a thrombin-​ enzyme system purified from the venom of the com- like serine protease purified from the venom of the mon lancehead pit viper (Bothrops atrox). The enzyme Brazilian lancehead pit viper (Bothrops moojeni) that system includes batroxobin and an SVMP that activates induces defibrinogenation4,21,118,119. This toxin is mar- factor X, which results in anti-​haemorrhagic activity. keted in China and Japan for the treatment of acute Haemocoagulase is approved for use in Japan, India and cerebral infarction, ischaemia caused by vascular South Korea to treat internal and external haemorrhages. a b α-​Cobrotoxin. α-​Cobrotoxin, which is purified from the venom of the Chinese cobra (Naja atra)21,121,122, is a 3FTx α-​neurotoxin that binds nicotinic acetylcholine receptors at the neuromuscular junction. α-​Cobrotoxin is approved for use in China as an analgesic for moder- ate to severe pain. However, its high bioactivity might lead to side effects, such as respiratory arrest. Drugs in preclinical and clinical trials Several compounds based on components from snake venom are in preclinical and clinical trials21,120,123. We focus on selected examples that are among the most promising and advanced in preclinical or clinical trials. Anfibatide. Anfibatide is an anticoagulant C-​type lectin-​ like protein purified from the venom of the sharp-​nosed viper (Deinagkistrodon acutus). The protein is heterod- c d imeric, comprising α-​subunits and β-​subunits linked by seven disulfide bonds. The anticoagulant activity of anfibatide is due to its strong binding to human plate- let glycoprotein Ib α-​chain (GPIbα), which inhibits the binding of GPIbα with von Willebrand factor (VWF) and thrombin124,125 (Fig. 6a). The binding of GPIbα and VWF is key to triggering platelet adhesion and throm- bosis, particularly under the high shear stress conditions at sites of arterial stenosis126, which lead to myocardial infarction and stroke. In addition, anfibatide decreases thrombus volume and stability124. Recombinant anfibatide was produced at a pilot scale in yeast127, avoiding issues relating to quality control and the limited supply of snake venom. Anfibatide might become the first drug to target GPIbα, which would be Fig. 6 | Drugs derived from snake venoms in clinical or preclinical trials. a | Anfibatide a game-​changer for anticoagulant therapy, as anfibatide (blue cartoon) is a snake C-​type lectin-​like protein that is predicted to bind to platelet does not interfere with haemostasis and thus does not seem glycoprotein Ib α-​chain GPIbα (orange surface)124 at a site that partially overlaps with to cause the haemorrhages that currently marketed drugs the GPIbα–von Willebrand factor binding surface (PDB ID: 1SQ0), thus inhibiting the do. So far, anfibatide has passed phase I clinical trials124. association of von Willebrand factor and consequently platelet aggregation. Anfibatide is a promising anticoagulation candidate that has passed phase I clinical trials. b | Crotamine is an amphipathic and highly basic defensin that penetrates cells and is resistant to Crotamine. Another toxin with tremendous therapeu- proteolysis. Crotamine exhibits antiproliferative, antinociceptive and analgesic activity tic potential is crotamine. This toxin is a small defen- in vivo upon oral administration. Cationic residues are shown as sticks and the disulfide sin purified from the venom of some populations of bonds are shown in yellow. c | Dendroaspis natriuretic peptide (DNP) from the eastern the South American neotropical rattlesnake (Crotalus green mamba (ochre tube with the disulfide bond in yellow) bound to the dimeric durissus)128. Although the venom is very toxic, crotamine particulate guanylyl cyclase A receptor (shown as a lime surface and a green transparent has low myotoxicity and neurotoxicity129. Crotamine is a cartoon) (PDB ID: 7BRI). Cenderitide is a natriuretic peptide chimaera resulting from the very basic (with a pI of 10.3 and a charge of +8) amphip- fusion of human C-​type natriuretic peptide (CNP) to DNP and co-​activates both DNP and athic 42-​residue peptide with three disulfide bridges and CNP transmembrane receptors. d | The three-​finger toxins mambalgin-1 and mambalgin-2 structural folds similar to those of human α-​defensins bind to the acid-​sensing ion channels 1a and 1b, locking the channels in the closed state and impairing their function, with an analgesic effect as potent as that of morphine and β-​defensins60,130 (Fig. 6b). but with much lower toxicity in rodents. The complex of mambalgin-1 (green) with the Crotamine is a cell-​penetrating peptide that is rap- transmembrane (light yellow) acid-​sensing ion channel 1a (violet) is shown (PDB ID: 7CFT). idly internalized into almost all cell types131. The pri- The mambalgins are promising scaffolds for the development of a new generation mary cytotoxicity mechanism is accumulation in and of analgesics. disruption of lysosomes132. Crotamine has very high 462 | July 2022 | volume 6 www.nature.com/natrevchem 0123456789();:  Reviews Natriuresis selectivity for actively proliferating cells133, such as pathophysiological processes, such as ischaemic strokes The process of excretion cancer cells, making it a promising antitumour agent132. and tumour growth148. Mambalgins inhibit ASIC1a and of sodium in the urine. Its anti-​melanoma activity was demonstrated in mice134 ASIC1b in the central and peripheral nervous systems without toxicity to healthy cells and it can even be with nanomolar affinity, both in vitro (rat and human) and administered orally129,135 owing to its excellent resistance in vivo (rat)146,149,150. In rodents, administration of mam- to proteolysis and its cell-​penetrating ability. balgins into the peripheral or central nervous systems Among other bioactivities (Table 1) , crotamine strongly abolishes acute and inflammatory pain146,151,152, also exhibits antinociceptive activity (and is 500 times with an analgesic effect as potent as that of morphine more potent than morphine (mol mol–1))136 and anti- but with much less tolerance and without the respiratory inflammatory activity in in vivo mouse models and arrest typical of morphine and toxic side effects146. Thus, upon oral administration129,135 without toxic side effects. mambalgins represent molecular scaffolds for a new Furthermore, its chemical and recombinant syntheses generation of strong, non-​toxic analgesics. were recently reported137–139, which are fundamental Mambalgin-1 and mambagalin-2 have been chem- steps required for upscaling crotamine production. Thus, ically synthesized and their structures determined149,150. the therapeutic future of crotamine looks promising. These mambalgins represent a new family of 3FTx; they share the core of a typical 3FTx but with short first Cenderitide. Cenderitide is a natriuretic peptide based and third fingers and an elongated middle finger. The on one purified from the venom of the eastern green structure of the human ASIC1a channel, both free and mamba (Dendroaspis angusticeps) and is under clinical bound to mambalgin-1, was determined in 2020 (ref.153). trials for the treatment of heart failure55,140. Experimental and computational studies154 used this Natriuretic peptides are regulators of body fluid vol- structure to refine earlier proposals for the working model ume and induce natriuresis, diuresis, vasodilation and of ASIC inhibition, that is, the locking of the channel in hypotension, as well as inhibiting fibrosis, among other the closed state (Fig. 6d). The recent wealth of activity bioactivities. Natriuresis and diuresis are essential for and structural data have laid a solid foundation for the the treatment of heart failure141. Three natriuretic pep- structure-​based rational design of mambalgin analogues tides (atrial natriuretic peptide (ANP), brain natriuretic with favourable delivery routes. peptide (BNP) and C-​type natriuretic peptide (CNP)) are endogenous to humans. They are small peptides Toxins targeting SARS-​CoV-2 virus. We conclude this of 28, 32 and 22 (or 53) residues, respectively, with a section with a review of early-​stage in vitro tests of toxins highly conserved 17-​residue cyclic structure (Fig. 6c). that target the SARS-​CoV-2 virus, which have directed Natriuretic peptides exert their effects by activating the considerable attention to the medicinal potential of particulate guanylyl cyclase (pGC)-​A and pGC-​B trans- sna