Snake venom disintegrins update: insights about new findings

ABSTRACT Snake venom disintegrins are low molecular weight, non-enzymatic proteins rich in cysteine, present in the venom of snakes from the families Viperidae, Crotalidae, Atractaspididae, Elapidae, and Colubridae. This family of proteins originated in venom through the proteolytic processing of metalloproteinases (SVMPs), which, in turn, evolved from a gene encoding an A Disintegrin And Metalloprotease (ADAM) molecule. Disintegrins have a recognition motif for integrins in their structure, allowing interaction with these transmembrane adhesion receptors and preventing their binding to proteins in the extracellular matrix and other cells. This interaction gives disintegrins their wide range of biological functions, including inhibition of platelet aggregation and antitumor activity. As a result, many studies have been conducted in an attempt to use these natural compounds as a basis for developing therapies for the treatment of various diseases. Furthermore, the FDA has approved Tirofiban and Eptifibatide as antiplatelet compounds, and they are synthesized from the structure of echistatin and barbourin, respectively. In this review, we discuss some of the main functional and structural characteristics of this class of proteins and their potential for therapeutic use.


Background
Snake venom is a secretion produced in the glands located on both sides of the animal's upper jaw.Its evolutionary function includes the defense and survival of the snake, as well as the immobilization and digestion of prey, aiding in its feeding.It is a complex cocktail, as its composition is formed by the mixture of various compounds, predominantly proteins, peptides, amino acids, nucleic acids, carbohydrates, lipids, and metals [1,2].After its production in pairs of homologous glands, venom is secreted into the base of the fangs, which can be located in the posterior region (opisthoglyphous) or anterior region of the animal's mouth, with the latter case having either short and fixed fangs (proteroglyphous) or long and movable fangs (solenoglyphous) [2,3].
Snakebite envenomation is considered a Neglected Tropical Disease with high incidence and severity, mainly affecting poverty regions [4].It is estimated that around 5.4 million snakebites occur worldwide each year, resulting in 1.8 to 2.7 million cases of envenomation and approximately 81,000 to 138,000 deaths [5].Snake venom exhibits a highly complex composition, and due to the diverse toxins with a wide range of biological functions, various clinical manifestations resulting from envenomation are observed, including local and systemic effects [6].However, beyond its toxic action, snake venom is also recognized for its high therapeutic potential, as its composition contains approximately 100 to 500 pharmacologically active compounds capable of acting on different target sites.For this reason, many studies have been conducted in the search for alternative therapies for various diseases [7].
In this context, snake venomics has demonstrated great relevance for the more detailed analysis of venom components [8].By using this strategy, which combines advances in proteomics and transcriptomics, it is possible to isolate venom compounds, estimate the content of toxins, as well as understand their biological and toxicological aspects [9].Advances in these techniques have allowed the characterization of up to 20 families of proteins in the venom of a single snake, with some of these families containing up to 80 different toxins [10].Despite the fascinating variability of compounds, most snake venoms are composed of four dominant protein families: phospholipase A 2 (PLA 2 ), three-finger toxins (3FTx), snake venom serine protease (SVSP), and snake venom metalloprotease (SVMP), along with secondary protein families, such as cysteine-rich secretory protein (CRISP), Kunitz peptides, L-amino acid oxidase (LAAO), natriuretic peptides, C-type lectins (CTL), disintegrins, among others [11].
In this review, we present the functional and structural aspects of disintegrins found in snake venom, as well as the evolutionary history of their emergence.We also discuss the potential applications of this class of peptides and the drugs already approved for therapeutic use.

What are snake venom disintegrins?
Snake venom disintegrins comprise a family of highly homologous, non-enzymatic polypeptides rich in cysteine (Cys).Their presence is described in the venom of snakes from the families Viperidae, Crotalidae, Atractaspididae, Elapidae, and Colubridae [12].This family of small proteins interacts specifically with integrins, a group of cell adhesion receptors on the surface of certain cells, including platelets, vascular endothelial cells, and some tumor cells [13,14].This way, disintegrins, by preventing such binding, interfere in intercellular and cell-matrix interactions, as well as signal transduction [12,14].

Integrins: a family of heterodimeric receptors
Integrins are transmembrane receptors that regulate or trigger different cellular processes upon binding to specific extracellular ligands [15].They are heterodimeric proteins formed by the noncovalent association of α and β chains.In vertebrates, at least 18 α subunits and 8 β subunits have been identified, which can form a total of 24 different heterodimers.The α and β subunits of integrins do not have detectable homology between them, but there are conserved regions among different α subunits (approximately 30% identity) and among β subunits (around 45%) [16].
Integrins can recognize ligands from the extracellular matrix, cell surfaces, and other soluble ligands, with the αβ pairings of integrin subunits being determinants for binding specificity [16,17].Structurally, each integrin subunit consists of an extended multidomain extracellular region (up to 1104 residues in the α subunit and 778 residues in the β subunit), a transmembrane helix, and a short cytoplasmic tail (with 20 to 70 amino acids).The N-terminal portions of each subunit, located in the extracellular region, combine to form a globular ligand-binding "head" (Figure 1) [18,19].
Integrins are present on the surface of many cell types and enable cell-cell interactions and interactions between cells and extracellular matrix proteins, including fibronectin, collagen, and laminin-1 [20].These interactions are related to a wide range of biological effects, so the role of integrins is associated with physiological events such as cell adhesion [21], wound healing [22], regulation of neuronal connectivity [23], and synapses [24], as well as pathological effects as inflammation [17], tissue fibrosis [25], atherosclerotic plaque development [26], They also interfere in various stages of cancer development and progression, including survival, proliferation, angiogenesis, migration, invasion, survival in circulation, extravasation, and metastatic growth [12,15,17,[27][28][29][30][31].

Snake venom disintegrins: evolution from metalloproteases
Snake venom disintegrins are peptides derived from the proteolytic processing of snake venom metalloproteinase (SVMP) precursors and carry in their structure the recognition motifs for integrins RGD, KGD, WGD, VGD, MGD, RTS, KTS [13,32].SVMPs are found in large quantities in snake venom and are the main components responsible for the hemorrhagic action after snakebite, interfering with the victim's hemostatic system [33,34].They are divided into different subclasses based on size and domain structure.Class P-I SVMPs contain only the typical metalloproteinase domain (M), composed of the pro-domain and proteolytic domain, and have a molecular mass of 20 to 30 kDa.Class P-II SVMPs have a molecular mass of 30 to 60 kDa and are structurally composed of pro-domain, proteolytic domain, and disintegrin-like domain (DI).Class P-III SVMPs (hemorrhagins) have a molecular mass between 60 to 100 kDa and are composed of a pro-domain, proteolytic domain, a disintegrin-like domain, and a cysteine-rich domain (C).In general, the hemorrhagic activity of these toxins depends on the M domain, but the DI and C domains are also important for their biological function.Thus, class P-III is recognized for its ability to induce higher and more diverse hemorrhagic activity when compared to class P-I and P-II SVMPs [33,35,36].
Evidence from molecular phylogenetics suggests that SVMPs evolved from a gene that encodes an A Disintegrin And Metalloprotease (ADAM) molecule, likely from an ancestral ADAM 7 or ADAM 28, belonging to the adamalysin family.Evolutionarily, SVMPs were recruited to the snake venom gland at the base of the advanced snake radiation, after the divergence of Pareatidae from the remaining Caenophidians, during the Paleogene period of the Cenozoic Era.The evolutionary history of SVMPs shows the loss of the cysteine-rich domain in class P-III, forming the SVMPs-PII, followed by the loss of the disintegrin-like domain and the formation of class P-I [35,37].
Regarding domain organization and sequence, important similarities are observed between ADAMs and P-III SVMPs, including the presence of the pro-domain, proteolytic domain, disintegrin-like domain, and cysteine-rich domain.Regarding structural differences, ADAMs have an EGF domain, a transmembrane domain, and a cytoplasmic tail, which are not present in SVMPs [38].
The evolutionary history of disintegrins occurred through positive Darwinian selection, and their presence in snake venom results from the proteolytic processing of P-II metalloproteinases or translation of short messenger RNAs without the metalloproteinase coding region [39][40][41][42].Thus, the presence of both free metalloproteinases and disintegrins can be observed in the venom [43].

Discovery and distribution of snake venom disintegrins
Snake venom disintegrins emerged in the scientific community in 1987, when Stefan Niewiarowski and Tur-Fu Huang isolated a low molecular weight non-enzymatic protein from the venom of Trimeresurus gramineus.The researchers observed that the protein, called trigramin, could block the binding of fibrinogen to stimulated GPIIb/IIIa receptors on platelets, thus inhibiting platelet aggregation.Although introduced in Toxinology in 1987, the term "disintegrin" was first used in 1990 when it was described as a new class of peptides isolated from snake venom, rich in the amino acid cysteine and containing an RGD domain in their structure [44,45].Since then, numerous studies have been conducted searching for this class of compounds in snake venom (Table 1).Approximately ten years after its discovery, non-RGD disintegrins were identified, challenging the concept of the obligatory presence of the Arg-Gly-Asp amino acids, and paving the way for the future discovery of different integrin recognition motifs [46,47].
Initially, disintegrins were studied for their inhibition of platelet aggregation due to the ability to interact with the transmembrane GPIIb/IIIa receptors (or αIIbβ3 integrin) present on the surface of platelets [39,[48][49][50].Fibrinogen is a bivalent molecule capable of simultaneously binding to the activated GPIIb/IIIa receptor on two different platelets, forming bridges between the activated platelets [51][52][53][54].Thus, disintegrins inhibit platelet aggregation by preventing the interaction of the αIIbβ3 integrin with fibrinogen.

Structural characterization of snake venom disintegrins
Snake venom disintegrins can be structurally classified into two major groups: monomeric and dimeric (Figure 2).Monomeric disintegrins are composed of three classes [73].The first class consists of short disintegrins with 41 to 51 amino acid residues and four disulfide bonds.The second class comprises medium disintegrins with approximately 70 amino acids and six disulfide bonds.The third class of monomeric disintegrins contains long disintegrins with about 84 residues and seven disulfide bridges [74].The second group of disintegrins is the dimeric disintegrins, which are further classified as homo-or heterodimers when the subunits are identical or different, respectively [73].The subunits of dimeric disintegrins are composed of around 67 residues with ten cysteines, which are involved in forming four intrachain and two interchain disulfide bonds [74].
Regarding binding specificity, the correct pairing of cysteine residues is essential for exposing the motif that mediates the interaction with integrins and determining their inhibition [74].In this context, the family of snake venom disintegrins can be divided into seven groups, each with a specific pattern of sequence and disulfide bond formation between cysteine residues (Figure 4).

Function and potential applications of snake venom disintegrins
Snake venom disintegrins can selectively bind to integrins, which are strongly tied to the specific motifs found in their structure [90] (Figure 5).This way, during envenomation, they exhibit a wide array of functions, serving various crucial roles, like binds to platelet receptors, impeding their aggregation, and resulting in the onset of bleeding disorders [91].Consequently, disintegrins contribute to disrupting hemostatic processes (Table 2).Some snake venom disintegrins can inhibit bone resorption in vitro [92] and can also be used as a diagnostic tool.An example, we cite bitistatin, which can potentially be used in molecular imaging of thromboembolic diseases [93].
It has also been demonstrated that disintegrins can interfere with the chemotaxis of human neutrophils to sites of inflammation and tissue injury [55] and exhibit antiparasitic activity against Leishmania infantum promastigotes [56].
Intriguingly, certain disintegrins have demonstrated notable anti-tumor and anti-angiogenic properties (Table 3).This remarkable feature opens up new possibilities for their utilization as potential therapeutic agents in cancer treatment, and by targeting tumor growth and impeding blood vessel formation, these disintegrins exhibit promising potential in medical research and innovation.

Snake venom disintegrins: from lab bench to market
Animal venoms are rich mixtures of components that may have important pharmacological actions.Many of these components have already been extensively studied to become drugs, and after approval by the Food and Drug Administration (FDA), turned into widely used molecules [94].
A very important example of a drug derived from animal toxins is captopril (Capoten®, Bristol-Myers Squibb, New York, NY, EUA), which is widely used against hypertension [95].This was the first animal-derived drug approved by the FDA in 1981, which mechanism is responsible for inhibiting the angiotensin-converting enzyme (ACE).Thus, the production of angiotensin II is also inhibited, reducing hypertension effects, and increasing the hypotensive action of bradykinin, known as a bradykinin potentiating factor (BPF) [96][97][98][99].Although it is a very effective natural molecule, the captopril used in medicaments is a synthetic molecule based on the miniaturization of the original molecule and chemically modified to be administered orally [94,100].In sequence, in 1985, the FDA approved Enalapril (Vasotec®, Merck, Darmstadt, Germany), which was also used to treat hypertension and congestive heart failure [94,101].
Some disintegrins have been extensively studied and are nowadays FDA-approved drugs as well.Tirofiban (Aggrastat®, Table 2. Snake venom disintegrins that can act on the hemostatic system.
Since the approval of the first venom-derived drug and the beginning of disintegrins' saga in Toxinology [44], it took over 10 years of research and effort for the first medication derived from snake venom disintegrins also to be approved (Figure 6).However, it was already known that venoms and their components could cause modifications in the human body, and their applicability in clinical settings had been recognized.
Currently, a product based on snake venom toxins has been attracting attention: Heterologous Fibrin Sealant.This sealant is composed of a thrombin-like enzyme from Crotalus durissus terrificus venom and fibrinogen-rich cryoprecipitate extracted from the blood of Bubalus bubalis buffaloes.It can be used for the treatment of chronic venous ulcers, as demonstrated in phase I/II clinical trials, highlighting its effectiveness and safety [108].While there are currently no clinical studies using snake venom disintegrins, human disintegrins, especially ADAMs, have been targeted for the therapy of other pathological conditions in clinical trials, such as cirrhosis and portal hypertension (NCT04267406), epithelial dysfunction (NCT00898859), idiopathic pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension (NCT05478226), among others [109].

Conclusion
Snake venom disintegrins' saga was started in 1987 and classified these molecules as small peptides that can inhibit the function of integrins, which are cell surface receptors involved in various cellular processes like cell adhesion, migration, and signaling.
Integrins are important for cell adhesion to extracellular matrix proteins, mediating cell-cell interactions, and interfering in integrin-mediated processes, as snake venom disintegrins can have various effects on cells and tissues.Among their unique properties, snake venom disintegrins can inhibit platelet aggregation, i.e., bind to integrins on platelets, preventing their aggregation and potentially disrupting the clotting process.Consequently, two important antiplatelet drugs were based on disintegrins from snake venoms, and they are on the market nowadays.
Moreover, snake venom disintegrins have shown anti-cancer properties by targeting integrins that are overexpressed in specific cancer cells and blocking integrin-mediated signaling pathways.These disintegrins can also inhibit tumor growth and metastasis.Notably, although snake venom disintegrins possess therapeutic potential, they exhibit high potency and can manifest toxicity.Thus, rigorous investigation is required before contemplating snake venom disintegrin use in medical applications.

Figure 1 .
Figure 1.Integrin structure.Conversion of integrin from its inactive low-affinity conformation to the active high-affinity conformation for the ligand through intra-or extracellular stimuli.

Figure 3 .
Figure 3. Multiple alignments among selected disintegrins from different structural classes.Cysteine residues are highlighted in gray.The integrin-binding RGD motif is represented in red, and non-RGD motifs are in blue.

Figure 5 .
Figure 5. Interaction of snake venom disintegrins motifs with different integrins.

Figure 6 .
Figure 6.Timeline of snake venom disintegrins, from the beginning of disintegrins' saga in Toxinology until their FDA approval.

Table 3 .
Discovery of snake venom disintegrins that can act as anticancer agents.