Discovery of potent calpain inhibitors based on the azolo-imidazolidenone scaffold.

A series of new azolopyrimidine-peptide hybrids and indolomethylideneimidazolones were obtained and evaluated as calpain inhibitors. The hybrid compounds were inactive, whereas some members of the initial azolomethylideneimidazolone series showed interesting calpain inhibitory activity. By using 4b as a hit compound, a new series of analogs were synthesized by an efficient synthetic procedure based on a multicomponent reaction followed by an unprecedented reaction at the methylene position of the molecule. The best inhibitor found for calpain I (IC50 = 20 nM) was about 20 times more potent than the hit compound. Studies on 4b showed that its inhibition is consistent with an uncompetitive inhibition mode. This compound did not exhibit cellular toxicity at any of the doses tested (0.1-10 µM) and further studies indicated that it was capable of blockading chemical ischemia induction of apoptosis by preventing sodium azide-dependent calpain activation in intact human kidney tubular epithelial cells. The results of molecular modeling studies rationalized the inhibitory activity found for this series and account, from a structural point of view, for the most active compound identified (4j).

Asymmetrical response of aminopeptidase A and nitric oxide in plasma of normotensive and hypertensive rats with experimental hemiparkinsonism.

Aminopeptidases and dopamine (DA) exhibit asymmetries in the brain that are reflected in the peripheral response to unilateral striatal DA depletions (experimental hemiparkinsonism). This might be due to asymmetries in the autonomic innervation of the peripheral vessels. Nitric oxide (NO) is released through vascular sympathetic activation. A similar pathway could be postulated for aminopeptidases. Angiotensin II, metabolized by aminopeptidase A (AP A), interacts with NO and dopamine in the control of blood pressure. Moreover, plasma AP A activity and NO concentrations are elevated in hypertensive rats in which sympathetic activity is increased. We hypothesize that plasma AP A activity and NO concentrations may reflect a central asymmetry of the sympathetic activity. Therefore, we analyzed the effect of unilateral depletions of brain DA by injecting 6-hydroxydopamine into the left or right striatum and measuring plasma AP A, NO and systolic blood pressure (SBP) in normotensive and hypertensive rats. Changes in plasma AP A and NO in opposite directions may reflect an asymmetry in the function of the nigrostriatal system. Our results also revealed an inverse correlation between AP A and NO, in normotensive rats lesioned or sham operated in the right side and hypertensive rats lesioned in the left one. We concluded that the observed changes in plasma NO and AP A after left or right striatal DA depletions may be due to asymmetries in the peripheral autonomic innervation of the vessels.

Papel de las aminopeptidasas en el control neuroendocrino de la presión arterial en animales de experimentación / Role of aminopeptidases in the neuroendocrine control of blood pressure in experimental animals

En el control de la presión arterial participan varias enzimas proteolíticas–incluidas en el llamado sistema renina-angiotensina– que producen diversos péptidos activos que son los agentes efectivos del sistema. El estudio de estas enzimas resulta esencial para conocer en profundidad el mecanismo de control de la presión arterial y puede ofrecer la posibilidad de controlar dicho sistema con fármacos. Una glutamato aminopeptidasa transforma la angiotensina II en angiotensina III. Ésta a su vez es transformada en angiotensina IV por la alanina o arginina aminopeptidasa. La angiotensina I, por acción de la aspartato aminopeptidasa, se transforma en angiotensina 2-10, a la que se han atribuido acciones contrapuestas a las hipertensivas de la angiotensina II. La angiotensina III es la forma más activa de las angiotensinas cerebrales y tiene un efecto estimulador tónico de la presión arterial. El estudio de la inhibición de la glutamato aminopeptidasa, por lo tanto, ha permitido el desarrollo de agentes que actúan eficazmente reduciendo la presión arterial. Asimismo, el desarrollo de activadores de la aspartatoaminopeptidasa constituye otro posible objetivo para el diseño de nuevos agentes antihipertensivos. Nuestro grupo de investigación ha observado que las lesiones unilaterales del sistema nigroestriatal en ratas da lugar a modificaciones simultáneas de la presión arterial y de la actividad aminopeptidásica cerebral y plasmática, curiosamente dependiente del lado de la lesión. Esta posible interacción entre presión arterial, actividad aminopeptidásica y asimetría cerebral, que daría lugar a una respuesta neuroendocrina diferenciada sobre el control de la presión arterial, podría ayudarnos a comprender el mecanismo íntimo por el cual el cerebro controla en la circulación la presión arterial (AU)

Control of blood pressure is partially accomplished by several proteolyticenzymes included in the renin-angiotensin system. These enzymes produce several peptides that form the active components of the system. Study of these enzymes is essential for a deep understanding of blood pressure control and could offer the possibility of controlling this system pharmacologically. Glutamyl aminopeptidase converts angiotensin II into angiotensin III, which in turn is converted into angiotensin IV by an alanylor arginyl aminopeptidase. Angiotensin I, through the action of aspartylaminopeptidase, is converted intoangiotensin 2-10, which may counteract the hypertensive actions of angiotensin II. Angiotensin III is the most active form of brain angiotensins and has a tonic stimulatory effect on blood pressure. Analysis of glutamyl-aminopeptidase inhibition has allowed the development of agents that effectively reduce blood pressure. Moreover, the development of as partyl-aminopeptidase activators could be another goal, with a view to designing new antihypertensive agents. Our group has observed that unilateral lesions of the nigrostriatal pathway in rat brain produce simultaneous modifications in blood pressure and aminopeptidase activities, both in brain and plasma, curiously depending on the side of the lesion. This possible interaction among blood pressure, aminopeptidase activities and brain asymmetry, which could produce a differentiated neuroendocrine response on blood pressure control, may help us to understand the deep mechanism by which the brain is able to control blood pressure peripherally (AU)

Role of aminopeptidases in the neuroendocrine control of blood pressure in experimental animals.

Control of blood pressure is partially accomplished by several proteolytic enzymes included in the renin-angiotensin system. These enzymes produce several peptides that form the active components of the system. Study of these enzymes is essential for a deep understanding of blood pressure control and could offer the possibility of controlling this system pharmacologically. Glutamylaminopeptidase converts angiotensin II into angiotensin III, which in turn is converted into angiotensin IV by an alanyl or arginyl aminopeptidase. Angiotensin I, through the action of aspartyl aminopeptidase, is converted into angiotensin 2-10, which may counteract the hypertensive actions of angiotensin II. Angiotensin III is the most active form of brain angiotensins and has a tonic stimulatory effect on blood pressure. Analysis of glutamyl-aminopeptidase inhibition has allowed the development of agents that effectively reduce blood pressure. Moreover, the development of aspartyl-aminopeptidase activators could be another goal, with a view to designing new antihypertensive agents. Our group has observed that unilateral lesions of the nigrostriatal pathway in rat brain produce simultaneous modifications in blood pressure and aminopeptidase activities, both in brain and plasma, curiously depending on the side of the lesion. This possible interaction among blood pressure, aminopeptidase activities and brain asymmetry, which could produce a differentiated neuroendocrine response on blood pressure control, may help us to understand the deep mechanism by which the brain is able to control blood pressure peripherally.

Brain aminopeptidases and hypertension.

The brain aminopeptidases that participate in the enzymatic cascade of the renin-angiotensin system play a major role in blood pressure (BP) control, and their study offers new perspectives for the understanding of central BP control and the treatment of hypertension. In this system, angiotensin II is converted to angiotensin III (Ang III) by glutamyl aminopeptidase (GluAP) and Ang III is further metabolised to angiotensin IV by alanyl aminopeptidase or arginine-aminopeptidase. It is now clear that Ang III is the key active form of the central angiotensins, exerting tonic stimulatory control over BP. Therefore, the development of GluAP inhibitors as potential antihypertensive agents offers new perspectives for therapy. Brain aspartyl aminopeptidase, which converts angiotensin I to angiotensin 2-10, is also a possible target for antihypertensive therapy because of its potential role in BP control. Finally, since changes in BP levels, that paralleled changes in brain and plasma aminopeptidase activities, were observed after unilateral lesions of the nigrostriatal system, brain asymmetry, aminopeptidase activities and BP control appear to be related, resulting their interplay in an asymmetrical neuroendocrine response that differentially affect BP control. The study of this interaction may contribute to our understanding of how the brain controls BP.

Atrial angiotensinase activity in hypothyroid, euthyroid, and hyperthyroid rats.

Thyroid dysfunction produces marked cardiovascular responses. Hypothyroidism and hyperthyroidism cause important changes in the circulating renin-angiotensin system (RAS). Modifications in cardiac RAS have also been involved in cardiovascular alterations. Studies have revealed that thyroid hormones activate some components of cardiac RAS. Angiotensin (Ang) peptides are regulated by the activity of several aminopeptidases (AP) called angiotensinases. Previous results in our laboratory have demonstrated that thyroid dysfunction altered angiotensinase activities in hypothalamus, pituitary, and kidney. In the present study, we investigated the relationship between thyroid status and local angiotensinase activities in the atrium of hypothyroid, euthyroid, and hyperthyroid adult male rats. We have determined fluorometrically soluble and membrane-bound alanyl, glutamyl, and aspartyl aminopeptidase activities using naphthylamide derivatives as substrates. These activities have been, respectively, involved in the metabolism of Ang III to Ang IV, Ang II to Ang III, and Ang I to des-Asp Ang I. Hyperthyroidism was induced with subcutaneous injections of tetraiodothyronine (300 microg/kg/day), and the hypothyroid rats were obtained with 0.03% methimazole via the drinking water. Compared with that in euthyroid rats, a highly significant increase (by 50%) of soluble aspartyl aminopeptidase activity (P < 0.001) was observed in the atrium of hyperthyroid and hypothyroid animals. In membrane fractions, T4 treatment produced an increase in alanyl aminopeptidase (37%; P < 0.05) and aspartyl aminopeptidase activities (30%; P < 0.01). These results suggest higher formation of des-Asp Ang I in both hypothyroid and hyperthyroid rats but also suggest higher metabolism of Ang III to Ang IV in hyperthyroid animals, which is in agreement with the described alterations of cardiac RAS after thyroid dysfunction.

Angiotensinase activities in the kidney of renovascular hypertensive rats.

In spite of the well-known contribution of angiotensin II (Ang II) in the pathogenesis of Goldblatt two-kidney one clip (G2K1C) hypertension, the importance of other Ang peptides, such as Ang III, Ang IV or Ang 2-10, is scarcely understood. The functional status of these peptides depends on the action of several aminopeptidases called angiotensinases. The metabolism of Ang III to Ang IV by aminopeptidase M (AlaAP) and of Ang I to Ang 2-10 by aspartyl aminopeptidase (AspAP) was evaluated in the renal cortex and medulla of normotensive (Sham-operated) and hypertensive (G2K1C) rats, treated or not with the AT(1) receptor antagonist valsartan. The results demonstrated a highly significant increase of membrane-bound (MEMB) AlaAP in the cortex of the non-ischemic kidney of G2K1C rats compared with the kidney of normal rats and with the clipped kidney of G2K1C rats. This suggests an increased formation of Ang IV in the non-clipped kidney of G2R1C rats. Valsartan reduced MEMB AlaAP and AspAP activities in the renal cortex of normotensive and in the clipped kidney of hypertensive rats. The reduced metabolism of Ang III may prolong its half-life in valsartan-treated animals. These results suggest a role for AlaAP in renovascular hypertension. In addition, the higher AspAP activity of the renal cortex compared to medulla reflects its relative functional difference between both locations.

Aminopeptidase activity in renovascular hypertension.

BACKGROUND: Renovascular hypertension is accompanied by increased renin-angiotensin system activity. Angiotensin II (Ang II) is degraded by aminopeptidases into various metabolites. Increased Ang II production and decreased Ang II degradation may have pathological consequences in maintaining high tissue/plasma Ang II levels. MATERIAL/METHODS: We report the effects of renovascular hypertension on alanyl- (AlaAP), arginyl- (ArgAP), cystinyl- (CysAP), aspartyl- (AspAP), glutamyl- (GluAP) and pyroglutamyl- (pGluAP) aminopeptidases, using arylamides as substrates. The enzymatic activities were analyzed in plasma, right atrium, lung, left ventricle and aortic ring of rats, normotensive (sham-operated) and hypertensive (Goldblatt two-kidney one-clip, G2K1C), treated or not with the AT1 receptor antagonist valsartan. All determinations were performed six weeks after surgery. RESULTS: Whereas the atrium exhibited an increase, the lung, ventricle and aorta showed a decrease of aminopeptidases in G2K1C rats. Except in the aorta of normotensive rats, valsartan did not affect aminopeptidases in the groups studied. CONCLUSIONS: The present study may imply reduced metabolism of angiotensin II in the lung and aorta of G2K1C rats. This down-regulation could prolong the half-life of Ang II and contribute to the maintenance of hypertension. Changes in AP activities did not appear to be part of the action mechanism of AT1 receptor blockade in hypertensive rats