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

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

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

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

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-

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.

and discovery of the biochemical pathways involving these targets.

antihypertensive drugs.

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M1), in vasodialtion were demonstrated [23].

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 of small animals, for instance mammalian preys.

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‐ peptidases, carboxypeptidases, and also endopeptidases [33].

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

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].

The pharmacological evaluations revealed that even acting in the same pathway, they can show distinct biological activities compared to BK, including potentiating its effects by inhibition of its degradation or by acting on receptors and/or molecules involved in the BK signaling pathway, including activating or blocking the BK receptors [10, 35]. As such, the BRPs also include BPPs and the Bradykinin Inhibitor Peptides (BIPs) [13, 29, 32, 36].

accelerated evolution hypothesis suggested by Ohno and colleagues [42]. According to this hypothesis, the more frequent occurrence of nucleotide nonsynonymous substitutions in the coding regions compared to the untranslated regions (UTRs) of the genes allows specific genes to evolve in an accelerated fashion to attain unique physiological activity. On the other hand, despite the consequent changes in the BRPs sequences observed, both the high content of proline residues and the biological activities correlated to BK effects are still maintained (Figure 1). Another highly conserved region involves the sequence of the NPs always present in C-

The NP system consists of three types of hormones [atrial NP (ANP), brain or B-type NP (BNP), and C-type NP (CNP)], and three types of receptors [NP receptor (R)-A, NPR-B, and NPR-C]. Both ANP and BNP are circulating hormones secreted from the heart, whereas CNP is basically a neuropeptide. The NP system plays pivotal roles in cardiovascular and body fluid homeo‐ stasis. The ANP is secreted in response to an increase in blood volume, and acts on various

are always regulated in the same direction. Vertebrates expanded their habitats from fresh water to the sea or to land during evolution. The structure and function of osmoregulatory

Members of the NPs family have been detected in several snake venoms, and they have been shown to be located in the same precursor protein containing multiple BRPs sequences. While this organization was demonstrated for many species of viperid snakes, including members of the genera *Bothrops, Crotalus, Lachesis, Agkistrodon,* and *Trimeresurus* [12, 13, 15, 30, 31], it may extend to some other taxa such as *Bitis gabonica*, that was also shown to have BPPs in their venom [12, 13, 15, 30, 31, 44]. The presence of NPs in some elapid snakes venom (*Dendroaspis*) has also been described [45]. It has been shown that the venom-derived NP precursors from helodermatid lizard have a structural organization similar to that found in many BRPs precursors from viperid snake venoms. However, the additionally encoded tandem-repeat peptides are non-canonical BPPs, based on their primary structural characteristics or in terms of the amino acid cleavage site, presenting characteristics of a recognition site typical of propeptide convertase enzymes, that eventually might be the potential responsible for the release of the mature BIPs from the respective biosynthetic precursors [36]. However, the BPP/ CNP biosynthetic precursors of the bushmaster (*Lachesis muta*), the tropical rattlesnake (*Crotalus durissus terrificus*), and the massasauga rattlesnake from desert (*Sistrurus catenatus ewardsi)* showed that, in addition to the classical BPPs and a NP sequence, they all also encode single copies of a BIP exhibiting a closer structural similarity, and a propeptide convertase cleavage site that allows the release of the BIP helokinestatins, whose sequences are [TPPAGPDVGPR] or [TPPAGPDGGPR] [36] (Figure 1). On the other hand, putative heloki‐ nestatins peptides could not be identified in the BPP/CNP precursor of snakes as *Bothrops jararaca, Bothrops jararacussu*, and *Agkistrodon blomhoffi* (Figure 1). The phylogenetic analysis

hormones have also undergone evolution during this ecological evolution [43].

, resulting in restoration of blood volume. The family

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and water in tetrapods, in which both


terminus extremity of all known BRPs precursor proteins [12-15, 30, 31, 34, 36].

**2.3. BRPs and NPs**

organs to decrease both water and Na+

depleting hormones promoting the excretion of both Na+

of NPs were originally Na+

### **2.2. BPPs and BIPs**

Helokinestatins are a family of proline-rich peptides (PRPs) found originally in the lizard venom (*Heloderma suspectum*) that display the function of inhibiting the BK actions on the vascular smooth muscle [35]. Synthetic replicates of all helokinestatins were found to antag‐ onize the relaxation effect observed following BK application to a rat arterial smooth muscle preparation, and hence, represent a family of BRPs also known as BIPs [34].

In contrast, BPPs firstly described and isolated from the venom of the Brazilian snake *Bothrops jararaca* are mainly known due to their ability to potentiate the biological effects of BK [3, 26]. These BRPs are one of the most outstanding group of PRPs, as they were used as structural and functional template/model for the development of a drug employed up to now for the treatment of human hypertension [6].

Although functionally related to the BK and also present with the NPs in the same precursor protein, the helokinestatins are quite different from the snake venom BPPs [12, 30, 31, 37-39]. PRPs with the same BK inhibitory characteristics have also been described in the 'venomous' secretion of two species of anguid lizards, the *Texas alligator* (*Gerrhonotus infernalis*) and the *Giant Hispanolian galliwasp* (*Celestus warreni*) [38]. Although the primary structural variation of the peptides from these species, they share several common features [34]. For instance, they are peptides rich in prolyl residues (30-50%), which confer rigidity and order to the spatial structure features, and also a measure of resistance to generalized proteolysis. They all possess a Pro-Arg dipeptide motif at the C-terminus, which is quite different from the C-terminal Ile/ Val-Pro-Pro motif present in most BPPs C-terminus extremity [12]. The high degree of conservation of these structural core features across phylogeny suggest a fundamental biological function for this group of peptides in the lizards venoms. Among the two closelyrelated species of helodermatid lizards, several helokinestatins have fully-conserved primary structure, while several others present different sequences. Similarly to the BRPs from amphibian skin [40] and snakes venom [13, 14, 30], helokinestatins compose tandem repeat domains in their respective precursor proteins, probably reflecting discrete exons within the genomic DNA. As already mentioned, some tandem repeats are composed by identical primary structure, while some others exhibit significant amino acid substitutions. prominent And this process of exon multiplication might facilitate the molecular diversity, by permitting the expression of site-mutated isoforms, which is a phenomenon often described for bioactive peptide-encoding genes, as also observed for the glucagon gene in vertebrates [41].

Cloning and alignment of cDNAs encoding BRPs precursors from the venom gland and brain of a pit viper have allowed observing a higher degree of sequence consevation for the regions not including the bioative peptides, and a higher variation in the primary structure of these biological active peptides [14]. These results were shown to be in good agreement with the accelerated evolution hypothesis suggested by Ohno and colleagues [42]. According to this hypothesis, the more frequent occurrence of nucleotide nonsynonymous substitutions in the coding regions compared to the untranslated regions (UTRs) of the genes allows specific genes to evolve in an accelerated fashion to attain unique physiological activity. On the other hand, despite the consequent changes in the BRPs sequences observed, both the high content of proline residues and the biological activities correlated to BK effects are still maintained (Figure 1). Another highly conserved region involves the sequence of the NPs always present in Cterminus extremity of all known BRPs precursor proteins [12-15, 30, 31, 34, 36].

### **2.3. BRPs and NPs**

The pharmacological evaluations revealed that even acting in the same pathway, they can show distinct biological activities compared to BK, including potentiating its effects by inhibition of its degradation or by acting on receptors and/or molecules involved in the BK signaling pathway, including activating or blocking the BK receptors [10, 35]. As such, the

Helokinestatins are a family of proline-rich peptides (PRPs) found originally in the lizard venom (*Heloderma suspectum*) that display the function of inhibiting the BK actions on the vascular smooth muscle [35]. Synthetic replicates of all helokinestatins were found to antag‐ onize the relaxation effect observed following BK application to a rat arterial smooth muscle

In contrast, BPPs firstly described and isolated from the venom of the Brazilian snake *Bothrops jararaca* are mainly known due to their ability to potentiate the biological effects of BK [3, 26]. These BRPs are one of the most outstanding group of PRPs, as they were used as structural and functional template/model for the development of a drug employed up to now for the

Although functionally related to the BK and also present with the NPs in the same precursor protein, the helokinestatins are quite different from the snake venom BPPs [12, 30, 31, 37-39]. PRPs with the same BK inhibitory characteristics have also been described in the 'venomous' secretion of two species of anguid lizards, the *Texas alligator* (*Gerrhonotus infernalis*) and the *Giant Hispanolian galliwasp* (*Celestus warreni*) [38]. Although the primary structural variation of the peptides from these species, they share several common features [34]. For instance, they are peptides rich in prolyl residues (30-50%), which confer rigidity and order to the spatial structure features, and also a measure of resistance to generalized proteolysis. They all possess a Pro-Arg dipeptide motif at the C-terminus, which is quite different from the C-terminal Ile/ Val-Pro-Pro motif present in most BPPs C-terminus extremity [12]. The high degree of conservation of these structural core features across phylogeny suggest a fundamental biological function for this group of peptides in the lizards venoms. Among the two closelyrelated species of helodermatid lizards, several helokinestatins have fully-conserved primary structure, while several others present different sequences. Similarly to the BRPs from amphibian skin [40] and snakes venom [13, 14, 30], helokinestatins compose tandem repeat domains in their respective precursor proteins, probably reflecting discrete exons within the genomic DNA. As already mentioned, some tandem repeats are composed by identical primary structure, while some others exhibit significant amino acid substitutions. prominent And this process of exon multiplication might facilitate the molecular diversity, by permitting the expression of site-mutated isoforms, which is a phenomenon often described for bioactive

peptide-encoding genes, as also observed for the glucagon gene in vertebrates [41].

Cloning and alignment of cDNAs encoding BRPs precursors from the venom gland and brain of a pit viper have allowed observing a higher degree of sequence consevation for the regions not including the bioative peptides, and a higher variation in the primary structure of these biological active peptides [14]. These results were shown to be in good agreement with the

BRPs also include BPPs and the Bradykinin Inhibitor Peptides (BIPs) [13, 29, 32, 36].

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preparation, and hence, represent a family of BRPs also known as BIPs [34].

**2.2. BPPs and BIPs**

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treatment of human hypertension [6].

The NP system consists of three types of hormones [atrial NP (ANP), brain or B-type NP (BNP), and C-type NP (CNP)], and three types of receptors [NP receptor (R)-A, NPR-B, and NPR-C]. Both ANP and BNP are circulating hormones secreted from the heart, whereas CNP is basically a neuropeptide. The NP system plays pivotal roles in cardiovascular and body fluid homeo‐ stasis. The ANP is secreted in response to an increase in blood volume, and acts on various organs to decrease both water and Na+ , resulting in restoration of blood volume. The family of NPs were originally Na+ -extruding hormones in fishes; however, they evolved to be volumedepleting hormones promoting the excretion of both Na+ and water in tetrapods, in which both are always regulated in the same direction. Vertebrates expanded their habitats from fresh water to the sea or to land during evolution. The structure and function of osmoregulatory hormones have also undergone evolution during this ecological evolution [43].

Members of the NPs family have been detected in several snake venoms, and they have been shown to be located in the same precursor protein containing multiple BRPs sequences. While this organization was demonstrated for many species of viperid snakes, including members of the genera *Bothrops, Crotalus, Lachesis, Agkistrodon,* and *Trimeresurus* [12, 13, 15, 30, 31], it may extend to some other taxa such as *Bitis gabonica*, that was also shown to have BPPs in their venom [12, 13, 15, 30, 31, 44]. The presence of NPs in some elapid snakes venom (*Dendroaspis*) has also been described [45]. It has been shown that the venom-derived NP precursors from helodermatid lizard have a structural organization similar to that found in many BRPs precursors from viperid snake venoms. However, the additionally encoded tandem-repeat peptides are non-canonical BPPs, based on their primary structural characteristics or in terms of the amino acid cleavage site, presenting characteristics of a recognition site typical of propeptide convertase enzymes, that eventually might be the potential responsible for the release of the mature BIPs from the respective biosynthetic precursors [36]. However, the BPP/ CNP biosynthetic precursors of the bushmaster (*Lachesis muta*), the tropical rattlesnake (*Crotalus durissus terrificus*), and the massasauga rattlesnake from desert (*Sistrurus catenatus ewardsi)* showed that, in addition to the classical BPPs and a NP sequence, they all also encode single copies of a BIP exhibiting a closer structural similarity, and a propeptide convertase cleavage site that allows the release of the BIP helokinestatins, whose sequences are [TPPAGPDVGPR] or [TPPAGPDGGPR] [36] (Figure 1). On the other hand, putative heloki‐ nestatins peptides could not be identified in the BPP/CNP precursor of snakes as *Bothrops jararaca, Bothrops jararacussu*, and *Agkistrodon blomhoffi* (Figure 1). The phylogenetic analysis

> presented here separates NPs precursor of different species into three distinct groups, those which contain in their precursor sequence (i) only BPPs, (ii) only BIPs, and/or (iii) both BRPs, *i.e.* BPPs and BIPs (Figure 1 and 2), suggesting that mutations in the coding regions of BRPs

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**Figure 2. Phylogenetic tree based on BRP/NPs precursors.** The phylogenetic analysis was performed using T-Coffee - Multiple Sequence Alignment avaiable at http://www.ebi.ac.uk/Tools/msa/tcoffee/. In this analysis, protein sequen‐ ces of BRP/CNP precursors from reptiles were used. The NPs precursor of *A. blomhoffi. B. jararaca,* and *B. jararacussu* contain only BPP sequences; *C. warreni, G. infernalis, H. suspectum,* and *H. horridum* contain only BIP sequences and, *C. durrissus*, *L. mutta*, and *S. catenatus* contain both BRPs sequences, *i.e.* BPP and BIP. Despite the aligned *B. martensii* sequence was partial, and therefore does not contain the sequence coding for BRPs, this species was found to express the closest related precursor sequence to those containing only BIPs and to the *B. jararaca* non-coding RNA homolo‐

Here, we take BPP/CNP and helokinestatin/CNP precursors as examples to illustrate the evolutionofagene,sinceBPP/CNPprecursorisalsoexpressedinothertissuesof*Bothropsjararaca* besides the venom gland, incluing brain and spleen [14, 15]. A high similarity was oberved for the BPP/CNP cDNAs isolated from brain and venom gland, although they are not identical to each other as it should be expected [14]. Three out of the five BPP isoforms present in the brain precursor (BPPs of 5, 10 and 13 amino acid residues) were identical to those found in the venom glandprecursor[14].Moreover,mostofinsertions/deletionsandpointmutationswereobserved within the BPP/CNP coding region, suggesting an effect of a Darwinian-type accelerated evolutionfrequentlyobservedintra-specie [42, 46]Thisprocesshasbeenwidelyobserved, since a number of neuropeptides and hormones, such as the NPs [15, 47] and the vascular endotheli‐

um growth factor [48] evolved into toxins in the venom gland of poisonous animals.

It is believed that BRPs from the venom may be considered the toxin counterparts of endoge‐ nous peptides. It has also been suggested that both CNP and BPPs could be physiologically associated, to perform fluid homeostasis and regulation of the vascular tonus, since BPP/CNP precursor are present in regions of the snake brain showed to be involved in the control of

gous to BPP/CNP precursor.

**2.4. Molecular evolution of genes encoding BRPs**

these activities, as described for the mammalian CNS [14].

were important for the adaptative changes along evolution of the venom system [40].


**Figure 1. Alignment of the amino acid sequences of BRPs precursors**. Organization of the BPP/CNP and helokines‐ tatin/CNP precursor from venoms, indicating the mature BPPs (grey), helokinestatins, BIPs (red), and CNP (underlined) Note that precursors of *C. durissus*, *L. muta*, and *S. catenatus* present both mature BPPs and BIPs in their sequences. The conserved amino acid seqeunces compared to fragments involving the CNP region are highlighted (pink) and the proline residues are indicated by boxes.

presented here separates NPs precursor of different species into three distinct groups, those which contain in their precursor sequence (i) only BPPs, (ii) only BIPs, and/or (iii) both BRPs, *i.e.* BPPs and BIPs (Figure 1 and 2), suggesting that mutations in the coding regions of BRPs were important for the adaptative changes along evolution of the venom system [40].

**Figure 2. Phylogenetic tree based on BRP/NPs precursors.** The phylogenetic analysis was performed using T-Coffee - Multiple Sequence Alignment avaiable at http://www.ebi.ac.uk/Tools/msa/tcoffee/. In this analysis, protein sequen‐ ces of BRP/CNP precursors from reptiles were used. The NPs precursor of *A. blomhoffi. B. jararaca,* and *B. jararacussu* contain only BPP sequences; *C. warreni, G. infernalis, H. suspectum,* and *H. horridum* contain only BIP sequences and, *C. durrissus*, *L. mutta*, and *S. catenatus* contain both BRPs sequences, *i.e.* BPP and BIP. Despite the aligned *B. martensii* sequence was partial, and therefore does not contain the sequence coding for BRPs, this species was found to express the closest related precursor sequence to those containing only BIPs and to the *B. jararaca* non-coding RNA homolo‐ gous to BPP/CNP precursor.

### **2.4. Molecular evolution of genes encoding BRPs**

**Figure 1. Alignment of the amino acid sequences of BRPs precursors**. Organization of the BPP/CNP and helokines‐ tatin/CNP precursor from venoms, indicating the mature BPPs (grey), helokinestatins, BIPs (red), and CNP (underlined) Note that precursors of *C. durissus*, *L. muta*, and *S. catenatus* present both mature BPPs and BIPs in their sequences. The conserved amino acid seqeunces compared to fragments involving the CNP region are highlighted (pink) and the

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proline residues are indicated by boxes.

Here, we take BPP/CNP and helokinestatin/CNP precursors as examples to illustrate the evolutionofagene,sinceBPP/CNPprecursorisalsoexpressedinothertissuesof*Bothropsjararaca* besides the venom gland, incluing brain and spleen [14, 15]. A high similarity was oberved for the BPP/CNP cDNAs isolated from brain and venom gland, although they are not identical to each other as it should be expected [14]. Three out of the five BPP isoforms present in the brain precursor (BPPs of 5, 10 and 13 amino acid residues) were identical to those found in the venom glandprecursor[14].Moreover,mostofinsertions/deletionsandpointmutationswereobserved within the BPP/CNP coding region, suggesting an effect of a Darwinian-type accelerated evolutionfrequentlyobservedintra-specie [42, 46]Thisprocesshasbeenwidelyobserved, since a number of neuropeptides and hormones, such as the NPs [15, 47] and the vascular endotheli‐ um growth factor [48] evolved into toxins in the venom gland of poisonous animals.

It is believed that BRPs from the venom may be considered the toxin counterparts of endoge‐ nous peptides. It has also been suggested that both CNP and BPPs could be physiologically associated, to perform fluid homeostasis and regulation of the vascular tonus, since BPP/CNP precursor are present in regions of the snake brain showed to be involved in the control of these activities, as described for the mammalian CNS [14].

It is known that in the process of evolution, several mutations may occur in the genes, some of which not affecting the mature protein sequence, while others might lead even to the genera‐ tion of messenger RNAs that are not translated into proteins. In 2000's it was first shown that non-coding RNAs can be involved in several roles including repression of genes, catalysis, regulation of the development process, among others [49]. A non-coding mRNA showing a sequence similarity to the BPPs precursor of the pit viper *Bothrops jararaca* was cloned by us from the venom gland [Genbank Acc. No. AY310916.1]. This long RNA sequence does not encode a protein, since several stop codons were observed for all possible reading frames (Figure 3).

Nevertheless, it is also possible that non-coding RNA of 3.5 Kb [GenBank Acc. No. AY310916.1] may also ensures the stability of the functional coding messenger RNA, since the stability of

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**Figure 4. Partial sequence alignment of the***Bothrops jararaca* **BPP/C NP-related pseudogene mRNA and mRNA coding for BPP/CNP precursor.** Alignment of the nucleotide sequences of a segment of the pseudogene mRNA (non-coding: upper sequence) and the mRNA coding for BPP/CNP precursor (BPP-coding: lower sequence) was per‐ formed using the Clustaw W program, available at http://www2.ebi.ac.uk/clustalw/. Identical nucleotides are indicat‐ ed by "\*" and insertions or deletions are represented by gaps (-).The boldface type letters indicate the region with

higher similarity between the RNA sequences, corresponding to about 97% identity in this region.

messenger RNAs is preferably controlled by factors present in the 3' UTR region [52].

**Figure 3.** Potential deduced amino acid sequences from the all possible frames of the non-coding RNA homologous to the BPP/CNP precursor coding RNA both from *Bothrops jararaca*. Total mRNA obtained from *Bothrops jararaca* ven‐ om gland higher than 2 Kb length was used to construct the long cDNA library. The complete cDNA sequence of the BPP/CNP precursor (clone NM 96) was used as template to screen about 2 x 106 clones, allowing the identification of four independent positive clones containing identical inserts of approximately 3.5 Kb [15]. All potential frames of the *Bothrops jararaca* BPP/CNP-related pseudogene mRNA presents a high content of stop codons (bold), as shown by the representative amino acid deduced sequence from one of the possible reading frames. No signal peptide could ob‐ served, although several methionine (Met) residues (underlined) that does not seem to represent an initial of transla‐ tion were found.

These non-coding RNAs are usually transcribed by a gene known as a pseudogene, which are often found in the genomes of several life forms, including bacteria, plants, insects, and vertebrates [50]. The pseudogene is a sequence that is present in the genome, and it is typically characterized by presenting high similarity with one or more functional gene paralogs. The pseudogene can be derived from gene duplication occurred by two different pathways: retrotransposition or duplication of genomic DNA [50].

The regulation of the expression of a functional gene showing sequence similarity to a pseudogene has been reported [51]. Generally, the sequence similarity between functional genes and pseudogenes is observed at the 5' UTR fragment. However, for the comparison of the non-coding RNA and the RNA coding for BPP/CNP precursor, a high similarity was observed only for the 3' UTR sequence (Figure 4). Moreover, the non-coding RNA is of approximately 3.5 Kb, while the RNA coding for the BPPs precursor is of about 1.8 Kb, and its RNA expression was found to be about 6-fold higher than that of the 3.5 Kb non-coding RNA. Nevertheless, it is also possible that non-coding RNA of 3.5 Kb [GenBank Acc. No. AY310916.1] may also ensures the stability of the functional coding messenger RNA, since the stability of messenger RNAs is preferably controlled by factors present in the 3' UTR region [52].

It is known that in the process of evolution, several mutations may occur in the genes, some of which not affecting the mature protein sequence, while others might lead even to the genera‐ tion of messenger RNAs that are not translated into proteins. In 2000's it was first shown that non-coding RNAs can be involved in several roles including repression of genes, catalysis, regulation of the development process, among others [49]. A non-coding mRNA showing a sequence similarity to the BPPs precursor of the pit viper *Bothrops jararaca* was cloned by us from the venom gland [Genbank Acc. No. AY310916.1]. This long RNA sequence does not encode a protein, since several stop codons were observed for all possible reading frames (Figure 3).

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**Figure 3.** Potential deduced amino acid sequences from the all possible frames of the non-coding RNA homologous to the BPP/CNP precursor coding RNA both from *Bothrops jararaca*. Total mRNA obtained from *Bothrops jararaca* ven‐ om gland higher than 2 Kb length was used to construct the long cDNA library. The complete cDNA sequence of the BPP/CNP precursor (clone NM 96) was used as template to screen about 2 x 106 clones, allowing the identification of four independent positive clones containing identical inserts of approximately 3.5 Kb [15]. All potential frames of the *Bothrops jararaca* BPP/CNP-related pseudogene mRNA presents a high content of stop codons (bold), as shown by the representative amino acid deduced sequence from one of the possible reading frames. No signal peptide could ob‐ served, although several methionine (Met) residues (underlined) that does not seem to represent an initial of transla‐

These non-coding RNAs are usually transcribed by a gene known as a pseudogene, which are often found in the genomes of several life forms, including bacteria, plants, insects, and vertebrates [50]. The pseudogene is a sequence that is present in the genome, and it is typically characterized by presenting high similarity with one or more functional gene paralogs. The pseudogene can be derived from gene duplication occurred by two different pathways:

The regulation of the expression of a functional gene showing sequence similarity to a pseudogene has been reported [51]. Generally, the sequence similarity between functional genes and pseudogenes is observed at the 5' UTR fragment. However, for the comparison of the non-coding RNA and the RNA coding for BPP/CNP precursor, a high similarity was observed only for the 3' UTR sequence (Figure 4). Moreover, the non-coding RNA is of approximately 3.5 Kb, while the RNA coding for the BPPs precursor is of about 1.8 Kb, and its RNA expression was found to be about 6-fold higher than that of the 3.5 Kb non-coding RNA.

retrotransposition or duplication of genomic DNA [50].

tion were found.

Applications

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**Figure 4. Partial sequence alignment of the***Bothrops jararaca* **BPP/C NP-related pseudogene mRNA and mRNA coding for BPP/CNP precursor.** Alignment of the nucleotide sequences of a segment of the pseudogene mRNA (non-coding: upper sequence) and the mRNA coding for BPP/CNP precursor (BPP-coding: lower sequence) was per‐ formed using the Clustaw W program, available at http://www2.ebi.ac.uk/clustalw/. Identical nucleotides are indicat‐ ed by "\*" and insertions or deletions are represented by gaps (-).The boldface type letters indicate the region with higher similarity between the RNA sequences, corresponding to about 97% identity in this region.

In the BPP/CNP precursor of *Bothrops jararaca*, the pentapeptide BPP-5a [QKWAP] that was used as template for the development of the antihypertensive drug captopril, is found duplicated, *i.e.*, there are two copies of the same peptide in a single precursor protein. It is believed that this peptide might have a special importance in the venom of snakes belonging to the *Bothrops* genus, since it is also found repeated three times in isoform 1 [GenBank Acc. No. AY310914.1], and four times in isoform 2 [Genbank Acc. No. AY310915.1] of the precursors isolated from *Bothrops jararacussu* venom glands (Figure 5). In fact, BPP-5a is a potent poten‐ tiator of the BK effects in isolated guinea pig ileum, and also *in vivo* [29].

At that time, among the identified peptides were BPP-9a, under the generic name of teprotide, and the BPP-5a, which was also one of first BPP to be characterized. Assays using these peptides showed that BPP-9a was more effective and had a longer lasting effect in blood pressure compared to BPP-5a [53]. Therefore, BPP-9a was used in the first clinical demonstra‐ tion of the potential use of BPPs for the hypertension control in humans, showing a significant

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

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However, on that time it was demonstrated that the therapeutic utility of BPP-9a was limited by the lack of activity by oral administration and the high cost of its synthesis [54, 56, 57]. Therefore, the pharmaceutical development of a non-peptide inhibitor of ACE orally effective was essential. Thus, molecular structure of the BRPs, namely BPP-5a and BPP-9a, were studied by Cushman and Ondetti [3, 58], who suggested specific interaction of the proline, present at the C-terminal of these peptides, with the ACE active site [59]. Thus, captopril was synthetized by simple addition of a chelator radical to a dipeptide containing a proline residue (BPP carboxy-terminal amino acid) [59]. Unodoubtely captopril was a blockbuster drug that

inspired the creation of generations of mimetic antihypertensive compounds [2].

**4.1. Interference of BRPs in the renin-angiotensin and kallikrein-kinin system**

The ACE (EC 3.4.15.1) is mainly expressed in vascular endothelium in epithelial cells of the proximal tubules of the kidney, brain, and intestinal cells [60]. This enzyme is responsible for conversion of angiotensin I (Ang I) to angiotensin II (Ang II), and for the degradation of BK. Therefore, this enzyme has roles in both renin-angiotensin and kallikrein-kinin system [61]. The renin-angiotensin system (RAS) is composed by a set of peptides, enzymes, and receptors, that are involved in the control of the extracellular fluid and blood pressure [62]. The formation of the effector peptide of this system occurs initially by the action of the renin released by the kidneys [62] that acts on the angiotensinogen produced in the liver [63]. This leads to the generation of the decapeptide Ang I, which then is cleaved by ACE to form the octapeptide Ang II, a potent antihypertensive molecule [64]. Ang II actions is mediated by the angiotensin receptors AT1 and AT2. The binding of Ang II to the AT1 receptor triggers several cellular processes, among them vasoconstriction, protein synthesis, cell growth, regulation of renal function, and electrolyte balance [65]. Ang II also acts as a neurotransmitter and as a neuro‐ regulador, modulating the central control of the blood pressure, influencing the sympathetic

The kallikrein-kinin system (KKS) is a metabolic cascade in which the tissue and plasma kallikrein release vasoactive kinins from both high and low molecular weight kininogens. The nonapeptide BK, derived from the cleavage of the high molecular weight kininogen by

Kinins are involved in various physiological and pathological processes, including vasodila‐ tion, increased vascular permeability, release of plasminogen activator of tissue type (t-PA),

kallikrein, is the major plasma kinin playing a role in the KKS [66].

antihypertensive effect [54, 55].

**4. Biological activities of BRPs**

activity, salt appetite, and thirst [65].

**Figure 5. Partial nucleotide and amino acid sequences of the BPP/CNP of precursor from***Bothrops jararacussu* **(isoform 1 and 2).** Shaded in grey, the amino acid sequences of the C-type natriuretic peptide (CNP). Sequences of new putative BPPs are shown in green, and the underlined sequences correspond to other previously known BPPs. In red, the pentapeptide BPP-5a that was found in duplicate in the pit viper precursor and, triplicate and quadruplicate in the isoforms 1 and 2, respectively, of the precursor from *Bothrops jararacussu*. Symbol "M" represents the initial me‐ thionine and (-) the stop codon.

## **3. BRPs as structural model for drug development**

The discovery of the potential inhibitory action of BPPs on ACE brought a great interest in these natural peptides, since the importance of ACE in blood pressure control and the urge to develop a therapy for cardiovascular disease, as hypertension, was iminent [2].

At that time, among the identified peptides were BPP-9a, under the generic name of teprotide, and the BPP-5a, which was also one of first BPP to be characterized. Assays using these peptides showed that BPP-9a was more effective and had a longer lasting effect in blood pressure compared to BPP-5a [53]. Therefore, BPP-9a was used in the first clinical demonstra‐ tion of the potential use of BPPs for the hypertension control in humans, showing a significant antihypertensive effect [54, 55].

However, on that time it was demonstrated that the therapeutic utility of BPP-9a was limited by the lack of activity by oral administration and the high cost of its synthesis [54, 56, 57]. Therefore, the pharmaceutical development of a non-peptide inhibitor of ACE orally effective was essential. Thus, molecular structure of the BRPs, namely BPP-5a and BPP-9a, were studied by Cushman and Ondetti [3, 58], who suggested specific interaction of the proline, present at the C-terminal of these peptides, with the ACE active site [59]. Thus, captopril was synthetized by simple addition of a chelator radical to a dipeptide containing a proline residue (BPP carboxy-terminal amino acid) [59]. Unodoubtely captopril was a blockbuster drug that inspired the creation of generations of mimetic antihypertensive compounds [2].
