**4. Disintegrins**

Disintegrins are low molecular weight (4–14 kDa), nonenzymatic, disulfide-rich proteins derived from the proteolysis of snake venom metalloproteases (SVMPs). These molecules are phylogenetically related to human ADAMs (a disintegrin and a metalloprotease) [31]. SVMPs are divided according to their multidomain metalloprotease structure into P-I, P-II and P-III classes. Members of the P-I class are composed of a metalloprotease domain (MD), P-II class proteins have a MD and a disintegrin-like domain, and P-III have a cysteine-rich or disintegrin-like domain, and sometimes a lectin domain. Both P-II and P-III classes are subdivided according to their proteolytic processing and the ability to organize dimers [11, 31].

Depending on the type and processing, SVMPs yield different disintegrins structurally classified into monomers, homodimers and heterodimers. Monomeric disintegrins come from the processing by the P-IIa class of SVMPs, homodimers are derived from P-IId class and heterodimers from the P-IIe class. P-III SVMPs are derived from disintegrin-like proteins (not containing the RGD motif), which consist of a cysteine-rich "disintegrin-like" domain, covalently linked and with a molecular weight of 30 kDa [31]. Another classification name disintegrins as short, medium, long, homo and heterodimers, based on the number of amino acid residues and disulfide bonds [12, 26, 32].

#### **4.1 Disintegrins binding motifs**

Disintegrins interact with specific integrins depending on the presence of a particular integrin-binding motif located in the hairpin loop, modulating the

selectivity toward cellular responses. There are three common motifs identified in disintegrins: RGD, KGD and KTS [33–35]. Other disintegrins contain different tripeptide sequences but conserve aspartic acid such as the MVD, KGD and WGD motifs [31].

RGD disintegrins correspond to the largest and most investigated family, they can inhibit physiological functions of α3β1, α4β1, α5β1, α6β1, α7β1, α8β1, αvβ1, αvβ3, αIIbβ3 and αvβ5 integrins. There are some dimeric disintegrins with motif MLD which binding to α3β1 integrins, α4β1, α5β1, α6β1, α7β1, α9β1, αIIbβ3 and α4β7, whereas others with KTS motifs in their active site have shown selectivity toward the α1β1 collagen receptor [31].

## **4.2 Disintegrins and cancer**

The main activity of disintegrins is to interfere with integrin-ligand interactions [35], i.e., they block biding integrins with ECM proteins such as collagen, fibronectin, laminin, and fibrinogen, preventing intracellular signals, cellular adhesion, migration, and metastasis [36]. The biological activity of disintegrins has prompted the research with these peptides as inhibitors of cancer progression [31]. For example, eristostatin binds and blocks α4β1, an integrin associated with metastasis in human melanoma cells [37], jerdoni inhibits tumor growth and improves survival in a mouse model of melanoma [12], and rhodocetin, which prevents fibrosarcoma cell invasion by blocking α4β1 to collagen [38].

Isolation and purification involve arduous processes to extract components from snake venom having small yielding. The production of recombinant disintegrins is a strategy to obtain large quantities of these proteins, suitable for *in vitro* and *in vivo* testing. Different disintegrins have been produced thanks to recombinant technology como albolatin [39], obtustatin [40], rubistatin [22] and salmosin [41]. Hosts such as *Escherichia coli* and *Pichia pastoris* are widely used for recombinant protein production with high yielding and easy genetic manipulation [11].
