Renin preparation

A number of chemical approaches have been used in the design of renin inhibitors. In the absence of the purified enzyme, most of the early search for inhibitors was carried out using crude renin preparations. The amino acid sequences... 

Hypertensin is soluble in alcohol, glacial acetic acid, phenol, and water, and insoluble in ether (61). Because it is inactivated by tyrosinase it probably contains a catechol or phenol group, and by amine oxidase, an amine group on an a-carbon atom (Figure 2). Hypertensin is inactivated by certain phenolic, catecholic, and amine oxidases, by pepsin, trypsin, chymotrypsin, and carboxypeptidase, and by hypertensinase found in plasma. The nature of hypertensinase is unknown, but it is probably not an oxidative enzyme. Because it is heat-labile, hypertensinase can be removed from blood and renin preparations by heating hypertensin itself is heat-stable. Lack of pure preparations of hypertensin has delayed its further chemical identification. 

A number of chemical approaches have been used in the design of renin inhibitors. In the absence of the purified enzyme, most of the early search for inhibitors was carried out using crude renin preparations. The amino acid sequences of mouse, rat and human renin were obtained later on using either the traditional isolation and sequencing techniques or cDNA methodology. Various three-dimensional models of renin were constructed in the early stages, based on the x ray structures of other similar aspartyl proteases, for example endothia-pepsin and penicillopepsin. Later on, the X ray crystal structure of recombinant human renin was reported. The inhibitor design process has been based on some of these models. 

Renin Activity Determination. With crude renin preparations it is best to use a specific radioimmunoassay determination of angiotensin I produced by renin. However, for the modification of pure renin, the fluoro-metric method of Roth and Reinharz or the radioisotope method of Gregerman will be convenient. In the fluorometric method, the renin containing solution (10 il) from the above reaction mixture is mixed with 230 /111 of 0.05 M sodium pyrophosphate (pH 5.6), and the reaction... 

Contrary to previous reports, the porcine renin preparation thus obtained was quite stable. When a solution containing 0.17 mg of renin per ml in 0.02 M Na phosphate buffer, pH 6.3, containing 0.1 M NaCl, was stored at 4° or frozen at -20, no detectable loss of activity was observed over a period of eight weeks. It is likely that the chemical inhibition of contaminating proteases and the relatively high concentration of the enzyme protein contributed to the stability. 

Figure 7. Chromatographic elution pattern of porcine big-renin (BB), big renin (B) and small renin (S) from a Sephadex G-150 column. The renin preparation obtained after affinity chromatography on a pepstatin-aminobutyl-agarose gel was the starting material. The renin activity (- -) and protein concentration (—) were determined as in Figure 2 and Ref. (60). ...

Evidence has accumulated that there are differences in the speed with which renin preparations from various sources hydrolyze the same substrate. Whereas pig renin hydrolyzes dog substrate and rabbit substrate about equally well, as measured by pressor response in vivo, human renin seems to react much more slowly with rabbit substrate than with dog substrate. Much larger quantities of human renin had to be given to rabbits to produce the same rise in blood pressure as in dogs (156,157). That these observations are connected with reaction velocities and are not due to an increased destruction of human renin in rabbits was made probable by the finding that the effect of human renin on rabbits persisted much longer (up to one hour) than that of pig renin (157). 

III Not present in commercial renin preparation i lOOO (S.M.A. Corp.) ... 

In connection with the synthesis of cytohalasin B, a pyrrole derivative 13 was prepared from methyl (5)-3-aminophenylbutyrate (78JA7775). In connection with the synthesis of l,2,4-triazolo[4,3-a]pyrazine derivatives with human Renin inhibitor activity, a /3,y-diamino acid derivative was transformed into a pyrrolidin-2-one (91JMC151). 

Renin Inhibitors. Preparation of the renin inhibitor terlakiren (18-6)... 

These preparations cause alterations in the renin-angiotensin-aldosterone system. Plasma renin activity has been found to increase, and there is an increase in aldosterone secretion. 

The site of formation of renin is not known, although the indirect and circumstantial evidence favors slightly the juxtaglomerular apparatus rather than the tubules as a source (18). Crude renin, however, is extracted readily from renal cortex by saline extraction, acidification, and precipitation with ammonium sulfate and sodium chloride. Other active protein substances are likewise extracted, and their separation from renin is often a matter of considerable difficulty. The renin substrate, an arglobulin, is found in blood serum and is probably formed by the liver. It can be easily salted out of beef serum as a crude preparation. 

Amine oxidase also was found to have the ability of diminishing or abolishing the pressor action of renin. When an active preparation of the enzyme was injected intravenously into rats, subsequent injections of renin had little effect (Figures 4 and 5). Hypertensin, however, showed only a modified response in these preparations, probably because of the relative slowness of action of the enzyme. Similar results were found with other pressor agents. 

These preparations cause alterations in the renin-angiotensin-aldosterone system. Plasma renin... 

Characterization of HIV-1 protease as a member of the aspartic acid protease family provided the rationale for most of the efforts to design inhibitors (Kohl et al, 1988 Krausslich et al., 1988 Navia et al., 1989 Pearl and Taylor, 1987). Previous efforts to develop therapeutically useful inhibitors of the mechanistically related enzyme renin had demonstrated that potent inhibitors could be prepared by replacing the scissile amide bond of a substrate analogue with a nonhydrolyzable isostere to mimic the tetrahedral intermediate or transition state involved in amide hydrolysis (Greenlee, 1990). Although several dipeptide isosteres have been used to successfully generate highly potent HIV-1 protease inhibitors, a relatively small number have resulted in compounds that reached clinical development. 

M33a. Meulengracht, E., and Schipdt, E., Pepsin and renin activity of preparations of dried stomach from cardia, fundus and pylorus respectively. Acta Med. Scand. 82, 375-383 (1934). 

Scheme 121 N-Chlorocarbonyl morpholine in the preparation of the hypertensive renin inhibitor FK-906.

N-chlorocarbonyl morpholine has found some valuable applications to convert amino acids to N-morpholinocar-bonyl amino acids, for example N-morpholinocarbonyl-(L)-phenylalanine [Scheme 120] for the preparation of renin-inhibiting peptides. 

To obtain the maximum rate of renin activity, saturating amounts of the renin substrate, angiotensinogen, should be present in the reaction system. In most procedures, however, the only substrate provided is that present in the test plasma, and its concentration can be quite variable. According to some investigators, PRA is best estimated when the plasma specimen is incubated with an excess of exogenous renin substrate prepared from nephrectomized human subjects, oxen, or sheep. This type of assay is usually known as a plasma renin concentration assay rather than PRA assay. Unfortunately the measured renin depends on the source and concentration of the renin substrate. Synthetic peptides that resemble the M-terminal portion of angiotensinogen have also been used as renin substrates, but these substances can be hydrolyzed by nonspecific plasma proteases. 

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