Frederic Cumin

Structure-based design of aliskiren, a novel orally effective renin inhibitor

Hypertension is a major risk factor for cardiovascular diseases such as stroke, myocardial infarction, and heart failure, the leading causes of death in the Western world. Inhibitors of the renin-angiotensin system (RAS) have proven to be successful treatments for hypertension. As renin specifically catalyses the rate-limiting step of the RAS, it represents the optimal target for RAS inhibition. Several peptide-like renin inhibitors have been synthesized previously, but poor pharmacokinetic properties meant that these compounds were not clinically useful. We employed a combination of molecular modelling and crystallographic structure analysis to design renin inhibitors lacking the extended peptide-like backbone of earlier inhibitors, for improved pharmacokinetic properties. This led to the discovery of aliskiren, a highly potent and selective inhibitor of human renin in vitro, and in vivo; once-daily oral doses of aliskiren inhibit renin and lower blood pressure in sodium-depleted marmosets and hypertensive human patients. Aliskiren represents the first in a novel class of renin inhibitors with the potential for treatment of hypertension and related cardiovascular diseases.

Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin

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.

Aliskiren, a novel, orally effective renin inhibitor, lowers blood pressure in marmosets and spontaneously hypertensive rats

Objectives Aliskiren is a new renin inhibitor of a novel structural class that has recently been shown to be efficacious in hypertensive patients after once-daily oral dosing. We report the results of animal experiments performed in marmosets and rats in order to characterize aliskiren before its recent investigation in humans. Methods The effects of aliskiren were investigated in sodium-depleted marmosets (oral dosing) and in spontaneously hypertensive rats (dosing via subcutaneous osmotic minipumps). Blood pressure (BP) and heart rate were measured by radiotelemetry. Results In sodium-depleted marmosets, single oral doses of aliskiren (1-30 mg/kg) dose-dependently lowered BP. At a dose of 3 mg/kg, peak effects were observed 1 h after dosing (−30 ± 4 mmHg, n = 6) and the response persisted for more than 12 h. A single oral dose of 3 mg/kg aliskiren was more effective than the same dose of either remikiren or zankiren, two orally active renin inhibitors previously tested in humans. Aliskiren (10 mg/kg) was at least as effective as equal doses of the AT1-receptor blocker valsartan or the angiotensin-converting enzyme inhibitor benazepril. In spontaneously hypertensive rats, aliskiren dose-dependently (10-100 mg/kg per day) decreased BP. Aliskiren also potentiated the antihypertensive effects of low doses of valsartan or benazeprilat (1 or 3 mg/kg per day). Conclusions Aliskiren is an orally effective, long-lasting renin inhibitor that shows antihypertensive efficacy in animals superior to previous renin inhibitors and at least equivalent to angiotensin-converting enzyme inhibitors and AT1-receptor blockers. Aliskiren may therefore represent an effective, novel approach to the treatment of hypertension and related disorders, alone or in combination with other antihypertensive agents.

Aspartic Proteases in Drug Discovery

Aspartic proteases are the smallest class of human proteases with only 15 members. Over the past years, they have received considerable attention as potential targets for pharmaceutical intervention since many have been shown to play important roles in physiological and pathological processes. Despite numerous efforts, however, the only inhibitors for aspartic proteases currently on the market are directed against the HIV protease, an aspartic protease of viral origin. Nevertheless, several inhibitors including those targeting renin, BACE1 and gamma-secretase are in clinical or preclinical development, and some other aspartic proteases are discussed as potential drug target. The crystal structures of seven human aspartic proteases have now been solved and, together with a detailed kinetic understanding of their catalytic mechanism, this has greatly contributed to the design and discovery of novel inhibitors for this protease class. This review describes current aspartic protease drug targets and summarizes the drug discovery efforts in this field. In addition, it highlights recent developments which may lead to a new generation of aspartic protease inhibitors.

Prolonged angiotensin II antagonism in spontaneously hypertensive rats. Hemodynamic and biochemical consequences.

The present study examines the effects of prolonged angiotensin II antagonism in spontaneously hypertensive rats by using an angiotensin II receptor antagonist (DuP 753) that is devoid of agonistic properties and selective for the subtype 1 of the angiotensin II (AT,) receptor. The antihypertensive effects of DuP 753 and its effects on circulating parameters of the reninangiotensin system were compared with those of a converting enzyme inhibitor (benazeprilat). To minimize any influence of differences in the pharmacokinetic properties of the two blockers, administration was by continuous intravenous infusion. The experiments were performed in conscious, freely moving rats with continuous 24-hour monitoring of blood pressure. DuP 753 (10 or 30 mg/kg/day) lowered mean arterial pressure to the same extent as benazeprilat (3 or 10 mg/kg/day) during a 48-hour period. The antihypertensive effect was sustained when the treatment was extended to 7 days (DuP 753,10 mg/kg/day, benazeprilat, 3 mg/kg/day). Neither of the compounds affected the baseline or diurnal rhythm of heart rate. Plasma concentrations of renin and angiotensin II were increased sevenfold and 10-fold, respectively, in the rats treated with DuP 753. In rats treated with benazeprilat, plasma renin concentration increased threefold, whereas angiotensin II was unchanged. Heart weights were significantly reduced to a similar extent by DuP 753 and benazeprilat Both compounds also induced a smaller but significant decrease in blood pressure in Wistar-Kyoto rats. Our results indicate that the antihypertensive effects of converting enzyme inhibitors in spontaneously hypertensive rats are mainly due to the blockade of the renin-angiotensin system. In this rat model, angiotensin II appears to play an important role in the maintenance of hypertension that is mediated via the AT, receptor.

Removal of endogenous leptin from the circulation by the kidney

OBJECTIVE: This study was performed to test the hypothesis that the kidneys play a primary role in the clearance of endogenous leptin from the circulation of obese rats. DESIGN: Zucker (fa/fa) obese rats were anaesthetized and subjected to various surgical manipulations of the kidneys. One hour after surgery arterial blood samples were taken at 1 h intervals for times upto 8 h. Plasma leptin concentrations were determined by radioimmunoassay. RESULTS: Bilateral nephrectomy induced a rapid increase in plasma leptin concentrations above control values. In contrast, continuous intravenous re-injection of voided urine did not increase circulating leptin concentrations, indicating that leptin is not present in the urine in large quantities. This conclusion was confirmed by the very low levels of detectable leptin in urine. Leptin is not metabolized across the renal circulation and is extracted intact by the kidney. Simultaneous measurement of renal plasma flow established renal leptin extraction at approximately 59 ng/min for both kidneys. Following intravenous infusion of leptin, renal clearance and whole body clearance were equal. This finding indicates that the kidneys alone are responsible for the systemic elimination of leptin in Zucker rats. Seven hours after bilateral ureteral ligation, a procedure which lowers glomerular filtration, plasma leptin concentrations were elevated. The renal extraction of leptin did not change over a wide range of plasma leptin concentrations suggesting that renal leptin extraction is a high capacity, non-saturable process most probably glomerular filtration. CONCLUSION: Endogenous leptin is rapidly cleared from the circulation by the kidney by glomerular filtration followed by metabolic degradation in the renal tubules.

Renal actions of the angiotensin AT2 receptor ligands CGP 42112 and PD 123319 after blockade of the renin-angiotensin system.

The purpose of this study was to investigate whether the selective angiotensin AT2 receptor ligands, CGP 42112B (Nic-Tyr-(N alpha-benzoyloxycarbonyl-Arg)Lys-His-Pro-Ile-OH) and PD 123319 ((s)-1-[[4-(dimethylamino)-3-methyl-phenyl]methyl]-5-(diphenylacetyl+ ++)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]-pyridine-6-carboxylic acid) are agonists at angiotensin receptors influencing blood pressure and renal function in the enalaprilat-treated anesthetized rat. The agonist angiotensin II significantly increased blood pressure and renal vascular resistance. Glomerular filtration rate was unchanged by angiotensin II. Effective renal blood flow decreased significantly in response to angiotensin II leading to a significant increase in filtration fraction. Angiotensin II did not induce significant change in urinary potassium excretion or free water formation but significantly increased both urine volume and urinary sodium excretion. At doses up to 3 orders of magnitude greater than angiotensin II, CGP 42112B also significantly increased blood pressure, filtration fraction, glomerular filtration rate, urine volume and urinary sodium excretion, but did not significantly affect effective renal blood flow or renal vascular resistance. The selective angiotensin AT2 receptor ligand PD 123319 had no significant effects on blood pressure nor any measured parameter of renal function. The changes in blood pressure and renal function produced by angiotensin II and CGP 42112B could be completely blocked by the angiotensin AT1 receptor antagonist losartan. The results therefore only support a role for angiotensin AT1 receptors and not angiotensin AT2 receptors in the control of renal function in the rat and demonstrate that at high doses the angiotensin AT2 selective ligand CGP 42112B behaves as an agonist at angiotensin AT1 receptors.

Left ventricular wall stress and sarcoplasmic reticulum Ca2+-ATPase gene expression in renal hypertensive rats: dose-dependent effects of ACE inhibition and AT1-receptor blockade

BACKGROUND Cardiac hypertrophy is associated with altered Ca2+ handling and may predispose to the development of LV dysfunction and cardiac failure. At the cellular level, the re-expression of ANF represents a well-established marker of myocyte hypertrophy while the decreased expression of the sarcoplasmatic reticulum (SR) Ca(2+)-ATPase is thought o play a crucial role in the alterations of Ca2+ handling and LV function. We assessed the dose-dependent effect of chronic ACE inhibition or AT1 receptor blockade on cardiac function in relation to the cardiac expression of the SR Ca(2+)-ATPase and ANF. METHODS AND RESULTS Renal hypertensive rats (2K-1C) were treated for 12 weeks with three different doses of the ACE inhibitor benazepril, the AT1-receptor antagonist valsartan (each drug 0.3, 3, and 10 mg/kg per day i.p.) or placebo. LV dimensions, hypertrophy and wall stress were determined in vivo by magnetic resonance imaging and the gene expressions of ANF and SR Ca(2+)-ATPase were quantified by Northern blot. Low doses of both drugs did not affect blood pressure, hypertrophy, systolic wall stress and the ANF and SR Ca(2+)-ATPase gene expression. High doses of each drug reduced systolic blood pressure, wall stress, and LV hypertrophy to a similar extent and to values comparable to normotensive, age-matched rats. In addition, high dose treatment reduced LV end-systolic and end-diastolic volume as compared to untreated 2K-1C animals and normalized the mRNA levels of both ANF and SR Ca(2+)-ATPase (as compared to normotensive animals). CONCLUSIONS We conclude that in this model, high doses of ACE inhibition and AT1-receptor blockade are necessary to normalize systolic blood pressure, LV hypertrophy and systolic LV wall stress which, in turn, is associated with restoration of a normal cardiac phenotype with respect to SR Ca(2+)-ATPase and ANF and normalization of cardiac function.

Pharmacology of renin inhibitors and their application to the treatment of hypertension

Several different strategies have been followed to block the activity of renin, the enzyme catalysing the first and rate-limiting step in the renin-angiotensin cascade. The unique substrate specificity of this enzyme makes it an attractive target for specifically interfering with the renin-angiotensin system. Attempts to block the activity of renin in animals by an immunological approach, with either active or passive immunization against renin, have been successful. This approach has not been considered as a realistic therapy in humans for the treatment of hypertension or heart failure, but has provided useful tools for purifying and quantifying renin. Considerable efforts have been focused on the design of orally active, synthetic inhibitors of renin. This has resulted in the discovery of low molecular weight pseudo-tetrapeptide compounds that are resistant to enzymatic cleavage and are potent and selective inhibitors of renin. Studies in animal models and preliminary studies in humans indicate that renin inhibitors have the same therapeutic potential as angiotensin-converting enzyme inhibitors. However, the generally poor oral bioavailability and rapid elimination of currently available renin inhibitors have prevented their development as useful drugs. Inhibitors with better oral bioavailability and a long duration of action are needed to assess their full therapeutic potential and to determine whether they offer advantages over the angiotensin-converting enzyme inhibitors or the more recently developed angiotensin II-receptor antagonists.

Analysis of the Role of Angiotensinogen in Spontaneous Hypertension

Allelic variants at the human angiotensinogen locus have recently been reported to increase susceptibility to the development of essential hypertension. In this study we analyzed the role played by angiotensinogen in the elevated blood pressure of the spontaneously hypertensive rat (SHR). The SHR angiotensinogen locus (on chromosome 19) cosegregated with a significant (P = .003) and specific increase in pulse pressure in F2 rats derived from a cross of the SHR with the normotensive Wistar-Kyoto rat (WKY), accounting for 20% of the genetic (10% of total) variance in this phenotype. To identify potential mechanisms underlying the effect of the locus, we further examined angiotensinogen structure and expression in the two strains. Sequence analysis of the respective coding regions revealed no differences in the primary structure of angiotensinogen between the strains. Likewise, plasma angiotensinogen level did not differ in adult rats of the two strains. However, gene expression studies showed tissue-specific, age-related differences in angiotensinogen mRNA levels between SHR and WKY, particularly in the aorta. The findings suggest that pulse pressure, which significantly influences cardiovascular risk, has independent genetic determinants. They further suggest that the effect of the angiotensinogen locus on this phenotype in the SHR may be mediated through a tissue-specific abnormality of angiotensinogen gene expression.