**1. Introduction**

The kallikrein-kinin system is an extensively studied biological pathway and involves a multiprotein complex, which includes serine proteinases from tissue and plasma. These proteinases act on substrates as kininogens (high and low molecular weight), releasing the active kinins. The main kinin is the nonapeptide bradykinin (BK).

Several studies aiming to evaluate the biological activities of the kinins revealed that this peptide is implicated in diverse physiological processes as regulation of blood pressure, cardiac, and renal function. Due to its ability to increase the vascular permeability by acting on endothelial cells, BK is correlated to several pathological processes including inflammation. These actions have been observed and described in both mammals and rodents [1].

The knowledge on the role of BK in various biological pathways as coagulation cascade, blood pression regulation, and central nervous system modulation and signaling has been signifi‐ cantly improved, leading to the identification of BK receptors and posterior development of drugs targeting its pathways [2].

This research was mainly driven by scientific studies on animal venoms, which lead to the identification of the BK-related peptides (BRPs). The best, and maybe also the first, example of such contribution was the discovery of the bradykinin-potentiating peptides (BPPs), first described in *Bothrops jararaca* venom [3, 4]. The BPPs are proline-rich oligopeptides that inhibit the angiotensin-converting enzyme (ACE), and that are responsible for the hypotensive effect of the *Bothrops* genus snake venoms. The pharmacological effects of these peptides have been studied since 70's [3, 4], and allowed not only to the discovery of the neuropeptide BK [5], but also to the development of the first active site-directed inhibitor of ACE as drug for the

treatment of human hypertension [6]. In fact, several other drugs derived from venom toxins, with or without modifications, are also commercially available (*e.g*. Captopril, Ancrod, and Prialt) [7]. Moreover, the study of toxins has widely contributed to the identification of new targets with therapeutic potential in mammals, as well as it has allowed to the understanding and discovery of the biochemical pathways involving these targets.

In this book chapter, we intend to gather the most important results obtained up to now, thanks to the isolation and characterization of BPPs from diverse organisms and to the knowledge

Venom Bradykinin-Related Peptides (BRPs) and Its Multiple Biological Roles

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The main function of snake venoms is still believed to be the immobilization of preys to ensure feeding. The snake venoms are composed of a complex mixture of proteins and biologically active peptides [24, 25]. The study of the pathophysiological mechanisms of poisoning and molecular characterization of toxins from the venom of *Bothrops jararaca* resulted in many scientific contributions of great importance, and among them, stand out the discovery of BK [5] and the discovery of the first BRPs, more specifically the BPPs produced by the snake venom glands, [4, 26] whose synergistic action is capable of causing a sharp drop in blood pressure

The BPPs are molecules able to enhance some pharmacological activities of BK, as the action of contractile smooth muscle of guinea pig ileum evaluated in *ex vivo* assays [26], and also *in vivo*, acting in the CNS, cardiovascular, and antinociceptive systems [27, 28]. The isolation of the first BPPs expressed by the *Bothrops jararaca* venom glands was described in the early 60's, and they were initially coined as Bradykinin Potentiating Factors (BPF) due to their ability to potentiate the effects of BK ignoring at that time the fact that these molecules were composed by amino acid residues [26]. Only in early 70's, when their primary sequence were determined, which allowed to characterize them as peptide molecules, they were re-named as BPPs [3]. Since then, several peptides presenting similar structural characteristics have been identified from the venom of these snakes and also from other snakes belonging to several different genus [12, 13, 29-31]. Interestingly, they had also been described in wasps and frogs [8-11]. Typically, the BPPs are peptides of 5-14 amino acid residues [32]. In general, all known naturally occuring BPPs could be classified into two groups: (i) peptides of small molecular size like BPP-5a from the venom of *Bothrops jararaca*, whose structural characteristic is a pyroglutamic acid at the Nterminal and a proline residues at the C-terminal of molecule, and (ii) peptides consisting of about ten amino acid residues, with a pyroglutamic acid at the N-terminal and a notable high content of proline residues [32], which gives to them some resistance to hydrolysis by amino‐

The BK and its related peptides, *e.g*. the BRPs, are widely found in venomous animals, for instance in snakes, lizards, frogs, and insects [10, 13, 34]. In general, they include several sequences, either showing only one single amino acid substitution compared to BK or, in some cases, presenting just a frugal sequence similarity, but with unquestionable biological/ functional correlation, for instance, acting on the same pathway or even same target protein. In fact, these sequence variations were verified either by *de novo* sequencing of several BRPs found in snake venoms [32] or by analysis of the deduced amino acid sequences of cDNAs cloned from venomous glands [12, 14, 15], and in some cases by using both strategies [30, 34].

accumulated, while searching for new targets for these molecules.

**2. The discovery of the snake venom BK and BRPs**

of small animals, for instance mammalian preys.

peptidases, carboxypeptidases, and also endopeptidases [33].

**2.1. cDNA cloning, identification and characterization of BRPs**

Since then, the BPPs/BRPs have been found in several snake venoms, and also in wasps and frogs, by using either biochemical or/and recombinant DNA techniques [8-11]. For instance, molecular cloning studies using cDNA libraries of four species of snakes from Crotalinae family showed evidences that these bioactive peptides are expressed by orthologous genes [12]. The cloning of orthologous precursors from different snakes from *Bothrops* and *Crotalus* genus allowed the identification of several new BPPs sequences [13-15], and some of them was shown to display different specificity toward each active sites of the somatic ACE ectoenzyme [16]. This was believed to be a great opportunity for the development of a new generation of antihypertensive drugs.

The employment of recombinant DNA techniques were also fundamental to first determine the structure of the precursor protein of BPPs, which was found to contain several sequences of BPPs distributed as tandem repeats, followed by a C-type natriuretic peptide (CNP) at the C-terminus of this precursor molecule [15]. In contrast to other members of the natriuretic peptide (NP) family, CNP is synthesized in the brain and has hypotensive effect with no significant diuretic or natriuretic actions [17]. Moreover, Northern blot analysis of several snake tissues demonstrated the presence of similar BPPs-CNP precursor mRNA in nonvenomous tissues, such as the central nervous system (CNS) [14]. In *situ* hybridization studies also detected the presence of the BPP/CNP-precursor mRNA in regions of snake brain correlated with neuroendocrine functions, such as the ventromedial hypothalamus, paraven‐ tricular nuclei, paraventricular organ, and subcommissural organ [14]. Analogous CNP precursor mRNAs was also described in similar regions in rat and human brains [18].

These studies suggesting the potential expression of BPPs in snake CNS stimulated us to investigate the putative target(s) of these peptides. Based on the *in vivo* biodistribution studies showing the preferential accumulation of BPPs in the rat kidney, and also a significant presence in the brain, the first studies were conducted in theses tissues leading to the description of several completely new potential targets and pathways, as the nicotinic acetylcoline receptors [19], L-argininosuccinate synthase [20], and an orphan G protein-coupled receptor (GPCR) [20]. The importance of both NO release for the antihypertensive effects of BPPs [20-22], and also the involvement of the GPCRs, namely B2 receptor and M1 muscarinic receptor (mACh-M1), in vasodialtion were demonstrated [23].

Together all these data collected during the last decade showed the pharmacologial signifi‐ cance of the BPPs and, more importantly, that the biological effects of these peptides, although first believed, are not limited to the inhibiton of the somatic ACE [2]. The high variability of molecular structures of these peptides reflecting in different specifities is an indicative that there are still more to be discovered regarding the biological effects of this peptides family.

In this book chapter, we intend to gather the most important results obtained up to now, thanks to the isolation and characterization of BPPs from diverse organisms and to the knowledge accumulated, while searching for new targets for these molecules.
