RESUMEN
BACKGROUND: The aspartic proteinase renin plays an important physiological role in the regulation of blood pressure. It catalyses the first step in the conversion of angiotensinogen to the hormone angiotensin II. In the past, potent peptide inhibitors of renin have been developed, but none of these compounds has made it to the end of clinical trials. Our primary aim was to develop novel nonpeptide inhibitors. Based on the available structural information concerning renin-substrate interactions, we synthesized inhibitors in which the peptide portion was replaced by lipophilic moieties that interact with the large hydrophobic S1/S3-binding pocket in renin. RESULTS: Crystal structure analysis of renin-inhibitor complexes combined with computational methods were employed in the medicinal-chemistry optimisation process. Structure analysis revealed that the newly designed inhibitors bind as predicted to the S1/S3 pocket. In addition, however, these compounds interact with a hitherto unrecognised large, distinct, sub-pocket of the enzyme that extends from the S3-binding site towards the hydrophobic core of the enzyme. Binding to this S3(sp) sub-pocket was essential for high binding affinity. This unprecedented binding mode guided the drug-design process in which the mostly hydrophobic interactions within subsite S3(sp) were optimised. CONCLUSIONS: Our design approach led to compounds with high in vitro affinity and specificity for renin, favourable bioavailability and excellent oral efficacy in lowering blood pressure in primates. These renin inhibitors are therefore potential therapeutic agents for the treatment of hypertension and related cardiovascular diseases.
Asunto(s)
Antihipertensivos/síntesis química , Antihipertensivos/farmacología , Diseño de Fármacos , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Renina/antagonistas & inhibidores , Inhibidores de la Enzima Convertidora de Angiotensina/efectos adversos , Angiotensinógeno/análogos & derivados , Animales , Antihipertensivos/química , Antihipertensivos/metabolismo , Sitios de Unión/fisiología , Callithrix , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Unión Proteica/fisiología , Conformación Proteica , Renina/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The new neuropeptide Y (NPY) Y5 receptor antagonist CGP 71683A displayed high affinity for the cloned rat NPY Y5 subtype, but > 1, 000-fold lower affinity for the cloned rat NPY Y1, Y2, and Y4 subtypes. In LMTK cells transfected with the human NPY Y5 receptor, CGP 71683A was without intrinsic activity and antagonized NPY-induced Ca2+ transients. CGP 71683A was given intraperitoneally (dose range 1-100 mg/kg) to a series of animal models of high hypothalamic NPY levels. In lean satiated rats CGP 71683A significantly antagonized the increase in food intake induced by intracerebroventricular injection of NPY. In 24-h fasted and streptozotocin diabetic rats CGP 71683A dose-dependently inhibited food intake. During the dark phase, CGP 71683A dose-dependently inhibited food intake in free-feeding lean rats without affecting the normal pattern of food intake or inducing taste aversion. In free-feeding lean rats, intraperitoneal administration of CGP 71683A for 28 d inhibited food intake dose-dependently with a maximum reduction observed on days 3 and 4. Despite the return of food intake to control levels, body weight and the peripheral fat mass remained significantly reduced. The data demonstrate that the NPY Y5 receptor subtype plays a role in NPY-induced food intake, but also suggest that, with chronic blockade, counterregulatory mechanisms are induced to restore appetite.
Asunto(s)
Regulación del Apetito/fisiología , Naftalenos/farmacología , Neuropéptido Y/metabolismo , Pirimidinas/farmacología , Receptores de Neuropéptido Y/fisiología , Animales , Unión Competitiva , Glucemia/metabolismo , Composición Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Condicionamiento Psicológico/efectos de los fármacos , Diabetes Mellitus Experimental/fisiopatología , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Humanos , Hipotálamo/metabolismo , Hipotálamo/fisiología , Insulina/sangre , Insulina/farmacología , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/metabolismo , Triglicéridos/sangreRESUMEN
Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor (GPCR) superfamily and mediate several physiological responses, such as blood pressure, food intake, sedation and memory retention. To understand the interactions between the NPY Y1 receptor subtype and its ligands, computer modeling was applied to the natural peptide agonist, NPY and a small molecule antagonist, BIBP3226. An agonist and antagonist binding domain was elucidated using mutagenesis data for the Y1 receptor as well as for other GPCR families. The agonist and antagonist ligands which were investigated appear to share common residues for their interaction within the transmembrane regions of the Y1 receptor structure, including Gln120, Asn283 and His306. This is in contrast to findings with tachykinin receptors where the binding domains of the non-peptide antagonists have very little in common with the binding domains of the agonist, substance-P. In addition, a hydrogen bond between the hydroxyl group of Tyr36 of NPY and the side chain of Gln219, an interaction that is absent in the model complex between Y1 and the antagonist BIBP3226, is proposed as one of the potential interactions necessary for receptor activation.
Asunto(s)
Proteínas de Unión al GTP/química , Modelos Moleculares , Receptores de Neuropéptido Y/química , Secuencia de Aminoácidos , Animales , Arginina/análogos & derivados , Arginina/química , Sitios de Unión , Bovinos , Simulación por Computador , Humanos , Ligandos , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Neuropéptido Y/química , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Receptores de Neuropéptido Y/genética , Receptores de Neuropéptido Y/metabolismoRESUMEN
Our attempts to synthesize a potent inhibitor of rat renin have resulted in the discovery of CGP 44 099 A, a potent inhibitor of plasma renin from all subprimate species tested so far [IC50 (in nM): dog, 0.007; rabbit, 0.033; guinea pig, 0.34; mouse, 0.4; cat, 0.57; and rat, 1.3]. This compound is also a potent inhibitor of primate renins [IC50 (in nM): human, 0.3; and marmoset, 1.4]. It is less potent against other aspartic proteinases [IC50 (in nM): porcine pepsin, 26; and bovine cathepsin D, 230). CGP 44 099 A exhibited a competitive mode of inhibition against human renin (Ki, 0.12 nM). During i.v. infusion of CGP 44 099 A in sodium-depleted normotensive rats (0.1 mg/kg/min) blood pressure (BP) was lowered by about 25 mm Hg. Plasma renin activity, angiotensin I and angiotensin II were almost completely suppressed. The converting enzyme inhibitor enalaprilat (1 mg/kg i.v.) also lowered BP by about 25 mm Hg. No further fall in BP occurred when CGP 44 099 A (0.1 mg/kg/min) was infused after pretreatment with enalaprilat (1 mg/kg i.v.) and CGP 44 099 A did not lower BP when infused in bilaterally nephrectomized rats. These results indicate that the hypotensive response induced by CGP 44 099 A in sodium-depleted rats is specifically due to the renin inhibition. Compounds such as CGP 44 099 may therefore be useful for comparing the effects of renin inhibition in different species and for studying the role of renin in various models of cardiovascular disease in nonprimate species.
Asunto(s)
Oligopéptidos/farmacología , Renina/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Presión Sanguínea/efectos de los fármacos , Callitrichinae , Gatos , Perros , Relación Dosis-Respuesta a Droga , Cobayas , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/enzimología , Riñón/fisiología , Cinética , Masculino , Ratones , Datos de Secuencia Molecular , Conejos , Ratas , Ratas Endogámicas , Renina/sangre , Especificidad de la EspecieRESUMEN
CGP 38 560 is a low-molecular-weight (730) inhibitor of human renin that contains only one natural amino acid. In vitro, it is a potent inhibitor of human renin (Ki with tetradecapeptide, 0.4 X 10(-9) M). It has a high enzyme specificity (Ki values against human pepsin, gastricsin, and cathepsin D are 5 X 10(-6), 3 X 10(-6), and 0.6 X 10(-6) M) and is also species specific (IC50 values against human, marmoset, dog, and rat plasma renins are 7 X 10(-10), 7 X 10(-10), 7 X 10(-9), and 1 X 10(-6) M). In vivo, CGP 38 560 inhibits plasma renin activity (PRA) and lowers blood pressure (BP) after oral administration to conscious, normotensive, furosemide-pretreated marmosets. A dose of 10 mg/kg induces complete inhibition of PRA and a decrease in BP of 23 +/- 3 mm Hg (n = 4) after 30 min. These effects persist for up to 2 h. Blockade of the renin-angiotensin system appears to cause the hypotensive response since it is completely prevented by pretreatment with a converting-enzyme inhibitor. These findings demonstrate that the molecular size of renin inhibitors may be reduced to improve their oral activity without loss of potency or specificity.
Asunto(s)
Oligopéptidos/farmacología , Renina/antagonistas & inhibidores , Animales , Presión Sanguínea/efectos de los fármacos , Callitrichinae , Enalapril/farmacología , Femenino , Humanos , Hidralazina/farmacología , Técnicas In Vitro , Masculino , Peso Molecular , Inhibidores de Proteasas , Porcinos , Factores de TiempoRESUMEN
A series of renin inhibitors containing the dipeptide transition state mimics (2S,4S,5S)-5-amino-4-hydroxy-2-isopropyl-7-methyloctanoic acid (Leu (OH)/Val) and (2S,4S,5S)-5-amino-4-hydroxy-2-isopropyl-6-cyclohexylhexanoic acid (CHa /(OH)/Val) was prepared. A structure-activity study with Boc-Phe-His-Leu (OH)/Val-Ile-His-NH2 (8a) as starting material led to N-[(2S)-2-[(tert-butylsulfonyl)methyl]-3-phenylpropionyl]-His-Cha (OH)/ Val- NHC4H9-n (8i) which has the length of a tetrapeptide and contains only one natural amino acid. Compound 8i had an IC50 of 2 x 10(-9) M against human renin and showed high enzyme specificity; IC50 values against the related aspartic proteinases pepsin and cathepsin D were (8 x 10(-6) and 3 x 10(-6) M, respectively). In salt-depleted marmosets, 8i inhibited plasma renin activity PRA and lowered blood pressure for up to 2 h after oral administration of a dose of 10 mg/kg.