**3. Discussion**

Identified from the proteomic analyses on the venom of *Tityus serrulatus* (TsV), the hypotensins are peptides made up of 25 amino acid residues that contain two consecutive prolines in their C-terminal portion. Hypotensins are a family of peptides with small structural differences between them, with TsHpt-I being the best-studied member—both the natural molecule and its synthetic counterpart. Tests involving the natural or synthetic TsHpt-I demonstrated that this peptide was able to exert hypotensive activity in normotensive Wistar rats through bradykinin potentiation. The hypothesis about the vasodilation effect is related to NO release, which is a mechanism independent of ACE inhibition [14].

Focusing on clinical conditions related to blood pressure alterations observed in accidents caused by *T. serrulatus*, the present study investigated the possible interaction of TsHpt-I and -II with the metallopeptidases angiotensin converting enzyme (ACE) and neprilysin (NEP). ACE is considered an important molecule in the regulation of blood pressure, as it generates angiotensin II (Ang II) from the cleavage of angiotensin I (Ang I), in addition to degrading bradykinin (Bk) [17]. NEP also acts to control blood pressure through the excretion of Na+ and water, as it is capable of degrading natriuretic peptides (ANP, BNP and CNP) [18,19]. Thus, both ACE and NEP are known as vasopeptidases. The third vasopeptidase is the endothelin-converting-enzyme I (ECE-1, EC 3.4.24.71), a metallopeptidase capable of releasing endothelin-I (ET-I), a vasoconstrictor peptide, from the big-endothelin [20].

Regarding TsV, data from the literature describe the presence of an ACE-like one [9,21], capable of converting Ang I to Ang II, and degrading Bk. Hence, the presence of this vasopeptidase in TsV may collaborate with the hypertension observed in accidents involving humans. The possible presence of an NEP-like in TsV has also been described, as well as an inhibitor of this metallopeptidase, called [des-Arg1]-Proctolin [16]. This peptide was characterized as a competitive inhibitor of human NEP, presenting an inhibition constant of 0.94 μM [16]. Proteomic studies also reported the presence of an ECE-like enzyme in TsV [9]. Moreover, high levels of ET-I were observed in the sera of patients after envenomation with the scorpion *Androctonus australis hector*, indicating that molecules of scorpion venoms also have an effect on the endothelin axis. [22]. Therefore, the presence of vasopeptidases and their inhibitors in scorpion venoms may contribute to acute changes caused in the cardiovascular system observed in cases of envenomation [23].

Although the preferred prey of the *Tityus serrulatus* scorpion are insects, such as crickets and cockroaches, its venom is dangerous to humans. According to data from proteomics and transcriptomics studies [9], the toxic effect of TsV in humans may be the result of evolutionarily preserved molecules present in both insects and mammals. This suggestion may explain the presence of ACE-like, ECE-like and NEP-like enzymes in TsV, together with neprilysin inhibitors and hypotensins. [23].

Interestingly, both hypotensins were able to increase the catalytic activity of ACE, but in different ways. While TsHpt-I activated ACE by 64%, TsHpt-II increased by 46% the hydrolysis of the substrate Abz-RGFK-EDDnp. In fact, in studies on the determination of the hypotensive mechanism of TsHpt-I, ACE activation can also be observed; however, the results were not discussed [14]. Studies with NEP also indicated different interactions with hypotensins, and results with TsHpt-I showed that this peptide is a non-competitive inhibitor of NEP, with a Ki value of 4.35 μM. As TsHpt-I is the second NEP inhibitor described in the *T. serrulatus* venom, it is possible that there is a combined action between hypotensin I and [des-Arg1]-Proctolin [16], which may be related to the hypotension caused by the envenomation. In contrast, TsHpt-II displayed a low interaction with NEP, and the different results are, probably, the effect of a single difference between the primary structures of the hypotensins. However, future studies of circular dichroism will be needed to clarify this matter.

As hypotensins demonstrated new activities in vitro, cytotoxicity and possible pro- or anti-inflammatory actions were investigated in order to increase our knowledge of these molecules. Both hypotensins have immunomodulatory potential, with pro-inflammatory effects on murine peritoneal macrophages, when used at a concentration of 100 μg/mL. Interestingly, at this concentration, both hypotensins did not exert cytotoxic activity on the tested cells, which makes the two molecules even more interesting, due to their pharmacological potential for the long-term development of new immunostimulants and/or adjuvants. Pucca and collaborators [24] demonstrated the pro-inflammatory effect of three peptides derived from the TsV venom on a strain of murine macrophages, with increased production of IL-6. Similar to the results presented in this work, the effects were subtle compared to LPS, although significant in relation to the negative control. In our study, we observed increased TNF production in the presence of both peptides and increased IL-6 production in the presence of TsHpt-II. As for the mechanism of molecules as hypotensives, it is important to emphasize that both TNF and IL-6 induce vasodilation, and its massive release can even lead to shock.

Cassini-Vieira and colleagues suggested an anti-inflammatory role for TsHpt-I from *T. serrulatus* venom, based on its ability to reduce neutrophil infiltration and TNF production in a murine model of sponge implant-induced inflammation. On the other hand, increased macrophage infiltration was observed in this model, indicating a pro-inflammatory role, which demonstrates the need for further studies on the mechanisms of action of TsHpt-I [25].

Interestingly, both treatments with both peptides promoted a significant increase in the phagocytic index, demonstrating that the pro-inflammatory action of these peptides also affects the macrophages' biological function. This phenomenon is interesting, considering the possible development of immunomodulators. It is known that adjuvant and/or immunostimulant molecules generally induce a pro-inflammatory environment that favors the activation of antigen-presenting cells and, consequently, the development of adaptive immunity against specific antigens. Increased macrophages' phagocytic capacity by hypotensins may reflect increased microbicidal activity and/or antigen presentation. The anti-candida and anti-biofilm activities of TistH, a hypotensin present in the venom of the *Tityus stigmurus* scorpion, were recently described and confirmed [26], but functional antigen presentation assays are needed to deepen our knowledge of the immunomodulatory action of these peptides.

Despite the biotechnological potential of hypotensins, the activities already described for these peptides, and the new results showed in the present work, indicate that both molecules do not have a specific target or mechanisms of action. Considering that they are multifunctional toxins present in the *Tityus serrulatus* venom, new studies aiming at drug development should be very carefully carried out in order to minimize unexpected effects.

#### **4. Conclusions**

Despite the great similarity between the primary structures of hypotensins, different levels of interactions with the vasopeptidases ACE and NEP were observed. Hypotensins increase ACE activity at different levels while TsHpt-I is a non-competitive inhibitor of NEP, suggesting other hypotensive mechanisms for this peptide in addition to those already described. Furthermore, the release of some interleukins may suggest a role for these peptides in the inflammatory response induced by the venom. Hypotensins are multifunctional toxins, and further studies are needed to clarify the potential of these molecules for biotechnological use.

### **5. Material and Methods**

### *5.1.* Reagents

The synthetic peptides TsHpt-I and TsHpt-II were obtained by the solid-phase peptide synthesis method, and purchased from GenOne Biotechnologies (Rio de Janeiro, Brazil). Angiotensin Converting Enzyme (ACE) from rabbit lung, RPMI 1640 medium, LPS from *E. coli* 0127:B8, Trypan Blue, Giemsa stain and glutaraldehyde solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Neprilysin and the Fluorescence Resonance Energy Transfer (FRET) substrates, Abz-FRK (Dnp) P-OH and Abz-RGFK (Dnp)-OH, were

provided by Prof. Adriana Carmona, from the Department of Biophysics of UNIFESP-EPM, São Paulo, SP, Brazil. Acetonitrile and TFA used in RP-HPLC were acquired from J. T. Baker (Avantor, Radnor, PA, USA). Fetal cow serum (FCS) and penicillin and streptomycin antibiotics were purchased from Cultilab (Campinas, SP, Brazil). Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was purchased from Invitrogen (Waltham, MA, USA). DMSO was purchased from Merck (Darmstadt, Germany). BD Cytometric Bead Array Mouse Inflammation Kit was purchased from BD Biosciences (San Jose, CA, USA). The *Saccharomyces cerevisiae* suspension was obtained from washing and adjusting the concentration of bread yeast (Fleischmann – Petrópolis, RJ, Brazil) in RPMI.
