*1.2. Biodiversity*

Earth's existing biodiversity is a direct consequence of Darwin's Natural Selection, i.e., the survival of the fittest, in a constant struggle to survive [3]. With an estimated 8.7 million species inhabiting our planet, the mere 1.2 million (mostly insects) that have already been identified and described have all—or are still in the process of—adapted and evolved so that, after numerous breeding cycles, poorly suited individuals are filtered out by nature.

One particularly interesting adaptation which emerged millions of years ago was the biochemical weaponry utilized for defense and/or predation by some organisms as a means of guaranteeing survival [4]. These so called 'toxins' can be found in procaryotic species, such as *Staphylococcus aureus* and *Klebsiella pneumoniae* [5,6], plants (*Cicuta maculate* (Socrates committed suicide by drinking cicuta, circa 399 B.C.) and *Nicotiana tabacum* (homage to Jean Nicot de Villemain, who introduced snuff to the French court in 1560)) and, obviously, animals.

For animals, these toxins are believed to have originated from ancestral house-keeping genes that underwent variation and neofunctionalization [4,7], resulting in molecules displaying an 'increased' biological activity, normally targeted to major biological systems that when unbalanced may result in severe risk of death, such as the hemostatic-interfering molecules. The toxins were then specifically expressed in venom-secreting cells that eventually became specialized venom glands [8]. Such specialization became an evolutionary advantage, due to unique pharmacokinetic properties that these (typically) peptides and proteins granted to such animals [9,10].

#### *1.3. Toxins: Snakes, Spiders and Scorpions as Classical as It Can Be*

Toxinology has its origins long associated with venomous animals and not poisonous ones. There might be some controversy in this separation, but it is commonly accepted that venomous animals would possess an inoculating apparatus capable of delivering toxins into the prey/aggressor. On the other hand, poisonous animals would secrete toxins in their skin or body organs and would have to be actively eaten/beaten/attacked/poked/colonized (bacteria) in order for to the toxins exert their effect.

Nevertheless, mystical, magical, medical and/or lethal uses of some animals' venoms are well known throughout history. For example: Cleopatra may have committed suicide by letting herself be bitten by a snake (*Naja haje* probably). In the Bible there are nine verses citing scorpions (Luke 10:19 and 11:12, Kings 12:11 and 12:14, Deuteronomy 8:15, Ezekiel 2:6, Revelation 9:3, 9:5 and 9:10). Greek mythology presents us the Lernaean Hydra, a serpentine water monster with many heads (depending on the myth source) with poisonous breath and blood so virulent that even its scent was deadly, as well as the Medusa, one of the three monstrous Gorgons, generally described as winged human females with living venomous snakes in place of hair.

These venomous animals are still present in modern-day fiction, such as the famous Spiderman, whose superpowers derived from mutations resulting from the bite of a radioactive spider. Even Harry Potter was forced to deal with the Basilisk, a giant snake capable of instant kill just by gazing at the victim's eyes. There are also urban legends and local habits, such as the well-known North American arachnophobia.

On the other hand, poisonous animals share a less glamorous role in human history. They have participated, for example, in human (sacrificial) rituals and attempted pharmaceutical developments throughout history. There were Maya human bloodletting rituals that employed the sting of marine stingrays as blades, due to a 'more efficient' bleeding [11]. Hunters have long sought the Central and South American Dendrobatidae 'poison arrow frogs' (self-explanatory) to use their toxic skin secretion for hunting [12]. Traditional Chinese medicine uses the 'all healing' Chan'Su (dried *Bufo bufo* skin) for mostly any illness [13]. Amazon tribes traditionally used Kambo (or Kampum) in their purification rituals [14]. This medicine is extracted from *Phyllomedusa bicolor* skin secretion and has become known as the 'frog vaccine' in urban environments. The Bible also cites such animals in the infamous passage in Exodus 8:1–4, in which the "*great LORD says: Let my*

*people go, so that they may worship me. If you refuse to let them go, I will plague your whole country with frogs. The Nile will teem with frogs. They will come up into your palace and your bedroom and onto your bed, into the houses of your officials and on your people, and into your ovens and kneading troughs. The frogs will go up on you and your people and all your officials*". Unfortunately, the poisonous animals are presented from a more neglected, less charming perspective, as presented above.

All this glamour associated with venomous animals has led to the establishment of what can be considered the 'greatest-hits' of (classical) toxinology: snakes, spiders, and scorpions (the triad). Undoubtedly, studying these animals' venoms has yielded a myriad of relevant scientific papers [15–19] produced by highly committed international scientific groups. The molecular dissection of the venom constituents has made it possible that effective sera could be manufactured [20–22], thus reducing mortality and morbidity associated with envenomation [23,24]. Moreover, one of the world's most administered antihypertensives (Captopril) is a direct derivative of one viper toxin [25].

Another example is a tumor-labeling molecule (tozuleristide), currently undergoing clinical phase 1 studies, that is being used in surgeries as marker and diagnostics for glioma and other tumors. This molecule is an analogue of a chlorotoxin isolated from the venom of the scorpion *Leiurus quinquestriatus* [26–28].

It is noteworthy to mention that there is young blood trying to join the party. Even though the marine mollusks of the *Conus* genus do not belong to the classic triad, they are becoming more and more famous since the discovery of ziconotide (Prialt), the strongest analgesic ever described: a calcium channel blocker, purified from the *Conus magnus* venom [29]. These animals are discussed below.

However, even for such well-studied animals there are still 'neglected' molecules present in their venoms, such as L-amino acid oxidase, crotapotin, crotamine that 'simply' for not killing or harming the animal models are put aside, turning the spotlight to the super-toxic metallopeptidases, phospholipases (A2 and D) and ionic channel blockers.

Still, a number of other animals can (and do) cause accidents upon human encounters, displaying broad variation in terms of the clinical outcome. Marine animals are good examples: sea urchins can be solely painful [30] whereas mollusks can instantly kill [31]. Yet, for some reason, such animals have not been able to attract the attention of major research groups in toxinology, remaining in 'neglect' for the past couple of decades.

The modern reptiles are a group comprised of the Crocodila, Lepidossaura, Rhynohocephalia, Squamata, Testudines and Aves. With the exception of snakes, no other true venomous reptile (i.e., with a specialized venom inoculation apparatus) is currently known. The venomous living dinosaurs, i.e., birds pitohui, ifrita and rufous [32], and the Komodo dragon are considered to be poisonous [33,34].

However, in the end, snakes are the most classical venomous animals. Since ancient times, their behavior has been considered to be mischievous—even tempting—and their venom has been associated to magic spells and even cures. Not surprisingly, The Rod of Asclepius, i.e., the Medicine symbol (Figure 3A), is a snake serpentizing around a rod [35]. Nevertheless, the caduceus—the traditional symbol of Hermes—represented by two snakes serpentizing around a winged rod (Figure 3B) is often mistakenly used as a symbol of medicine instead of the Rod of Asclepius, especially in the United States, as a consequence of documented mistakes, misunderstandings and confusion in the late 19th and early 20th centuries. However, the two-snake caduceus design has ancient and consistent associations with trade, eloquence, negotiation, alchemy, and wisdom. Last, but not least, the current Butantan Institute logotype (created in 1983) is a clever design in which the capital 'I' and 'B' are fused and the 'B' serif becomes the snake serpentizing around the 'I', which serves as the rod (Figure 3C).

**Figure 3.** (**A**) Rod of Asclepius, (**B**) the caduceus and (**C**) Butantan Institute logotype.

Jumping a few centuries ahead, there is indeed current medicine based on snake venoms, such as Captopril [25,36,37], Aggrastat, Intergillin and Aggretin [30,33], proving that ancient wisdom may be old, but never outdated. Not only that, but this particular *Toxins* issue that celebrates the 120th anniversary of Butantan corroborates this. At the same time, one can easily note the iconic fascination that the snake has exerted over the local scientific community, that has—and still does—followed Vital Brazil's initial steps.

### *1.4. Lizards*

Lizards' biting has long been discussed among the toxinology field due to the lack of an inoculating venom apparatus. *Heloderma* bites have been reported since 1882 [38,39], and the first toxic activities were described in 1900–1950. At that time, authors were aware that such lizards' toxins included neurotoxins, causing respiratory depression. Inflammation, edema and pain have also been described. However, once this animal bites 'as strong as a bulldog' according to the authors, these symptoms may not be exclusively toxinderived [40]. Moreover, its hemolytic activity is mild, when compared to snakes, and seems to be species-specific [41].

Later, between 1950–1990, a wide range of biological activities were described, such as phospholipasic, hyaluronidasic, proteolytic [42], L-amino acid oxidase, fibrinolytic, [43] esterase, 5 -nucleotidase [44], secretagogue [45] and nerve growth factor activity [46]. Furthermore, new venom components (at the time) were isolated and identified, such as: kallikrein [47,48], Helospectins 1–2 (acting as secretagogues) [49], Gilatoxin (serine peptidase) [50], Helodermin (vasoactive peptide) [51], and Helothermine (CRISP) [52]. Hyaluronidase [53], a Phospholipase A2 [54], Helodermatin (hypotensive toxin) [55], and Exendin-3 (secretagogue) [56] were also described. Such myriad of toxins could, then, be correlated to the many established envenomation symptoms, such as hypotension and respiratory difficulties [57], smooth muscle contraction [58] and anticoagulant effect [59].

In 1992, Exendin-4 identification was a major event and *Heloderma* venom studies skyrocketed from this year onwards [60]. Several research projects have evaluated the antidiabetic potential of this molecule, which gave rise to exenatide, a new drug for the treatment of diabetes [61]. A few years later, the inhibition of platelet aggregation by a phospholipase isolated from a Helodermatid lizard was described [62]. Even though it was already known that *Heloderma* venom presents at least five anionic phospholipases, being the most abundant similar to *Apis mellifera* phospholipase [63], it was another important event.

In the following years, Helokinestatin, a toxin that acts as an antagonist of the bradykinin B2 receptor, was described [64]. Moreover, Helofensin was identified by Fry and co-workers by genetic and functional analysis [65], and classified together with a class of lethal toxins firstly described by Komori at al. in 1988 [66]. The presence of a natriuretic peptide in *Heloderma* venom was pointed out by different authors [67,68].

A work comparing the venom proteome of *Heloderma suspectum* with the venom of *H. exasperatum* and *H. horridum* presented an interesting result. Although *H. suspectum* was evolutionarily separated from the other species 30 million before, the venom composition was basically the same for the three species, presenting the same toxins with slight differences in their relative proportions [69]. Moreover, authors could also describe two new molecules: semaphorin and a bactericidal/permeability-increasing (BPI) molecule. Another study that characterized the *H. suspectum* venom proteome relates to the presence of a neuroendocrine convertase 1 homolog, and proposes that this protein is responsible for the cleavage of the proforms of exendins. In the same study, the authors also point out the high presence of phospholipase propeptides in the venom proteome [70]. Recent works allowed access to different classes of proteins, and also new biological activities from *Heloderma* venom. The venom gland transcriptome analysis from *H. horridum horridum* revealed the presence of metalloproteases, lipases, vespryns, waspryns, lectins, cystatins and serine peptidase inhibitors, but none of these proteins were actually isolated from the venom [71]. Furthermore, *Heloderma* contains neurotoxins in its venom, and these toxins are able to bind sodium and calcium channels [72]. An important work by Fry et al. [73] evaluated phylogeny between snakes and lizards and demonstrated that the venom delivery system of these animals could have evolved from the same common ancestor. This was the first study that biochemically evaluated the venom of a lizard from the Varanidae family. The crude venom from *Varanus varius* displays a hypotensive effect and an isolated PLA2 from the venom inhibits platelet aggregation, via adrenaline pathway. The LC-MS analysis indicates the presence of natriuretic peptide, PLA2, CRISP, and Kallikrein. cDNA libraries analyses indicated the presence of AVIT, cobra venom factor, cystatin, crotamine, nerve growth factor and vespryn. Later studies demonstrated that the venom of *V. komodensis* (Komodo Dragon) also induces a hypotensive action, and that the venom is composed of toxins, such as PLA2, kallikrein, natriuretic peptide, CRISP, and AVIT [74].

A cDNA libraries analysis conducted by Fry et al. [67], comparing different lizards, was able to reveal new classes of toxins presents in the Varanidae family, such as lectin, veificolin, hyaluronidase, Cholecystotoxin (binds to CCK-A), Celestoxin (hypotensive), epididymal secretory protein and Goannatyrotoxin (hypertensive/hypotensive effect).

Then, the venoms of *Lanthanotus*, *Varanus* and *Heloderma* genus were compared through proteomic approaches and enzymatic activities profiling [69]. Interestingly, the only ubiquitous protein was Kallikrein and, different from Heloderma, which presents a conservation of venom constitution and actions in different species, the Varanus genus presents a variability in venom proteins and enzymatic activities such as serine peptidases, phospholipase activity and differential potential to cleavage alpha and beta chains from fibrinogen.

Venoms from different species of the Varanus genus were evaluated for the ability to prevent blood clotting by thromboelastography, and the venoms differ regarding the activity; the most potent effects were found in arboricole species, probably due to the selective pressure, according to the authors [75]. Similar to *Heloderma*, Varanid lizards possess neurotoxins that are able to bind sodium and calcium channels [72].
