**2. Targets of proline-rich oligopeptides from Bothrops jararaca**

Recently, argininosuccinate synthase (AS) was identified as another target for the *Bj*-PROs, which both *in vitro* and *in vivo* positively modulates the activity of this enzyme [14] which leads to L-arginine synthesis [22]. L-arginine is a nonessential amino acid under normal con‐ ditions as it is obtained from the breakdown of proteins or synthesized de novo from citrul‐ line in the kidneys by AS (EC 6.3.4.5) and argininosuccinate lyase (ASL, EC 4.3.2.1). AS catalyses the reversible condensation of citrulline with aspartate with consumption of ATP to form argininosuccinate; ASL catalyzes the conversion of the argininosuccinate to fuma‐ rate and L-arginine, which is released into the circulation [22].

In the liver, enzymes involved in the anabolism of L-arginine, AS and ASL are present; how‐ ever, there is not a net production of L-arginine due to arginase activity (EC 3.5.3.1) as part of the urea cycle, catalyzes the hydrolysis of L-arginine into L-ornithine and urea. The urea is then excreted in the urine and L-ornithine is recycled back into the cycle [23]. Further‐ more, AS is the rate-limiting enzyme of the citrulline-nitric oxide (NO) cycle for the supply of L-arginine which is then metabolized by NO synthase (NOS) to form NO and citrulline [24-26]. Citrulline, through the reactions catalyzed by AS and ASL may cycle back to argi‐ nine, constituting the citrulline-NO cycle [27, 28]. In summary, AS activity contributes to three major different functions in the adult organism depending on the cell/tissue consid‐ ered: (i) ammonia detoxification in the liver, (ii) L-arginine production for the whole organ‐ ism by kidney and (iii) L-arginine synthesis for NO production in many other cells [22].

for "rational drug design", a concept much heralded today and made possible by computer

Many studies on structure–activity of *Bj*-PROs showed that a simple analogous structure to Ala–Pro was optimal for binding to the active site of ACE. Replacement of the carboxyl by a sulfhydryl group enhanced the inhibitory activity of the analogue by 1,000-fold. This com‐ pound proved to be one of the most potent competitive inhibitors of ACE and, therefore, turned into a useful drug to treat human hypertension (reviewed by [12]). Captopril was a blockbuster drug and inspired the creation of several generations of similar antihyperten‐

However, due to structural diversity of *Bj*-PROs [13] other mechanisms besides inhibiting ACE were proposed. In fact, some of these peptides augmenting argininosuccinate synthase (AS) activity *in vitro* and *in vivo*, can also induce rises in free intracellular calcium concentra‐

or reversal inhibition of nicotinic acetylcholine receptor [19]. These novel mechanisms of ac‐ tion, recently identified for *Bj*-PROs explain their anti-hypertensive effects [14-17, 20, 21]. Therefore, investigation of *Bj*-PRO-induced effects through acting on different targets opens possibilities of applications for these peptides in the treatment of several pathologies lacking

Here, we describe the targets of various *Bj*-PROs and their potential use to treat different target-related pathologies, as well as discuss chemical properties of these peptides for ob‐

Recently, argininosuccinate synthase (AS) was identified as another target for the *Bj*-PROs, which both *in vitro* and *in vivo* positively modulates the activity of this enzyme [14] which leads to L-arginine synthesis [22]. L-arginine is a nonessential amino acid under normal con‐ ditions as it is obtained from the breakdown of proteins or synthesized de novo from citrul‐ line in the kidneys by AS (EC 6.3.4.5) and argininosuccinate lyase (ASL, EC 4.3.2.1). AS catalyses the reversible condensation of citrulline with aspartate with consumption of ATP to form argininosuccinate; ASL catalyzes the conversion of the argininosuccinate to fuma‐

In the liver, enzymes involved in the anabolism of L-arginine, AS and ASL are present; how‐ ever, there is not a net production of L-arginine due to arginase activity (EC 3.5.3.1) as part of the urea cycle, catalyzes the hydrolysis of L-arginine into L-ornithine and urea. The urea is then excreted in the urine and L-ornithine is recycled back into the cycle [23]. Further‐ more, AS is the rate-limiting enzyme of the citrulline-nitric oxide (NO) cycle for the supply of L-arginine which is then metabolized by NO synthase (NOS) to form NO and citrulline [24-26]. Citrulline, through the reactions catalyzed by AS and ASL may cycle back to argi‐ nine, constituting the citrulline-NO cycle [27, 28]. In summary, AS activity contributes to

**2. Targets of proline-rich oligopeptides from Bothrops jararaca**

rate and L-arginine, which is released into the circulation [22].

) by acting on muscarinic acetylcholine, BK or yet unidentified receptors [15-18]

imaging and genome science [11].

sive compounds.

460 Drug Discovery

tion ([Ca2+]*<sup>i</sup>*

efficient treatment options.

taining an oral pharmaceutical formulation.

Three isoforms of NOS catalyze the reaction: the endothelial constitutive NOS (eNOS), the neuronal constitutive NOS (nNOS) and the inducible NOS (iNOS), reviewed in [29, 30]. NO is a gaseous molecule capable of interacting with many intracellular targets for triggering a series of signal transduction pathways, resulting in a stimulatory and inhibitory signals. NO plays roles in cardiovascular, immune and neuronal control. It is directly involved in arterial tension control since it regulates the local and systemic resistance of vascular walls, as well as the sodium balance [31]. The NO produced by endothelial cells reaches neighboring smooth muscle cells where it activates two types of K+ channels, ATP-sensitive and Ca2+-de‐ pendent [32, 33], and thereby induces the relaxation of blood vessels and brings about vas‐ cular dilation leading direct consequences in processes like erection, arterial pressure systemic or organ-specific [34].

Due to the great physiological importance of the NO, compounds revealing properties aspo‐ tential NO donors have been protected by patents. They are based on the fact that there are many pathological states related to NO deficiency (reviewed by [35]). However, a major problem of NO donors is to achieve a therapeutic dose without reaching a threshold of tox‐ icity. Mostly, if NO is produced in excess around of cells in a pro-oxidant state, NO could react with reactive oxygen species (ROS) such as superoxide and hydrogen peroxide form‐ ing peroxynitrite and nitrogen oxide III, which has been linked to pathogenesis of neurode‐ generative disorders [35].

The superoxide anion is produced by uncoupling of NOS due to the lack of its natural sub‐ strate L-arginine or tetrahydrobiopterin (BH4) [36], an important cofactor for NOS. Excessive production of superoxide is explained by increased activity and expression of the enzyme arginase, which competes with eNOS for its substrate L-arginine [37]. Other possible sources for elevated concentrations of ROS include increased expression and activity of NADPH and reduced superoxide dismutase activity [38, 39].

In order to compensate for the deficiency of NO production without induction of toxicity, addition of exogenous L-arginine in the maintenance of NO production has been investigat‐ ed. The inefficiency of swallowed L-arginine in promoting increase of NO can be explained by its low availability, due to the first pass effect, since the viability of L-arginine as sub‐ strate for NOS is reduced by the activity of arginase in the liver. Several studies have shown that induction or activation of arginase may lead to impaired NO production and endothe‐ lial dysfunction (reviewed by [35]).

Thus, NO presents challenges and opportunities to intervene and promote human health. The study of regulation of NO production becomes important for understanding the mecha‐ nisms which maintain NO levels in a safe range and not injurious to the body.

An important mechanism for control and maintenance of NO levels is achieved by its recy‐ cling via the NO-citrulline cycle. The obtained L-arginine provided by the citrulline-NO cy‐ cle is then directed to sustain NO production, sparing bulk intracellular L-arginine for other metabolic roles [24]. In view of that, compounds that increase AS activity and sustain tightly NO production avoiding an excess production will ensure adequate bioavailability for prop‐ er physiological functioning. Guerreiro and colleagues demonstrated that a *Bj*-PRO pro‐ motes activation of AS, assayed in the presence of the substrates ATP, citrulline, and aspartate, thus leading to NO production by endothelial cells [14]. More recently, we have demonstrated that other *Bj*-PROs induce NO production by activation of AS or kinin-B2 re‐ ceptors as well as by M1 muscarinic acetylcholine activation, thereby inducing vasodilata‐ tion *in vivo* [16, 17].

PH in these patients is unclear. Hemolysis may result in a nitric oxide deficient state through free hemoglobin scavenging of nitric oxide and release of erythrocyte arginase,

Applications of Snake Venom Proline-Rich Oligopeptides (Bj-PROs) in Disease Conditions Resulting from...

http://dx.doi.org/10.5772/52509

463

Nitric oxide is synthesized from terminal nitrogen of L-arginine by NOS. All three NOS iso‐ forms are expressed in the lung and are distinguished by regulation of their activities, as well as by specific sites and developmental patterns of expression [46]. The isoform eNOS is expressed in vascular endothelial cells and is believed to be the predominant source of NO production in pulmonary circulation [40]. This hypothesis is corroborated by the fact that NO inhalation in premature newborns with severe respiratory failure due to PH provides

Although large well-designed studies paved the way to Food and Drug Administration (FDA) approval of therapeutic NO inhalation, it is equally important to note that inhaled NO did not reduce the mortality, length of hospitalization, or the risk of significant neurode‐ velopmental impairment associated with persistent PH in newborn children [40]. It is known that at excessive levels NO can react with reactive oxygen species (ROS) such as su‐ peroxide and hydrogen peroxide. Such increase in ROS was observed in the smooth muscle and adventitia of pulmonary arteries from lambs with chronic intrauterine PH [47, 48],

Inhaled NO is usually delivered with high concentrations of oxygen. Whereas hyperoxic ventilation continues to be a mainstay in the treatment of PH, little is known about the side effects of oxygen supply together with NO. The extreme hyperoxia routinely used in PH management may in fact be toxic to the developing lung due to ROS formation [39, 50, 51]. Superoxide may react with arachidonic acid to increase concentrations of isoprostanes and may also combine with NO to form peroxynitrite [52] with possible induction of vaso‐ constriction, cytotoxicity, and damage to surfactant proteins and lipids. Moreover, peroxy‐ nitrite has been shown to directly induce NOS uncoupling. New data indicate that even brief (30 min) periods of exposure to 100% O2 are sufficient to increase reactivity of pulmo‐ nary vessels in healthy lambs [53, 54], to diminish the response of the pulmonary vascula‐ ture to endogenous and exogenous nitric oxide [54], and to increase the activity of cGMPspecific phosphodiesterases [51]. Inhaled NO would theoretically benefit patients with chronic primary or secondary pulmonary hypertension, but its therapeutic application in this setting has been limited by the risk of causing rebound pulmonary hypertension, if it is inadvertently discontinued, and the lack of practical home-based continuous delivery

Therefore, we have proposed an alternative approach for controlled induction of NO pro‐ duction. We believe that cytosolic L-arginine provides a major NO donor. Arginine con‐ centrations subject to metabolic fine-tuning controls will assure that the amino acid is kept in a homeostatic concentration range. These effects could be achieved by the action of *Bj*-PRO on AS, a target unexplored by the pharmaceutical industry. Compounds inducing an increase in AS expression and activity are promising for the treatment of diseases related

improvement of symptoms, accompanied by marked increase in oxygenation [34].

forming peroxynitrite, an anion with deleterious tissue-oxidant effects [49].

devices.

with deficient NO production.

which limits L-arginine, a substrate for nitric oxide synthesis [45].

The patent entitled "Proline-Rich Peptides, Pharmaceutical Composition, use of one or more peptides and method of treatment" was deposited to protect the use and application of *Bj*-PROs and analogous molecules [patent: BR2007/ 000003]. All applications contemplated by patent BR2007/ 000003 are consistent with the use of PROs as prototype molecules for the development of new drugs aiming to treat a range of pathological states related to deficien‐ cy in NO production and AS activity, e.g. lung hypertension, preeclampsia, essential hyper‐ tension, coagulopathies and citrullinemia. Some of these applications will be discussed below.
