**1. Introduction**

The image of the scorpion has long been connected to human history, being represented in cults, legends, philosophy and arts, as it is one of the oldest animals on the planet. Dating from the Silurian period, more than 400 million years ago, scorpions are organisms that have long intrigued human beings [1,2].

The order Scorpiones is represented by 2200 species and, through taxonomic studies, have been grouped into 20 families and 165 genera of scorpions. The most dangerous, and capable of causing fatal accidents in humans, belong to the Buthidae family, represented by the following genera: *Androctonus* and *Leiurus* (North Africa and Middle East), *Centruroides* (Mexico and the United States) and *Tityus* (South America and Trinidad) [3].

In Brazil, the scorpions *Tityus serrulatus*, *T. bahiensis* and *T. stigmurus* are the animals responsible for serious accidents. Among these, the *T. serrulatus* scorpion, popularly known as the "Brazilian yellow scorpion", is the one with the highest Medical and Scientific relevance in Brazil. It is mainly distributed among the states of Bahia, Goiás (including the Federal District), Paraná, Espírito Santo, Rio de Janeiro, Minas Gerais and São Paulo [4].

*T. serrulatus* reproduces by parthenogenesis, and each female is able to generate about 70 offspring during its life. They are commonly found in sewers, cemeteries and wastelands, where they find safe shelter and plenty of food. Therefore, in addition to the potency of its venom, their adaptation to urban centers may explain the significant increase in the number of accidents caused by this scorpion in Brazil [4–6].

In this scenario, accidents caused by *T. serrulatus* stings are considered a public health problem in Brazil due to its potential to cause severe clinical manifestations, which might bring a prognosis of death, especially on children aged from 0 to 14 years. Although most of the cases have been classified as mild, the biggest concern is related to the high number of cases that are reported annually in Brazil, since scorpion stings represent 41% of all venomous animal accidents, including snakes, spiders, bees and others, as reported in 2016 [7].

Generally, the *T. serrulatus* venom (TsV) is composed of mucus, inorganic salts, lipids, amines, nucleotides, enzymes, kallikrein inhibitors, natriuretic peptides, high molecular weight proteins, peptides, amino acids and neurotoxins [8]. Current studies carried out by Oliveira and colleagues [9] involving the transcriptome of the venom glands have shown that more than 30% of the venom is made up of enzymes and, approximately, 40% of peptides. The peptides present in the TsV can be classified as structured—which are stabilized by disulfide bonds—or linear [8]. The so-called structured peptides have been the most studied components, classified as neurotoxins that interact with ion channels (Na+ and K+) and are related to the most serious effects caused by the venom. On the other hand, linear peptides, although found with some abundance in the venom, are still poorly characterized. The peptidomic analysis performed by Rates and colleagues [10] demonstrated the existence of a great diversity of peptides in the TsV, all of them not yet characterized. Many could not be found in the database, and as this information is scarce, this requires de novo sequencing. Other studies using "omic" techniques have demonstrated the complexity of the venom in relation to linear peptides from post-translational modifications of larger proteins, generating lists with hundreds of components [11–13].

Among the linear peptides we have the hypotensins (TsHpt), identified from TsV proteomic analyzes. Both peptides are made up of 25 amino acid residues that contain two consecutive prolines in their C-terminal portion and a punctual difference between TsHpt-I and -II, which is the residue at position 15, being a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Studies with TsHpt-I, also known as Ts14, showed that this peptide was able to exert hypotensive activity in normotensive Wistar rats by potentiating bradykinin. The hypothesis is that the vasodilation effect is related to the release of NO by an independent mechanism of ACE inhibition [14].

In order to increase knowledge about hypotensins and their biological activities, the present work demonstrates, for the first time, the interaction of these peptides with human vasopeptidases, ACE (EC 3.4.15.1) and NEP (EC 3.4.24.11), alongside with cellular assays, which were carried out in order to verify the possible action of hypotensins as inflammatory or anti-inflammatory peptides.

#### **2. Results**

#### *2.1. Modulation of ACE and NEP Activities by Hypotensins*

For the initial kinetic tests, the synthetic hypotensins were incubated with the metallopeptidases ACE and NEP and their fluorescent substrates, Abz-FRK(Dnp)P-OH and Abz-RGFK (Dnp)-OH, respectively. These enzymes were chosen for the studies because they are considered of medical importance, where their modulations caused by venom peptides may be related to some symptoms present in serious accidents, such as hypotension/hypertension. As shown in Table 1, the hypotensins showed different activities in relation to the modulations of the peptidases studied.

**Table 1.** Modulations of ACE and NEP activities by interaction with synthetic hypotensins.


The results were obtained by incubating the enzymes with their FRET substrates (10 μM) in a final volume of 100 μL. For the ACE assays, the used buffer was Tris HCl 100 mM, NaCl 50 mM and ZnCl2 10 μM, pH 7.0. For NEP, the assays were made in Tris HCl 50 mM, pH 7.5 buffer. All reactions occurred at 37 ◦C, in a Victor 3 fluorimeter (Perkin– Elmer) adjusted for excitation and emission readings at 320 and 420 nm, respectively, for 15 min (one reader per minute). Results were obtained in triplicate and analyzed on GraFit 5 software.

Both peptides increased the catalytic activity of ACE, and TsHpt-I was more effective than TsHpt-II in activating the enzyme. Contrary to that observed with ACE, the results involving the peptides tested with NEP revealed the presence of inhibitors of this enzyme. As a highlight, TsHpt-I was able to reduce by 75% the hydrolysis of the FRET substrate used.

As already mentioned, the suggested mechanism of action on hypotension caused by TsHpt-I in vivo is the release of NO, together with the agonistic effect on bradykinin B2 receptors [15]. Taken together, our results with TsHpt-I seem to indicate a second hypotensive mechanism for this peptide, which involves the inhibition of NEP. Interestingly, TsHpt-I showed a strong inhibition of NEP's catalytic activity, in contrast with TsHpt-II. This fact is probably the result of a single difference between the primary structures between the hypotensins; that is, the presence of glutamine at position 15 in TsHpt-I, instead of a glutamic acid present in TsHpt-II.
