Why to Study Peptides from Venomous and Poisonous Animals?

Venom and poison peptides are powerful biological weapons and have proven immense pharmacological potential because of their high binding affinity to a wide range of molecular targets. Nonetheless, many of these peptides cannot directly be used as medicines due to their toxicity but their derivatives are very valuable to explore and can be a great treasure trove for the development of novel drugs. This review presents a detailed overview of venom peptides present in reptiles, amphibians, arachnids, gastropods, clitellatas, fish, insects, and mammals. We address the most recent findings that underline their therapeutic potential against a wide variety of diseases from cancer to vascular, autoimmune, and inflammatory diseases.


Venoms vs. Poisons
First, it is important to conceptualize and distinguish the two terms: venom and poison. Both can lead to the victim death but are two different biological weapons. The major difference between venom and poison depends on how the toxins are secreted and transferred to the prey or to the victim. However, according to Jared and colleagues (Jared et al. 2021), in practice there is no consensus among theorists and naturalistic biologists regarding the use of both terms.
The authors support a more complete definition of both terms, in which, in addition to conventional and functional interpretation, include: (i) The type of chemical defensive behavior of the organism, whether passive or active; (ii) Venomous animals inject their toxins directly into the tissues and/or organs of their victims. Poisonous animals, on the other hand, usually release venom through their mucous membranes; (iii) The glands of active or passive organisms seem to show anatomical and morphological differences. In active defense, the glands communicate with teeth or fangs and are surrounded by muscles that assist the rapid injection of the venom. In passive defense organisms (such as in amphibians), the action promoted by aggression, for example through bites, provokes the ejection or release of the poison that first reaches the mucous membranes or the skin, indirectly accessing the blood circulation. In this sense, for this editorial we will consider both terms in an integrated manner, as previously proposed.

Venomous and Poisonous Animals
Venomous and poisonous animals are those capable of producing toxic secretions for both protection and predation. These animals are widely distributed throughout the world, but envenomation accidents occur mainly in tropical and subtropical regions where the abundance of species is greater .
Snakes and scorpions are the envenoming protagonists responsible for the most serious accidents. Snakes alone cause an estimated 2.7 million envenomings every year and up to 138 thousand deaths (Chippaux 2008). As a result, the World Health Organization (WHO) has included snake bite envenomation in the list of neglected tropical diseases, noting that a new approach to preventing and treating snake bite envenomation is urgently needed (Chippaux et al. 2019;Basnyat and Shilpakar 2022).
Each animal venom is unique. Venoms consist of a species-specific heterogeneous complex mix, often a mixture of peptides, enzymes, low molecular weight organic molecules, and inorganic salts (Simoes- Silva et al. 2018). All molecules present in the venom have physicochemical and biological properties that result from an evolutionary process, providing such animals an efficient mechanism of subjugation and feeding, which also acts as a defense weapon (Guimaraes et al. 2014;Estevao-Costa et al. 2018;Fischer and Riedl 2022).

Venoms: Villains or Heroes?
The mere sight of venom animals strikes terror in the hearts of millions of people, but venom has been used in traditional medicine for centuries. It is true that accidents with venomous animals, even mild ones, can lead to death. However, because of their pharmacologic potential, venom peptides (and other molecules) are also of great use to humanity and save many more lives than they take (Almeida et al. 2016;Correa et al. 2016;Charitos et al. 2022;Oliveira et al. 2022;Diniz-Sousa et al. 2023). Peptide toxins have high affinity to various molecular targets such as enzymes, transmembrane receptors, and cell membranes. They destabilize the victims or prey physiological systems which are essential for their survival (Calvete 2018). As a result, venom peptides exhibit hemotoxic, cytotoxic and/or neurotoxic effects, among others, achieved by specific interactions between the toxin and its target (Warrell 2017(Warrell , 2019Akef 2018;Simoes-Silva et al. 2018;Almeida et al. 2019). Due to the ability of toxins to interact with key physiological targets, which are fundamental to homeostasis, many peptides from animal venoms have the potential to produce prototype therapeutic molecules that can help organisms restore their normal functions.
Thus, it is easily conceivable that such molecules can be used as powerful therapeutics in appropriate doses and in the right context (Herzig et al. 2020). In fact, many isolated peptides from venom toxins showed therapeutic potential exhibiting antitumor, antimicrobial, anticoagulant, or analgesic activities ( Fig. 1) (Correa et al. 2016;Diniz-Sousa et al. 2018;Simoes-Silva et al. 2018;da Silva Caldeira et al. 2021).
With the advent of protein structural techniques and the refinement of isolation and characterization methods, it became possible to clarify the structure and pharmacological activities of these bioactive molecules and to better understand venom toxins. Such studies have helped elucidate toxin interactions with specific molecular targets and led to effective drugs for various pathologies (Almeida et al. 2017(Almeida et al. , 2018(Almeida et al. , 2019Utkin 2017;Mendes et al. 2019).
It is important to mention that while snakes and lizards have large venom glands that easily produce enough venom quantities for biochemical, pharmacological, structural, and functional studies, other venomous animals such as spiders, scorpions and bees produce much smaller amounts, which has long been a challenge to study. Current sequencing technologies, peptide engineering and the latest computational tools have greatly overcome these obstacles. In fact, after 1980, many drugs were developed from animal venom toxins and approved by the Food and Drug Administration ( FDA) and the European Medicines Agency ( EMA) (Ma et al. 2017;Bordon et al. 2020;Coulter-Parkhill et al. 2021) (Table 1). This review summarizes the most relevant animal venom peptides that have led to powerful drugs or are currently being evaluated in clinical or preclinical trials.

Snakes
Animal venom-derived peptides are a potential source of inspiration to produce new drugs and therapies. Toxins isolated from snake venom are a great example of molecules that led to important drugs in clinical practice. In fact, there are eight small synthetic drugs inspired by snake venom toxins approved by the FDA and EMA, which mainly aim to treat cardiovascular disease, a global public health problem that causes more than 18 million deaths a year. Captopril ( Fig. 3; Table 1) was the first drug to be developed from snake venom. It is an antihypertensive molecule used to control blood pressure by inhibiting the angiotensin-converting enzyme (ACE) (Natesh et al. 2004;Camargo et al. 2012;Harvey 2014). Captopril is a drug derived from bradykininenhancing peptides isolated from the venom of the Brazilian jararaca (Bothrops jararaca), see Fig. 5A. The drug was approved by the FDA in 1981 and in Europe in 1984. Since Fig. 1 Examples of peptides derived from animal venoms that present therapeutic potential. In the figure are indicated 10 animals: snakes, spiders, scorpions, bees, amphibians, leeches, lizards, fish, shrews, and snails, and their molecular and cellular targets (A to F) A neuronal cells; B muscular cells; C cancer cells; D blood cells; E bacterial cell; and F pancreatic cell. Figure created with BioRender.com (2020) (https:// app. biore nder. com/ biore nder-templ ates) Table 1 FDA and EMA approved drugs and therapies for human clinical therapeutics use (Information based in (Mohamed Abd El-Aziz et al. 2019;Bordon et al. 2020;Coulter-Parkhill et al. 2021) Molecules Cleaves Aα chain of fibrinogen Acute cerebral infarction; Unspecific angina pectoral; Sudden deafness Straub and Harder (1971), Latallo and Lopaciuk (1973), Stocker and Barlow (1976), Jiang et al. (2000) Batroxobin (Plateltex-Act®) Bothrops atrox (Common lancehead snake) Cleaves Aα chain of fibrinogen Gelification of blood for topical applications Mazzucco et al. (2008) Harris and Smith (1981) and Jump (1982) Cobratide  Yantis et al. (2009) and Zakian et al. (2022) Page 5 of 39 76 then, it has become a blockbuster drug with an annual turnover of more than $1 billion. After all these years, captopril, and its derivatives, such as enalapril, ramipril, and lisinopril, remain among the four most prescribed drugs worldwide for hypertension (Table 1) (Mohamed Abd El-Aziz et al. 2019;Bordon et al. 2020).
Other relevant snake venom component with therapeutic interest are the disintegrins. These small cysteine-rich peptides are often found in viper venom and can develop severe toxic effects due to their high affinity for the α and ß subunits of the integrins, a cellular membrane receptor directly associated with the integrity of mammalian tissues. Disintegrins act as powerful integrin antagonists because they recognize integrins based on a tripeptide motif located on a specific binding loop of disintegrins. Well-known examples of the tripeptide recognition motifs are: the RGD motif, present on Echistatin and Bitistatin, the KGD motif of Barbuorin, and KTS motif of Obtustatin. Other similar tripeptide motifs present on disintegrins are: MLD, VGD, MGD, WGD, MVD and RTS. (Almeida et al. 2016;Mohamed Abd El-Aziz et al. 2019;Boldrini-Franca et al. 2020;Trim et al. 2021;Chan et al. 2022;Siigur and Siigur 2022). Yet, the RGD motif deserves particular attention due to its ability to bind 8 out of the 24 known integrins-αIIbβ3, α5β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8-which comprises not only the integrins that bind fibrinogen, glycoproteins, fibronectin and thrombospondin, but also the 4 collagen receptors, 4 laminin receptors, and 8 leukocyte receptors (Notni 2022). In contrast, the KGD motif recognizes specifically the αIIbβ3 on the platelet surface (Bachmann et al. 2019), and KTS motif recognizes the α1β1 integrin receptor (Khamessi et al. 2022).
Onwards, we will discuss two disintegrins used to develop drugs for the treatment of acute coronary syndrome, namely tirofiban and eptifibatide (Frangieh et al. 2021;Vasconcelos et al. 2021). The first, tirofiban ( Fig. 3; Table 1), is a nonpeptide drug antagonist that recognizes and competes with the fibrinogen to bind to the αIIBß3 integrin on platelets, which hinders its aggregation. Tirofiban was inspired by the echistatin structure (Fig. 4A), a short monomeric disintegrin, that present a RGD motif and was isolated from the Echis carinatus venom (Saw-scaled viper, Fig. 5B) (Gan et al. 1988;Garsky et al. 1989;Lazarovici et al. 2019;Coulter-Parkhill et al. 2021).
Tirofiban and eptifibatide are examples of pharmaceutical success based on inhibition of adhesion processes by integrins. Adhesion processes are fundamental to the production of the tumor microenvironment and the development of cancer. The ability of disintegrins to inhibit adhesion processes has encouraged researchers to devote enormous efforts and financial resources to understanding how ophidic disintegrins can be used against cancer. In fact, disintegrins induce antitumoral effects such as antiproliferative, cytotoxic, proapoptotic, antiangiogenic, antiinvasive and antiadhesive effects. They also inhibit migration, metastasis and invasion in lung, breast, pancreatic, prostate and melanomas (Mullooly et al. 2016;Scharfenberg et al. 2020). Yet, a deep knowledge about disintegrins and their involvement in cancer-associated cell mechanisms is needed to explore their potential and produce novel anticancer drugs (Calderon et al. 2014;Huang et al. 2019;Zhang et al. 2022;Zhou et al. 2022). Some of those examples are shown in Table 2. Boldrini-Franca et al. (2019) produced recombinant Collinein-1 (Fig. 1), a snake venom serine protease (SVSP) from the venom of Crotalus durissus collilineatus expressed in the Pichia pastoris system. Collinein-1 blocks the human etherà-go-go ion channel (hEAG1), a voltage-gated potassium (Kv) subfamily, which is overexpressed in cancer cell lines and is implicated in the progression of tumor cells (Boldrini-Franca et al. 2019). Collinein-1 reduced the viability of the human breast cancer cell line MCF7, which exhibits high expression of hEAG1. However, the toxin has no effect on Disintegrins derived drugs that have been approved by the FDA and EMA. The figure shows on the top a diagram of the peptides primary structure, where is highlighted the integrin recognition motif and the sulfur bridges, characteristic of this peptide family. It is also shown the secondary structure of these Viperidae toxins, where the residues pinpointed in the diagram are represented as spheres and sticks. On the bottom is shown the chemical structure of the medicines and the moieties that replicate the biopolymer. A Echistatin from Echis Carinatus (PDB ID. 2ECH) binds specifically to the αIIBβ3 integrin by the RGD motif and inspired the synthesis of Tirofiban. The (S)-NHSO 2 C 4 H 9 group increases the binding affinity of tirofiban for its target. B Barbourim from Sistrurus miliarius barbourin (Alpha fold model AF-P22827-F1) also binds the αIIBβ3 integrin and inspired the synthesis of eptifibatide. The last accomplishes maximum selectivity by melding two motifs into the unnatural homoRDG motif HepG2 (human liver cancer) and MCF10A (human breast cancer) cell lines that are low in expression of this ion channel. These findings suggest that the reduction in cell viability is related to hEAG1 by Collinein-1 (Bordon et al. 2020).
Crotamine (CTa) is a highly cationic and cysteine-rich peptide isolated from the venom of Crotalus durissis terrificus ( Fig. 1), which shows membrane translocation capabilities, penetrates the cell and presents cytoplasmic, vesicular and nuclear distribution (Almeida et al. 2017;Pompeia et al. 2022;Si et al. 2022). CTa can be specifically uptaken by melanoma and lymphoma cells during their proliferation and permeates several lines in vitro (Si et al. 2022). Furthermore, CTa and its analogs have been tested in tumor cell lines and have demonstrated aptitude to be used as selective delivery tools for anticancer molecules (Mambelli-Lisboa et al. 2018). CTa is also a potential tool for in vivo cancer cell identification (Kerkis et al. 2014).
Another example is the chimeric natriuretic cenderitide (CD-NP), produced by the association of 15 amino acids of the C-terminal segment of a Dendroaspis angusticeps natriuretic peptide and 22 residues of the human C-type natriuretic peptide. CD-NP is indicated in congestive heart failure because it activates natriuretic peptide receptors, leading to hypotension. Cenderitide showed natriuretic, diuretic, antiproliferative, and antifibrotic proprieties. The drug is being evaluated in phase I/II trials for stable chronic heart failure and moderate renal impairment (Table 6) (Lee et al. 2016;Kawakami et al. 2018;Zhang et al. 2018a;Ichiki et al. 2019).

Spiders
Spiders are the biggest group of venomous animals. These animals are able to produce small peptide-rich venoms; each spider species can have thousands of different toxic peptides, which, given the sheer number of spider species (100-300,000 species), gives us a glimpse of the hidden richness of spider venom (Diego-Garcia et al. 2010;Peigneur et al. 2021). Since proteinaceous spider toxins are an invaluable resource for pharmacologists, neuroscientists and medical chemistry, scientists have created the Arach-noServer, a database that collects molecular targets, structural and biological information.
Most spider venom-derived act as analgesics because they are allosteric modulators of the Na v channels and are classified in twelve categories-NaSpTx 1-12-based on their primary sequence and cysteine positions (Goncalves-Machado et al. 2016;Luddecke et al. 2022;Trevisan and Oliveira 2022). A typical spider toxin has three disulfide bonds, which forms the Inhibitor Cysteine Knot (ICK) motif (C1-C4, C2-C5, and C3-C6), present in 9 out of the 10 NaSpTxs that specifically bind the Na v channels. (Esteves et al. 2020;Lima et al. 2021). However, spider toxins can also form 4 or even 5 disulfide bonds, as it is the case of the ω-agatoxin and the PnTxs, respectively. This structural aspect allows the peptides to interact with other   Huang et al. (1987), Knudsen et al. (1988), Chiang et al. (1994Chiang et al. ( , 1995, and Swaim et al. (1996) r-viridistatin 2  (1996); Danen et al. (1998); Tian et al. (2007), and Hailey et al. (2013)   Page 11 of 39 76 channels involved in pain transmission, such as the acidsensing ion channel (ASICs), calcium channels, opioids and purinergic receptors (Estrada et al. 2007;Kabanova et al. 2012;Pan et al. 2012Pan et al. , 2021Utkin et al. 2019). This is indeed the case of the ω-agatoxins and the PnTxs which act as Cav channel modulators, blocking the presynaptic calcium channels, and thus, decreasing the calcium influx. Table 3 sums up the main spider toxins that exhibit analgesic properties.

RGD
Below we describe the pharmacological relevance of other peptides present in spider venoms and their supporting studies for putative medical applications, many of which not only have analgesic properties, but also antimicrobial and anticancer activities (Perumal Samy et al.
(2) The venom of the Chinese tarantula (Chilobrachys jingzhao) expresses a family of peptides named Jingzhaotoxins (JzTx). The JzTx-34 peptide inhibits Nav1.7 channels, exhibiting antinociceptive properties with higher affinity compared to all other subtypes of Nav (Zeng et al. 2018). (3) Gomesin is a peptide isolated from the venom of Acanthoscurria gomesiana (São Paulo Black Tarantula) ( Fig. 1). It has antimicrobial activity and reduces the viability and proliferation of human melanoma cells by inducing cell cycle arrest Ikonomopoulou et al. 2018). (4) LyeTxl-b is a potent antibiotic derived from the LyeTxl peptide found in the venom of Lycosa erythrognatha (Araña Lobo de Vientre Negro) ( Fig. 1). It has proven antibacterial efficacy against Gram-positive and Gramnegative bacteria, in vitro and in vivo, and has delivered promising results against resistant bacteria strains. LyeTxl-b also reduced biofilm viability (Cardoso et al. 2022;de Avelar Junior et al. 2022). (5) Hi1a is a peptide isolated from Hadronyche infensa venom (Australian blue mountain funnel-web spider). Hi1a attenuates brain damage after a stroke and can be used in brain ischemic injuries (Chassagnon et al. 2017). Therefore, it is considered an important lead for the development of neuroprotective agents (Ren et al. 2018). Hi1a showed partial inhibition of ASIC1a but does not inhibit ASIC1b. The total venom of the Hadronyche infensa spider also contains biopesticides that paralyze insects associated with crop damage while being non-toxic against bees, birds and mammals (Kilham and Isbister 2020).

Scorpions
Scorpions are predatory arthropods and one of the oldest animals on Earth. They can be found all over the world, except Antarctica, but live mainly in deserts. Their venom is rich in neurotoxic peptides that interact with many ion channels, causing paralysis and other neurotoxic effects. Therefore, these peptides have been investigated to understand whether they can be used as prototype analgesic drugs.  (Lippens et al. 1995;Jouirou et al. 2004;Morel et al. 2022). This peptide has the ability to bind to different targets including chloride channels, potassium channels membrane type-2 matrix metalloprotease (MMP-2) and annexin A2 ( Fig. 1) (Kuzmenkov et al. 2018;Tajti et al. 2020;Zhu et al. 2020;Gandomkari et al. 2022). To date, CTx is the only scorpion venom toxin undergoing clinical trials due to its ability to bind to glioma cells, leading to the development of cancer diagnosis methods and treatment (Delinois et al. 2020;Zhao et al. 2020;Vannini et al. 2021;Dardevet et al. 2022).
TM-601 is a synthetic derivative of CTx with proved antitumor characteristics. This drug candidate has undergone phase I and II clinical trials, for patients with recurrent malignant glioma and solid tumors (Jacoby et al. 2010;Kesavan et al. 2010;Ojeda et al. 2016;Dardevet et al. 2022). Another example is Tozuleristide (BLZ-100), also inspired on the CTx from the Leiurus quinquestriatus quinquestriatus scorpion (Fig. 6A). This CTx indocyanine green conjugate showed ability to bind to tumor cells. BLZ-100 can detect metastases (in mouse models) from prostate and intestinal cancers, malignant glioma and sarcoma medulloblastoma (Butte et al. 2014;Fidel et al. 2015;Parrish-Novak et al. 2017;Yamada et al. 2021). Furthermore, Hemilipins Hl-P11 and HL-P13 ( Fig. 1), present in the venom of the scorpion Hemiscorpius lepturus, inhibit angiogenic activity and can be cytotoxic to breast and colorectal cancer cell lines (Jridi et al. 2015(Jridi et al. , 2017Kazemi-Lomedasht et al. 2017;Parrish-Novak et al. 2017;Yamada et al. 2021;Rezaei et al. 2022). Many relevant toxin peptides present in the venom of various scorpion species can be used against channelopathies (ion channel associated pathologies), shown antimicrobial, antiangiogenic, anticancer, tumor binding or insecticide activities (Cohen-Inbar and Zaaroor 2016; Pucca et al. Table 3 Peptide toxins from spiders with analgesic potential ( Analgesic potential in the neuropathic and inflammatory effects Both peptides block and inhibition of P-type channels Nishio et al. (1993), Kuwada et al. (1994), Wang et al. (2001), and Sitges and Galindo (2005) ω-agatoxin TK (  (Fu et al. 2007). (4) Kaliotoxin (Ktx) or potassium channel toxin alpha-KTx 3.1 is a peptide isolated from the venom of Androctonus mauretanicus mauretanicus (Mauritanian scorpion, Fig. 6C) and is capable of preventing bone loss through osteoclastogenesis of periodontal disease, in rat models. Ktx-based drugs could be promising candidates for the treatment of periodontal disease and rheumatoid arthritis (Valverde et al. 2004). (5) ImKTX88 potassium channel toxin is a peptide toxin that was isolated from the venom of Isometrus maculatus (lesser brown scorpion). The peptide improves the pathological severity (in vivo) of autoimmune encephalomyelitis (Huang et al. 2017). (6) HsTX1 (potassium channel toxin alpha-KTx 6.3) is a peptide isolated from the venom of Heterometrus spinifer (Asian forest scorpion) with proven ability to reduce (in vivo) inflammation and arthritis (Tanner et al. 2017). (7) OSK1 (potassium channel toxin alpha-KTx 3.7) was isolated from Orthochirus scrobilosus venom (Central Asian Scorpion). This peptide can block Kv1.3, but (in vivo) OSK1 was shown to be neurotoxic in the mouse brain (Mouhat et al. 2005;Veytia-Bucheli et al. 2018). (8) The Ts6 and Ts15 peptides (potassium channel toxin alpha-KTx 12.1 and alpha-KTx 21.1, respectively) were isolated from the venom of Tityus serrulatus (Brazilian yellow scorpion). Both peptides inhibit the proliferation of memory effector T cells and reduce inflammation in a mouse model (Pucca et al. 2016a).

Bees
Bees are winged insects known for their role in pollination and, in the case of western bee species, honey production. There are more than 16,000 known bee species.  El-Seedi et al. 2020;Badawi 2021;Khalil et al. 2021). Melittin (Mlt) (Fig. 11A) is a peptide with 26 residues that weights 40% to 60% of the total bee venom (Yaacoub et al. 2022). Mlt contains hydrophilic and hydrophobic properties; a C-terminal with positively charged amino acids and a N-terminal of hydrophobic character. These characteristics are related to significant antibacterial and antitumor properties (Table 4) Soares-Silva et al. 2022;Tang et al. 2022;Wang et al. 2022;Xie et al. 2022).
Apamin (Apm) is a peptide with 18 amino acids present in 2% to 3% of dry weight of bee venom (Gu et al. 2020). Apm has a disulfide bond that shapes its highly stable and compact chemical structure (Nguyen and Lee 2021). The application of Apm has demonstrated potential benefits in antiatherosclerosis, antiheart failure, and in the improvement of neurological disorders (  (El-Seedi et al. 2020Kim 2021). Furthermore, they also showed an effect on arthritis (osteoarthritis, psoriatic arthritis, and inflammatory arthritis) (Tang et al. 2021(Tang et al. , 2022, wound healing (Kurek-Gorecka et al. 2020a, 2020band antiangiogenic properties (suppression of the formation of new vascular tubes).

Amphibians
Amphibians are ectothermic vertebrates that mainly live in terrestrial, arboreal or freshwater aquatic environments. Although they have lungs, amphibians use the skin as a secondary respiratory system. The peptides stored in the granular glands of the amphibian skin are released, at high concentrations, when amphibians are stressed or injured (Varga et al. 2018). Therefore, it is not surprising that the most important amphibian toxins studied to date are those found on their skin. Amphibian peptides are mainly cationic and amphoteric, and display antimicrobial, antioxidant, antiviral and antitumor characteristics (He et al. 2013;Grayfer and Robert 2016;Casciaro et al. 2020;Feng et al. 2021a;Soltaninejad et al. 2021;Chianese et al. 2022).
Bombinin was the first short antimicrobial peptide identified (17-20 residues) (Fig. 8). It was isolated from the amphibian skin of Bombina variegate. The antimicrobial bombinin family is divided into three categories: bombinins, bombinin-like peptides (BLPs) and bombinin-H (Fig. 1). Bombinins are effective against Gram-positive and Gramnegative bacteria, as well as fungi, but they proved to be ineffective in hemolysis tests (Peng et al. 2018;Patocka et al. 2019). BLPs (BLP-1, BLP-2, and BLP-3) have high homology with other antimicrobial peptides and were isolated from Bombina orientalis skin secretions (Fig. 7A). Bombinin-H was also isolated from Bombina orientalis skin secretions and can be found in Bombina maxima secretions (Mangoni et al. 2000a;Simmaco et al. 2009;Peng et al. 2018). BHL-bombinin and Bombinin-BO1 are two members of the Bombinin family that show antibacterial activity against S. aureus and E. coli. However, Bombinin-H has little antibacterial activity, and showed very toxic effects because it promotes erythrocyte lyses (Xiang et al. 2017;Peng et al. 2018).
Esculentins are circular antimicrobial peptides isolated from the skin secretions of two frogs: the North American Rana esculenta, which secretes esculentin-1; and the Asian frog Rana ridibunda, which produces esculentin-2. Esculentin-1 is a 46 amino acid peptide with antimicrobial activity against a variety of pathogenic fungi and bacteria such as C. albicans, E. coli, S. aureus and P. aeruginosa. Although, Esculetin-2 is a smaller peptide relative to Esculentin-1, this peptide contains 37 residues that also show antimicrobial activity against E. coli, S. aureus, and C. albicans (Wang et al. 2010;Patocka et al. 2019;Casciaro et al. 2020;Malik et al. 2021).
Like Esculentins, Brevinin-1 (24 amino acids) and brevinin-2 (33 amino acids) are two antibacterial peptides isolated from the Rana brevipoda porsa. The primary and secondary structures of Brevinin-1 family peptides are conserved. Studies suggest that the proline residue at the 14 th position plays a key role in the formation of a transmembrane pore, which in turn is essential for its antimicrobial activity against E. coli and S. aureus. In contrast, Brevinin-2 family peptides have at an equivalent position a Ser residue. Moreover, the C-terminal of this peptide family has a "Rana-box" disulfide motif-CKxxKxC-where x can be any amino acid and the cysteines are always in close proximity with a cationic residue, a characteristic of antimicrobial frog toxins. Brevinin-2 has a strong antibacterial activity against E. coli and S. aureus and antifungal activity against C. albicans (Savelyeva et al. 2014;Patocka et al. 2019;Jiang et al. 2020).
Ranatuerin-1 is a peptide isolated from the skin secretions of the Rana catesbeiana. Its primary sequence has 25 amino acids and forms a circular domain composed of 7 amino acids at the C-terminus. Ranatuerin-1 has strong antimicrobial activity and can inhibit the Streptococcus Group B, P. aeruginosa, S. aureus, E. coli and C. albicans (Sonnevend et al. 2004). Because of its broad-spectrum activity, it has been proposed to be an antibiotic candidate. In line, the Ranatuerin-2 peptide (ca. 30 amino acids), isolated from the skin of Rana aurora aurora, has high bacteriostatic activity against Gram-positive and Gram-negative bacteria and, as a great advantage, low hemolytic activity (Halverson et al. 2000). Ranatuerin-2 also has antimicrobial activities against: E. coli, P. aeruginosa, S. aureus, and the group B Streptococcus, like the Ranatuerin-1, but it also showed activity against K. pneumoniae, E. cloaca and S. epidermidis (Conlon et al. 2009;Zhou et al. 2019).
Ranacyclines are a family of short antimicrobial peptides (ca. 13 residues) that form a disulfide bridge between the Cys3 and Cys13, responsible for their cyclic form (Conlon et al. 2009;He et al. 2013). Both Ranacycline-B-RN1 and Ranacycline-B-RN2 have been isolated from Hylarana Nigrovittata skin and have specific antimicrobial effects against S. aureus. Two other members of this family with antimicrobial activity are Ranacycline E and Ranacycline T, isolated from Rana temporario and Rana esculenta, respectively (Patocka et al. 2019;Wang et al. 2020b). The latest have a potent antibacterial activity against Y. pseudotuberculosis YP III, B. megaterium Bm11, S. lentus, M. luteus, C. tropicalis, and C. guiller-mondii (Mangoni et al. 2003;Conlon et al. 2009;Wang et al. 2020b).
Dybowskins are short peptides isolated from Rana dybowskii skin secretions (Fig. 7C). Dybowskin-1 and Dybowskin-2 have high sequence similarity and differ only by two amino acids located at positions 7 and 14. On the other hand, Dybowskin-3 to Dybowskin-6 have different sizes and low conserved sequences (Kim et al. 2007c). Dybowskins-1 to -6 showed antimicrobial activity against Gram-positive and Gram-negative bacteria such as S. aureus, B. subtilis, S. epidermidis, S. dysenteriae, M. luteus, E. coli, K. pneumoniae and the fungi C. albicans. Dybowskin-1CDYa (13 residues) and Dybowskin-2 CDYa (18 residues) display only 5% of sequence identity and low sequence similarity to known antimicrobial peptides. Yet, both peptides have strong antimicrobial activity (E. coli   and Huang et al. (2023) and S. aureus strains) but little effect on the hemolysis of human erythrocytes. Peptides in the skin secretions of amphibians vary in structure and function. Therefore, it is not surprising that amphibians skin secretions activities are not limited to their antimicrobial role, and such peptides can be applied to other therapeutics of interest. In the following paragraphs, we briefly comment on amphibian peptides potential and how their development could be of great use in combating a wide variety of human pathologies.
Bombesin, a 14 amino acid peptide, was isolated from the skin of the Bombina bombina frog (European firebellied toad) (Fig. 8A) and showed a high affinity for gastrin-releasing peptide receptors (GRPR) (Tornesello et al. 2017b;Utkin 2019Utkin , 2021. Because GRPR is overexpressed in several human neoplasms, such as prostate, breast, and small cell lung cancer, the receptor could be a good target to inhibit tumors, and hence Bombesin, a potential drug candidate inspiration (Table 5) (Schwartsmann et al. 2002). Since Bombesin showed high toxicity, the peptide cannot be used in its native form; thus, scientists have tried chemical modifications to produce a stable synthetic Bombesin and increase its binding affinity and agonist/antagonist role (Cescato et al. 2008;Tornesello et al. 2015Tornesello et al. , 2017a. All bombesin-related peptides have a conserved C-terminal region characterized by the WAXGXXM-NH2 motif, but differ on N-terminal regions. To our knowledge, the only synthetic Bombesin/GRPR antagonist (RC-3095) evaluated to date showed tumor regressions (in vitro and in vivo) in murine and human tumor models. RC-3095 contains the bombesin residues 6 to 14, however, the N and C terminals suffered chemical modifications, resulting in the [D-Tpi6, Leu13 psi (CH2NH)-Leu14] bombesin-(6-14), as can be observed in Fig. 8B. Unfortunately, during phase I clinical trials, RC-3095 showed injection site toxicity, in patients with advanced solid malignancies, so its doses for phase II trials could not be defined (Schwartsmann et al. 2006;Umana et al. 2013;Baratto et al. 2018).
Antioxidant-rich peptides aroused scientific interest because of their ability to scavenge free radicals in the body and maintain homeostasis. Several peptides with antioxidant properties have been isolated from the skin secretions of several amphibians such as Rana plyuraden, Odorrana andersonii, Odorrana schmacker (Fig. 12F), and Odorrana lívida (Weber et al. 2005;Hwang and Daar 2017;Yang et al. 2022a). Additionally, OS-LL11 and OAVI12 peptides were isolated from the skin secretion of Odorrana schmackeri and Odorrana andersonii, respectively. Both directly reduce reactive oxygen species (ROS) levels (Calgarotto et al. 2007;Yin et al. 2019;Xie et al. 2021).
Bradykinin is a class of peptides in the kallikreinbradykinin system (Fig. 1). Bradykinins are involved in

RPI-MN (Pepteron®)
Naja naja atra (Taiwan cobra) (Fig. 2B) Connection to nicotinic acetylcholine receptors (nAChRs) and can protect cells due to its ability to inhibit viral replication Other important peptides isolated from Rana palustris and Bombina variegate skin are insulin-releasing peptides (Marenah et al. 2004;Abdel-Wahab et al. 2008;Manzo et al. 2015;Conlon et al. 2020), which regulate insulin secretion in vivo and hence the blood glucose levels. Tigerinin-1R, isolated from Hoplobatrachus rugulosus (Asian frog) (Fig. 1), is a potent non-toxic insulin-releasing peptide. It has demonstrated potential for the development of new therapeutic agents against type 2 diabetes mellitus. Another peptide, Pseudin-2, stimulates insulin secretion from BRIN-BD11 cells through a mechanism that involves the Ca 2+ -independent pathway. [Lys18]-pseudin-2 was identified as a peptide with the potential to develop valuable insulin drugs for the treatment of type 2 diabetes (Song et al. 2009;Srinivasan et al. 2014).
Some antimicrobial peptides from amphibian's skin secretion also showed activity to promote insulin-releasing peptides, as it is the case of Plasticin-L1 from Leptodactylus laticeps (Fig. 7D). Furthermore, strong anticarcinogenic peptides were also isolated from amphibian skin secretions. Among those we highlight the Dermaseptin-PS1 (Phyllomedusa sauvagei) and Aurein (Litoria aureus).
Along, the frog peptides Ranatensin, Litorin, and Phyllolitorin have shown activity in smooth muscle, constrict blood vessels, inhibit urine production, and are effective in the treatment of neurological diseases (Johnson et al. 1999;Cline et al. 2010;Laskowska et al. 2022).

Leeches
Leeches (Hirudinea family) are parasitic or predatory worms living in freshwater, marine, or terrestrial habitats. For centuries, mankind has used different species of leeches to draw blood from patients and to treat join diseases, extremity vein diseases, fever and rheumatic pain (Van De Car et al. 2010).
The discovery that leeches (Hiduro medicinalis) secrete a peptide called Hirudin, which prevents blood clotting, led the pharmaceutical industry to invest huge efforts in the development of thrombosis-inhibiting drugs (Fig. 9A). However, the native structure of hirudin secreted in the saliva of H. medicinalis native chemical structure resulted not to be efficient and safe for human use. However, Hirudin inspired the synthesis of two synthetic derivatives: Desirudin and Bivalirudin (Fig. 1, Fig. 9B and Fig. 9C), both acting as direct thrombin inhibitor drugs (Carswell and Plosker 2002;Warkentin and Koster 2005).
Recombinant Desirudin (Table 1) is the active principle of Iprivask® and Revasc®, used in the clinic for vein thrombosis prophylaxis. However, for commercial reasons, they were withdrawn from the market (Kong et al. 2014). Bivalirudin, a 20 amino acid peptide, led to Angiomax® Each amino acid is colored according to its polarity: grey was used for hydrophobic residues, polar residues are in cyan, special amino acids, such as glycines and prolines, are colored green, cysteines in yellow, and positive and negatively charged residues in blue and red, respectively. Non-natural amino acids are shown in squares (Alphafold model AF-P86994-F1). B Synthetic Bombesin/GRPR antagonist. In yellow/orange are highlighted the chemical modifications that transformed the natural Bombesin into the synthetic RC-3095 compound (PubChem CID 11948463) and Angiox® (Table 1) (Moen et al. 2005;Gargiulo et al. 2018). In 2000, the FDA approved the use of Angiomax® as a direct thrombin inhibitor, indicated for patients with percutaneous transluminal coronary angioplasty and unstable angina (Scholle and White 2010). Angiox® was used to prevent blood clot formation in cases of percutaneous coronary intervention and myocardial infarction, in adult patients. The last was withdrawn from the market and is no longer in use in the European Union also because of commercial reasons (Scholle and White 2010).

Lizards
The Gila monster (Heloderma suspectum) is a lizard whose bite is very painful and potentially fatal to humans. Its venomous saliva has several peptides with different functions, but exendin-4 is of particular interest (Table 1; Fig. 1). The primary sequence of exendin-4 shares about 53% of sequence identity with the glucagon-like peptide-1 receptor (Dhungat 2017; Lee et al. 2018;Coulter-Parkhill et al. 2021). The drug acts as a receptor agonist, promoting insulin secretion, and is used in the treatment of diabetes mellitus type 2, regulating insulin and glucagon levels in humans (Zhang et al. 2018b;Gentilella et al. 2019;Silva et al. 2019b;Graham et al. 2020;Coulter-Parkhill et al. 2021 Park et al. 2021).
Chemical modifications on exendin-4, which removed a proline and added six lysine residues, resulted in a potent 44-amino acid peptide drug-Lixisenatide (Adlyxin®) (Table 1; Fig. 1). The drug was approved by the EMA in 2013 and, later in 2016, by the FDA (Whyte et al. 2019;Graham et al. 2020;Candido et al. 2022).

Fish
Like other animals, fish venoms are also mixtures of proteins and peptides that interact with physiological processes of preys/predators causing immobilization, tissue's digestion, and death (Church and Hodgson 2002). In 2006, Sri Balasubashini and colleagues purified a peptide-FV peptide-from the venom of the red lionfish (Pterois volitans, Fig. 10B) that showed interesting antitumor effects (Fig. 1) (Sri Balasubashini et al. 2006a). The authors proved that the peptide decreased tumor volume and the number of viable Fig. 9 Leeches venom toxins and their derivative drugs. A Hirudin-a natural peptide isolated from the poison of Hiduro medicinalis (represented in green surface) is bound to Prothrombin (purple surface) (PDB. ID 7A0D). B 2D-representation of the chemical structure of Bivalirudin, here represented in ball and sticks (Pubchem CID 16129704). Under this figure is showed its primary sequence (spheres) and the non-natural amino acids are marked in square. The color scheme used for the amino acids follows the same pattern as showed in Fig. 8. C Desirudin (Pubchem CID 16129703) Ehrlich ascites carcinoma (ECA) cells. The exact anticancer mechanism of the FV peptide is not yet understood, but two hypotheses were suggested: 1) FV can act directly on the lytic action of the peptide; or 2) FV peptide can act indirectly, inducing apoptosis and DNA fragmentation by caspase activation in EAC (Sri Balasubashini et al. 2006b).
The SA-HT peptide is a hemorrhagic and nociceptive peptide cation-dependent peptide derived from the venom of Scatophagus argus Linn. The SA-HT toxin down-regulates the production of histamine, serotonin, cyclooxygenase, and lipoxygenase. These effects suggest that SA-HT interact with neurotransmitters, prostaglandins, and leukotrienes. Therefore, the toxin is of great interest for producing neurological or even anti-inflammatory drugs (Karmakar et al. 2004;Lopes-Ferreira et al. 2021).

Shrews
Shrews are venomous mammals found in northeastern North America. The submaxillary and sublingual glands of the Northern Short-tailed shrew (Blarina brevicauda, Fig. 10C) produce venomous saliva containing two very potent toxins (Bowen et al. 2013;Tanabe et al. 2022). A large toxin-Blarina-a 282 residues serine protease, capable of paralyzing its prey and killing small animals through paralysis of the respiratory center; and a small peptide of 54 residues, named Soricidin, that inhibits sodium channels and blocks nerve impulses in the prey (Fig. 1) (Brant and Orti 2002;Bowen et al. 2013). The peptide can inhibit the transient receptor potential vanilloid type 6 calcium channel (TRPV6). Because TRPV6 is overexpressed in a variety of cancers, Soricidin and its derivatives may be promising anticancer drug candidates (Bowen et al. 2013;Stewart 2020). In fact, SOR-C13®, a synthetic 13 amino acid peptide derived from the C-terminal region of Soricidin, suggested antitumor activity, leading to a reduction in solid pancreatic tumor (up to 27%) (Fu et al. 2017). So far, SOR-C13 has not revealed serious drug-related adverse events, and the FDA included the peptide in phase I clinical trial for the treatment of ovarian and pancreatic cancers (Table 5), (Fu et al. 2017).

Snails
Snail venoms, both marine and terrestrial, have a large amount and variety of attractive bioactive compounds for the cosmetic and pharmaceutical industries (Dhiman and Pant 2021). Their toxins can be useful in producing new formulations with reduced toxicity and side effects compared to current drugs (Lebbe et al. 2014).
Crude extracts, mucus and sludge contain many bioactive peptides which have been used, in traditional medicine, against viral lesions, warts and other dermatologic problems, pain, microbial infections, and tumors (Table 6)  Venoms from many species of cone snails (Conus ssp.) contain small cysteine-rich peptides (10 to 30 residues) (see Table 6). These peptides are associated with the Conotoxin family (CNTx) (Lewis 2009;Li et al. 2021b;Tran et al. 2022;Wiere et al. 2022) (Table 6 and Fig. 1). In particular, the ω-conopeptide isolated from the venom of the magic cone snail (Conus magus, Fig. 10A) is the most notable and famous peptide example from the Conotoxin family (Kristipati et al. 1994;Olivera et al. 1994;Favreau et al. 1999;Fedosov et al. 2013;Eisapoor et al. 2016;Chalil et al. 2021). This peptide was the inspiration to produce the synthetic ω-conotoxin MVIIA, named Ziconotide (Fig. 11C). It is a small cyclic peptide of 25 amino acids and 3 intramolecular disulfide bonds, with high affinity for N-type voltage-gated calcium channel. The drug was the first calcium channel inhibitor derived from venom with clinical application. It inhibits the release of nociceptive neurochemicals, resulting in severe chronic pain relief (Lugo-Fabres et al. 2021;Trevisan and Oliveira 2022;Wie and Derian 2022). This analgesic drug was first approved by the FDA (2004) and then by the EMA (2005), for severe chronic pain, which was until theses dates considered intractable, and is commercialized under the trade name Prialt® (Table 1), (Rigo et al. 2013;Loschner et al. 2021;Jergova et al. 2022;Wie and Derian 2022). Despite being 3 orders of magnitude more potent than morphine, it has low efficacy when administered orally and intravenously. To maintain its analgesic potential, the drug must be administered intrathecally, i.e. directly into the spinal fluid, which limits its regular use (Pope and (Fig. 11).

Perspective
Natural products and their derivatives have always contributed to a great extent to new approved drugs. Noble examples of their importance are the antibiotics, in particular, the penicillin obtained from Penicillium moulds. Today, about one-third of the FDA-approved drugs are natural products. Almost half of these are derived from mammals, one-quarter from plants, and one-quarter from microbes (Patridge et al. 2016).
From the chemical point of view, natural products are complex biomolecules that cannot be explored by smalldrug-like high-throughput virtual screening techniques. However, combinatorial chemistry techniques have succeeded in optimizing structures and have been extensively used in the transformation of many recently approved Fig. 11 Other venom peptides inspired the design of three approved drugs. (A) Apitox ® derived from melittin, a peptide from Bee venom used against knee and arthrosis pain (PDB ID. 2MLT) (Eisenberg et al. 1990)(B) The Gila monster derivative originated a drug used against type II diabetes due to its high similarity to the glucagon-like peptide Byetta® (PDB ID. 6GDZ) (Evers et al. 2018). C The ω-conotoxin of the cone snail led to ziconotide, an approved drug for pain treatment, commercialized under the trade name Prialt®. The human N-type voltage gated calcium channel CaV2.2 -alpha2/delta1-beta1 (represented in blue surface) is bound to ziconotide (in pink) (PDB ID. 7VFU) (Dong et al. 2021). The complex receptor-drug-POPC bilayer membrane (stick representation) was assembled using the Charmm-Gui webserver (Jo et al. 2008). Figures were created with BioRender. com (2020) (https:// app. biore nder. com/ biore nder-templ ates). Under each secondary structure figure is showed its primary sequence (spheres) and the. non-natural amino acids are marked in square. The color scheme used for the amino acids follows the same pattern as showed in Figs. 8 and 9 natural agents, improving drug stability and oral bioavailability. Since the 1930s, natural products have diminished, and synthetic and semisynthetic natural product derivatives have increased.
Despite the efforts of the scientific community to find new drugs, there are still big gaps in the treatment of diseases such as the neurodegenerative and cancer pathologies, among others. To date, there are no effective drugs that can reduce or rescue neurodegenerative debilitating symptoms or that can act as effective anticancer therapies, overcoming the cytotoxic effects of current chemotherapies. Moreover, there is an emergent need to develop new antibiotics against several resistant bacteria strains or new antiviral therapies (Kinch et al. 2014).
Natural products derived from animal venoms have been used in traditional medicine for eras. Despite lacking scientific explanation, they have been a cure for many years in traditional societies (Charitos et al. 2022). Recent advances in sequencing technologies, proteomic, structural, and analytical techniques not only explain theories, beliefs, and experiences, where alternative medicines are based, but now shed light on the molecular determinants that assist their pharmacological activities (Simoes- Silva et al. 2018). This breakthrough opens the use of venoms as a therapeutic and diagnostic source (Almeida et al. 2017). Allowing, the exploitation of a complete novel chemical space never studied before.
Animal venoms are truly near-unexplored mines of bioactive compounds. A common feature of animal venom is the presence of active peptides that can be used for biological purposes (Almeida et al. 2017). A characteristic of peptide toxins is that these molecules are highly selective and potent binders for a wide range of molecular targets and are associated with important pharmacological activities (Riciluca et al. 2021;Cao et al. 2022). A sustainable discovery strategy associated with biotechnology has allowed bioactive compounds derived from animal venoms, particularly peptide toxins, to stand out in the The drug discovery process is a long-term risk challenge that requires huge financial investments without a guarantee of success. This is a relevant fact regarding peptides derived from animal venoms (terrestrial and marine), which always require structural changes both to avoid their toxic effects and to obtain more efficient molecules. Although venoms are relevant sources of bioactive components for new leading molecules and powerful tools for the investigation of transmembrane receptors, ion channels, and enzymes, only a few of these toxins have reached the market. Captopril®, Integrilin®, Apitox®, Byetta®, and Prialt® are some successful examples of the use of animal peptides as lead compounds used in the development of new pharmaceuticals and medical therapies (other successful examples are shown in Table 1). Currently, many drugs based on toxins from different animals are being analyzed in preclinical (Table 2) or clinical phases (Table 3). As we saw in this review, worldwide interests, both scientific and business, are producing many drugs based on animal peptides, mainly due to the potential of their medical applications and bioactivities (Boldrini-Franca et al. 2017). The literature still shows how little we know about the jumbo biodiversity of the planet, from the biological, ecological, pharmacological, medicinal, and biotechnological points of view. Many more molecules, such as those discussed here, can be exploited sustainably, consolidating the bioeconomy. However, with the accelerated extinction species rate resources, we are losing many species without even knowing them. Those venoms might be among the endless sources of therapeutics that can alleviate the diseases afflicting humankind. Funding Open access funding provided by FCT|FCCN (b-on). Indicated in the item "Acknowledgments".

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