Design, structure-activity, and molecular modeling studies of potent renin inhibitory peptides having N-terminal Nin-For-Trp (Ftr): angiotensinogen congeners modified by P1-P1' Phe-Phe, Sta, Leu psi[CH(OH)CH2]Val or leu psi[CH2NH]Val substitutions

A structure-conformation-activity investigation of several angiotensinogen (ANG) based inhibitors of human renin modified by either Phe-Phe, Sta, Leu psi[CH2NH]Val, or Leu psi[CH(OH)CH2]Val at the P1-P1' clevage site and P5 Trp(Nin-For) (Ftr) was performed. Specifically, Ac-Ftr-Pro-Phe-His-Phe-Phe-Val-Ftr-NH2 (1) provided a potent (KI = 2.7 X 10(-8) M) P1-P1' Phe-Phe substituted renin inhibitor to initiate these studies. Substitution of the P1-P1' Phe-Phe in compound 1 by Sta effected a 1,000-fold increase in biological potency for the resultant octapeptide Ac-Ftr-Pro-Phe-His-Sta-Val-Ftr-NH2 (10; KI = 6.7 X 10(-11) M). Kinetic analysis of compound 10 showed it to be a competitive inhibitor of human renin catalyzed proteolysis of human ANG. Chemical modifications of the compounds 1 and 10 were evaluated on the basis of comparative human plasma renin inhibitory activities (IC50 values) in vitro. Carboxy-terminal truncation studies on compound 10 showed that the P2' Val and P3' Ftr residues could both be eliminated without significant loss (ca. 10-fold) in renin inhibitory activity as exemplified by the pentapeptide Ac-Ftr-Pro-Phe-His-Sta-NH2 (12; IC50 = 3.8 X 10(-9) M). In addition, the corresponding P1-P1' Leu psi[CH(OH)CH2]Val and Leu psi[CH2NH]Val derivatives of compound 12 were potent renin inhibitors: Ac-Ftr-Pro-Phe-His-Leu psi[CH(OH)CH2]Val-NH2 (13; IC50 = 3.1 X 10(-10) M) and Ac-Ftr-Pro-Phe-His-Leu psi[CH2NH]Val-NH2 (14; IC50 = 2.1 X 10(-8) M). The structure-conformation-activity properties of the N-terminal Ftr substitution of these human renin inhibitors was examined by (1) comparative analysis of several analogues of 1 and Ac-Ftr-Pro-Phe-His-Sta-Ile-NH2 (17; IC50 = 1.0 X 10(-10) M) having P5 site modifications by Trp, His, D-Ftr, and D-His, (2) deletion of the N-terminal Ftr residue from compounds 12 and 17, to provide Ac-Pro-Phe-His-Sta-Ile-NH2 (16; IC50 = 3.1 X 10(-8) M) and Ac-Pro-Phe-His-Sta-NH2 (15; IC50 = 5.6 X 10(-6) M), and (3) computer modeling and dynamics studies of compounds 1 and 17 bound to CKH-RENIN, a simulated human renin model, which were focused on identifying potential intermolecular interactions of their common P5-P2 sequence, Ac-Ftr-Pro-Phe-His, at the enzyme active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and renin inhibitory activity of angiotensinogen analogues having dehydrostatine, Leu psi [CH2S]Val, or Leu psi [CH2SO]Val at the P1-P1' cleavage site

The synthesis and in vitro renin inhibitory potencies of angiotensinogen (ANG) analogues having amide (CONH) bond replacements at P1-P1', the Leu-Val cleavage site, corresponding to Leu psi[CH2SO]Val, and the trans olefinic analogue of statine (Sta), 4(S)-amino-6-methyl-2(E)-heptenoic acid (dehydrostatine, Dhs), are reported. These are compared to P1-P1' Leu psi[CH2NH]Val-, Sta-, or Phe-Phe-substituted analogues of the same template. The Dhs pseudodipeptide was found to be an adequate mimic of a trans CONH bond and gave a peptide, H-Pro-His-Pro-Phe-His-Dhs-Ile-His-D-Lys-OH, approximately equal in potency to a Phe-Phe-containing inhibitor, but 200-fold less potent than its Sta-substituted congener. That the enhanced potency of the Sta-containing peptide most likely depends on hydrogen bonding as well as tetrahedral geometry is indicated by the 50-100-fold lower potency of the tetrahedral Leu psi[CH2S]Val and Leu psi[CH2SO]Val analogues as compared to the Leu psi[CH2NH]Val-containing congener.     

Inhibition of renin by substrate analogue inhibitors containing the olefinic amino acid 5(S)-amino-7-methyl-3(E)-octenoic acid

The olefinic dipeptide 5(S)-amino-7-methyl-3(E)-octenoic acid (1) was synthesized and used to make the olefinic peptides Leu psi [E-CH = CH]Gly-Val-Phe-OCH3 (2) and His-Leu psi [E-CH = CH]Gly-Val-Phe-OCH3 (3). These olefinic peptides were found to exhibit renin inhibitory activity against both hog kidney renin and human amniotic renin that was comparable to the substrate analogue inhibitors Leu-Leu-Val-Phe-OCH3 and His-Leu-Leu-Val-Phe-OCH3.     

Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P.

In addition to angiotensin I converting enzyme (ACE; EC 3.4.15.1) and carboxypeptidase N (CPN; EC 3.4.17.3), other peptidases contribute to bradykinin (BK) degradation in plasma. Rat plasma degraded BK by hydrolysis of the N-terminal Arg1-Pro2 bond, and the characteristics of hydrolysis are consistent with identification of aminopeptidase P (APP; EC 3.4.11.9) as the responsible enzyme. BK and BK[1-5] N-terminal hydrolysis was optimal at neutral pH, was inhibited by 2-mercaptoethanol, dithiothreitol, o-phenanthroline and EDTA, but was unaffected by the aminopeptidase inhibitors amastatin, puromycin and diprotin A, the endopeptidase-24.11 inhibitors phosphoramidon and ZINCOV, and the ACE and CPN inhibitors captopril and D,L-mercapto-methyl-3-guanidinoethylthiopropanoic acid (MERGETPA), respectively. Although kallidin (Lys-BK) was not metabolized directly by APP, conversion to BK by plasma aminopeptidase M (EC 3.4.11.2) resulted in subsequent degradation by APP. BK analogs containing N-terminal Arg1-Pro2 bonds, including [Tyr8-(OMe)] BK and [Phe8 psi(CH2NH)Arg9]BK (B2 agonists), des-Arg9-BK and [D-Phe8]des-Arg9-BK (B1 agonists), and [Leu8]des-Arg9-BK (B1 antagonist), were degraded by APP with Km and Vmax values comparable to those found for BK (Km = 19.7 +/- 2.6 microM; Vmax = 12.1 +/- 1.2 nmol/min/mL). In contrast, B2 antagonists containing D-Arg0 N-termini, including D-Arg[Hyp3,Thi5.8,D-Phe7]BK and D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, were resistant to APP-mediated hydrolysis. These data support a role for plasma aminopeptidase P in the degradation of circulating kinins, and a variety of B2 and B1 kinin agonists and antagonists. However, APP does not participate in the degradation of D-Arg0-containing antagonists.     

Pseudopeptide analogues of substance P and leucine enkephalinamide containing the psi (CH2O) modification: synthesis and biological activity.

The isosteric methyleneoxy psi (CH2O) function was employed as a novel peptide-bond surrogate and incorporated into sequences of two neuropeptides, substance P (SP) and enkephalin. A pseudopeptide analogue [pGlu6,Phe8 psi(CH2O)Gly9]SP6-11 (7) of SP related C-terminal hexapeptide [pGlu6]SP6-11 and two pseudopeptide analogues of [Leu5]enkephalinamide, [Tyr1 psi (CH2O)Gly2, Leu5] enkephalinamide (11) and [Gly2 psi (CH2O)-Gly3, Leu5]enkephalinamide (17), were synthesized. The N alpha-protected pseudodipeptidic units were incorporated in the appropriate peptide sequences by using conventional coupling methods in solution. Compound 7 was a potent agonist (EC50 = 4.8 nM) of substance P as compared to the parent peptide [pGlu6]SP6-11 (EC50 = 1.2 nM), in stimulating contraction of the isolated guinea pig ileum (GPI). Analogue 7 was more potent on the neuronal (NK-3) than on the muscular (NK-1) tachykinin receptors in the GPI as shown by the ratio of activities, EC50 (NK-1)/EC50 (NK-3) = 3.16, thus displaying an improved selectivity for the NK-3 tachykinin receptor subtype as compared to that of [pGlu6]SP6-11, EC50 (NK-1)/EC50 (NK-3) = 0.44. In the rat vas deferens (RVD) assay, a typical NK-2 system, the pseudopeptide analogue 7 was (EC50 = 2 microM) 10-fold more potent than the parent peptide and 20-fold less potent than eledoisin, an NK-2 selective tachykinin. The pseudopeptide enkephalin analogue 17 had low biological activity when tested in the electrically induced GPI (EC50 = 2.3 microM) and was inactive in the mouse vas deferens (MVD) assay. In the rat brain membrane (RBM) binding assay analogue 17 had low affinity (in the micromolar range) for both the mu and delta binding sites. In contrast, analogue 11 was a potent enkephalin agonist (EC50 = 30 nM), being equipotent to [D-Ala2, Leu5]enkephalinamide (DALE) in the GPI assay. In the MVD, analogue 11 showed a substantially reduced activity (EC50 = 92 nM), being about 10-fold less potent than DALE. In the RBM binding assay analogue 11 showed high affinity (in the nanomolar range) for both mu and delta binding sites with increased selectivity for the delta sites as shown by the ratio of the apparent affinities for both receptors, Ki (delta)/Ki (mu) = 2.1. The contribution of the modified peptide bonds in the mode of interaction of SP and enkephalin at their corresponding receptors is discussed.     

Analogues of growth hormone-releasing factor (1-29) amide containing the reduced peptide bond isostere in the N-terminal region

Previous peptide structure-activity investigations employing the psi[CH2NH] peptide bond isostere have produced antagonists when inserted into various sequences. These include bombesin, in which the incorporation of Leu13 psi[CH2NH]Leu14 produced a potent antagonist, and tetragastrin, with which Boc-Trp-Leu psi[CH2NH]Asp-Phe-NH2 is an antagonist. In this study, we chose to investigate the effect of this isostere on growth hormone-releasing factor (1-29) amide. Analogues were prepared by solid-phase synthesis and the isosteres incorporated by racemization-free reductive alkylation with a preformed protected amino acid aldehyde in the presence of NaBH3CN. The aldehydes were prepared by the reduction of the protected N,O-dimethyl hydroxamates with LiAlH4 at 0 degrees C. The purified analogues were assayed in a 4-day primary culture of male rat anterior pituitary cells for growth hormone (GH) release. Potential antagonists were retested in the presence of GRF(1-29)NH2. The following results were obtained: At position 5-6, a very weak agonist was produced with much less than 0.01% activity. Incorporation of the isostere in positions 1-2, 2-3, and 6-7 gave weak agonists with approximately 0.1% activity. Agonists with 0.39% and 1.6% activity were produced by incorporation at 10-11 and 3-4, respectively. The analogue [Ser9 psi[CH2NH]Tyr10]GRF(1-29)NH2 was found to be an antagonist in the 10 microM range vs 1 nM GRF and had no agonist activity at doses as high as 0.1 mM.     

Synthesis and use of 3-amino-4-phenyl-2-piperidones and 4-amino-2-benzazepin-3-ones as conformationally restricted phenylalanine isosteres in renin inhibitors.

The design of P2-P3 conformational restrictions in renin inhibitors by the use of a renin computer graphic model led to the synthesis of inhibitors containing N-Boc, N-acetyl, and N-phthalyl derivatives of 3(S)-amino-4(R,S)-2-piperidones and 4(S)-amino-2-benzazepinones in place of phenylalanine in the control compound N-acetyl-L-phenylalanyl-N-[4(S)-[(butylamino)carbonyl]-1(S)- (cyclohexylmethyl)-2(S)-hydroxy-5-methylhexyl]-L-norleuci namide (32). The piperidone inhibitors were prepared by utilization of the Evans chiral auxilliary to introduce the amino group with enantioselectivity and also to act as a leaving group in an intramolecular cyclization to the piperidone. The most potent inhibitor, 3(S)-(acetylamino)-alpha(S)-butyl-N-[4(S)- [(butylamino)carbonyl]-1(S)-(cyclohexylmethyl)-2(S)-hydroxy-5- methylhexyl]-2-oxo-4(R)-phenyl-1-piperidineacetamide (18, IC50 = 21 nM), was 25-fold less potent than the acyclic control 32. Considerable dependence of potency with the size of the P4 derivative was observed as had been expected based on the presynthetic modeling studies. Attempts to rationalize the observed potencies on the basis of further molecular modeling studies suggested that the loss in inhibitor potency was due to the conformational restrictions distorting the 3S center from the geometry present in the putative extended conformation present when the inhibitor is bound within the renin active site.     

A new class of high affinity ligands for the neurokinin A NK2 receptor: psi (CH2NR) reduced peptide bond analogues of neurokinin A4-10.

Analogues of [Leu10]NKA4-10 were synthesized in which each of the amide bonds was sequentially replaced with the reduced amide psi (CH2NH) bond to determine the effect of this structural modification on the antagonism of NKA binding to the HUB NK2 receptor. [psi (CH2-NH)9,Leu10]NKA4-10 (6) retained significant affinity for the NK2 receptor (IC50 = 115 nM) and showed weak partial stimulation of PI turnover (approximately 10-15% of NKA maximum). 6 behaves as a competitive antagonist of NKA-stimulated PI turnover with a pA2 = 6.7. The secondary amine of the psi (CH2NH) moiety of 6 was converted to a tertiary amine by alkylation. This modification was found to have a small effect upon receptor affinity but did result in attenuation of partial agonist activity. A combination of amino acid substitutions and psi (CH2NH) alkylation yielded [beta Ala8,psi (CH2N(CH2)2CH3)9,Phe10]NKA4-10 (21) which has very high affinity for the HUB NK2 receptor. This compound inhibited [125I]NKA binding with an IC50 = 1 nM which is equal to the receptor affinity of NKA. Compound 21 also shows very weak partial agonism of PI turnover (< or = 5% of NKA maximum) which makes this the most potent member of a new class of NKA ligands: psi(CH2NR)9-NKA4-10 analogues which potently antagonize NKA binding and possess minimal partial agonist activity.     

New reduced peptide bond substance P agonists and antagonists: effects on smooth muscle contraction.

Following the recent discovery of a new substance P (SP) competitive pancreatic acini cell receptor antagonist containing a reduced peptide bond in place of the C-terminal peptide bond, a new series of full chain and short chain (heptapeptide and hexapeptide) substance P analogues have been prepared in which one of the C-terminal-region peptide bonds has been replaced by CH2NH or CH2O groups. They were compared for their ability to recognize NK1 and/or NK2 tachykinin receptor binding sites on guinea pig ileum and rat duodenum smooth muscle preparations, respectively. It was found that all full sequence SP pseudopeptides were agonists with much reduced bioactivity in both tested systems and, in addition, [Gly9 psi(CH2NH)Leu10,Leu11]SP was found to be a relatively selective agonist for NK1 binding sites. Substitution of leucine at position 11 of SP heptapseudopeptides with phenylalanine generated a pseudopeptide with weak agonist activity when Gln at position 5 was replaced by D-Phe, or antagonists when this residue was replaced by D-Nal or D-Cpa. [Leu10 psi(CH2NH)Leu11]SP-(6-11) with Gln at position 6 substituted by D-Phe was a relatively stronger antagonist in both assay systems. These results suggest that, as with several other peptide systems of late, manipulation of the peptide bonds in SP can produce receptor antagonists which in some cases approach the potency of the classic spantide series and, furthermore, that the approach might be used to induce NK receptor specificity in both agonist and antagonist analogs.     

Renin inhibitors containing new P1-P1' dipeptide mimetics with heterocycles in P1'.

A series of renin inhibitors containing new P1-P1' dipeptide mimetics are presented. The P1-P1' mimetics were obtained from (4S,5S)-3-(tert-butoxycarbonyl)-4-(cyclohexylmethyl)-5-[(omega- mesyloxy)alkyl]-2,2-dimethyloxazolidines 5b, 9, and 11b by nucleophilic substitution of the mesylate groups with the sodium salts of mercapto- and hydroxyheterocycles. Removal of the protecting groups and stepwise acylations with amino acid derivatives provided renin inhibitors with a length of a tripeptide. Replacement of P2 histidine by other amino acids maintained or enhanced renin inhibitory potency. By alteration of P3 phenylalanine, compounds with IC50 values in the nanomolar range and stability against chymotrypsin were obtained. Finally, the effect of the C-terminal heterocycle on the renin inhibition was studied. Compound XVII was examined in vivo for its hypotensive effects. In salt-depleted cynomolgus monkeys, XVII inhibited plasma renin activity and lowered blood pressure after oral administration of a dose of 10 mg/kg.     

Synthesis and biological evaluation of novel potent antagonists of the bombesin/gastrin releasing peptide receptor.

This paper reports the synthesis and antagonist activity of 20 C-terminal analogues of gastrin releasing peptide (GRP). The ability of each analogue to inhibit bombesin (BN) stimulated amylase release from rat pancreatic acini was determined, and those showing antagonist activity were further evaluated for their ability to inhibit BN-stimulated [3H]thymidine uptake in serum-starved 3T3 cells. The assays also included two known peptide antagonists, C (Leu14,psi 13,14]BN) and H (N-pivaloyl-GRP20-25-(R)-2-methyl-4-nonylamide) as positive controls. On the basis of these assays we suggest that a des-Met27,Leu26-psi[CH2NHCOCH3]GRP C-terminal octapeptide imparts antagonist activity. The two most active compounds are peptides 14 ([D-Phe19,Leu26-psi(CH2NHCOCH3)]GRP19-26) and 18 ([D-Phe19,Gln20,Leu26-psi(CH2NHCOCH3)]GRP19++ +-26). In their ability to inhibit BN-stimulated [3H]thymidine uptake, the IC50 of peptides C, H, 14, and 18 were 43.2, 31.2, 2.7, and 32.5 nM, respectively. In conclusion, the novel C-terminal psi[CH2NHCOCH3] bond promises to be a useful peptide backbone modification for imparting antagonism in GRP/BN analogues.     

Short chain bombesin pseudopeptides with potent bombesin receptor antagonist activity in rat and guinea pig pancreatic acinar cells

A series of potent bombesin antagonists based on the reduced C-terminal peptide bond modification which in the past resulted in the first really potent antagonists are compared for effects on bombesin-stimulated amylase release from and binding to rat and guinea pig pancreatic acini. It was found that the original member of this series, [Leu13 psi (CH2NH)Leu14] bombesin, displayed partial agonist activity with 11% efficacy in the rat. More recent analogues of this type which were found previously to be even more potent pure antagonists in the guinea pig pancreas or 3T3 cells, exhibited similarly higher binding affinity for rat acini but displayed even higher residual partial agonist activity in the rat. For instance, [D-Phe6,Leu13 psi (CH2NH)Phe14]bombesin-(6-14) was one of the most potent bombesin antagonists known in the guinea pig and 3T3 cell systems but has 40% partial agonist activity in the rat. Several structural modification strategies were developed to remove rat partial agonist properties with retention of high antagonist potency in all systems tested. The most effective of these was the substitution of a Cl on the aromatic ring of the Phe residue (p-Cl-Phe, Cpa) in position 14 to give [D-Phe6,Leu13 psi (CH2NH)Cpa14]bombesin-(6-14). This had higher binding affinities for both rat and guinea pig pancreatic acini and was a pure antagonist on both cell types. Another effective method was alteration of the stereochemistry of the position 14 amino acid in [D-Phe6,Leu13 psi (CH2ND)D-Phe14]bombesin-(6-14) which had somewhat lowered binding affinities but pure antagonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate analogue renin inhibitors containing replacements of histidine in P2 or isosteres of the amide bond between P3 and P2 sites

Incorporation of beta-alanine or gamma-aminobutyric acid in position P2 of ACHPA or Leu psi [CHOHCH2]Val-based tetrapeptides gave highly active renin inhibitors (compounds V, VI, and XVII) with high specificity for renin and a remarkable stability against chymotrypsin. Replacement of the amide bond between P2 and P3 by isosteres (ketomethylenes, hydroxyethylenes, and the corresponding thio-insertion analogues) led to compounds (VIII-XIII, XVIII, and XIX) with renin inhibitory activity in the nanomolar range. Oral activity was achieved by incorporation of polar functionalities at the N-terminus of beta-alanine-containing tetrapeptides. One of these compounds (XXVIII) was chosen for further studies. This inhibitor demonstrated excellent efficacy and a long duration of action after intravenous and oral administration to cynomolgus monkeys.     

Renin inhibitors containing conformationally restricted P1-P1' dipeptide mimetics

A series of renin inhibitors containing lactam-bridged P1-P1' dipeptide mimetics based on the ACHPA (4(S)-amino-5-cyclohexyl-3(S)-hydroxypentanoic acid) design was studied. The inhibitors were obtained by aldol addition of various lactams with N alpha-Boc-L-cyclohexylalaninal, followed by Boc group removal and acylation with Boc-Phe-His. The aldol diastereomer having the S configuration at the two newly generated stereogenic centers gave optimal enzyme inhibition. Potency was further enhanced in the gamma-lactam ring series by substitution with small hydrophobic groups to mimic the P1' side chain of the renin substrate. Thus, 2(S)-[(Boc-L-phenylalanyl-L-histidyl)amino]-3-cyclohexyl-1(S)-hydroxyl-1 - (1,5,5-trimethyl-2-oxopyrrolidin-3(S)-yl)propane (34) has an IC50 of 1.3 nM in the human plasma renin assay. A variety of substituents on the lactam nitrogen are tolerated and can be used to vary the physical properties of the inhibitor. By using a model of the human renin active site, the conformation of 34 in the enzyme-inhibitor complex is proposed. This modeled conformation is very similar to the solid-state conformation of 2(S)-[(Boc-L-phenylalanyl-L-histidyl)amino]-3-cyclohexyl-1(S)-hydroxyl- 1-(1-methyl-2-oxopyrrolidin-3(S)-yl)propane (36), the structure of which was determined by single-crystal X-ray diffraction analysis. The most potent ACH-PA-lactam renin inhibitors show good selectivity when assayed against other types of aspartic proteinases. By varying the lactam ring substituents, potent and selective inhibitors of cathepsin D and cathepsin E can be obtained.     

1,2,4-Triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity. 1. Synthesis and biological properties of alkyl alcohol and statine derivatives

A series of 1,2,4-triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity, which incorporate (1S,2S)-2-amino-1,3-dicyclohexyl-1-hydroxypropane, statine (Sta), and (3S,4S)-4-amino-5-cyclohexyl-3-hydroxy-pentanoic acid (ACHPA) transition-state mimetics, have been prepared. Structure-activity relationships for renin inhibitory activity in the series are consistent with the 2-[8-isobutyl-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3-yl]-3-(3 pyridyl)propionic acid moiety 10b acting as a non-peptidic replacement for the P4-P2 (Pro-Phe-His) residues of the natural substrate angiotensinogen. Compounds 12m, 12o and 12q were potent inhibitors of partially purified human renin (IC50 values 1.7, 6.8, and 3.7 nM, respectively), and also effectively lowered blood pressure in anesthetized, sodium depleted marmosets following intravenous administration. On oral administration however, no blood pressure lowering activity could be detected, and absorption studies in bile duct cannulated rats indicate that this may be due primarily to poor oral absorption, rather than rapid biliary excretion. The reason for the observed poor oral activity is not clear, but it seems unlikely that poor aqueous solubility or metabolic instability to gut enzymes are rate-determining, and other factors such as high molecular weight may also be very important.     

Potent in vivo inhibitors of rat renin: Analogues of human and rat angiotensinogen sequences containing different classes of pseudodipeptides at the scissile site*

Using solid-phase methodology we have synthesised peptides based on the 8–14 or 6–14 human and rat angiotensinogen sequences, containing the following different isosteric units at the P₁-P₁’cleavage site: Leu-Ψ[CH₂NH]Leu; Leu-[CH(OH)CH₂]Val; Leu-Ψ[CH(OH)CH₂]Leu and Leu-Ψ[CH(NH₂)CH₂]Val. In vitro, peptide Piv-His-Pro-Phe-His-Leu-Ψ[CH(OH)CH₂]Leu-Tyr-Tyr-Ser-NH₂( XXI ) is the most potent inhibitor of rat plasma renin reported having an IC₅₀ of 0.21 nM; it is a much weaker inhibitor of human renin (IC₅₀ 45 nM). Peptide Boc-His-Pro-Phe-His-Leu-Ψ[CH(OH)CH₂] Leu-Val-Ile-His-NH₂ ( XX ) was a highly effective inhibitor of rat renin in vivo. When infused (1 mg/kg/h) into two-kidney, one-clip chronic renal hypertensive rats, it lowered blood pressure and suppressed both plasma renin and angiotensin II. When given as a bolus (1 mg/kg) there was a divergence between the rapid rebound of renin levels and blood pressure, which remained suppressed. These results indicate that potent in vivo inhibitors of rat renin could be useful not only in examining the role of circulating renin but also in elucidating the equally important involvement of extracirculatory renin pools.     

Synthesis and biological activity of potent,low molecular weight renin inhibitors*

A series of renin inhibitors containing the dipeptide transition state mimics (2R, 4S, 5S)-5-amino-4-hydroxy-2-methyl-6-cyclohexyl hexanoic acid (Cha-Ψ[CH(OH)CH₂]Ala) and (2R, 45, 5S)-5-amino-4-hydroxy-2-isopropyl-6-cyclohexyl hexanoic acid (Cha-Ψ[CH(OH)CH₂]Val) were prepared. A structure-activity study, using pseudopeptide (Boc-Phe-His-Leu-Ψ[CH(OH)CH₂]Val-Ile-His-OH) as our lead structure, led to a new series of inhibitors, which correspond to tripeptides and contain no natural amino acids. For example, R, S-Bpma-Ape-Cha-Ψ[CH(OH)CH₂]Ala-NH₂ (IC₅₀= 1.26 nM against human plasma renin at pH 6.0; molecular weight = 564) has only two thirds of the molecular weight but twice the potency of our original lead. This new class of low molecular weight renin inhibitor displays excellent specificity toward human renin versus the related aspartic proteinase pepsin and angiotensin-1-converting enzyme. Examples are given of selected inhibitors showing encouraging evidence for intestinal absorption after intracolonic and oral administration in male Sprague-Dawley rats.     

Vasorelaxant responses to endomorphin1[psi] and endomorphin2[psi], analogues of endomorphins, in rat aorta rings

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM2), the endogenous selective mu-opioid receptor agonists, can inhibit phenylephrine (PE) induced contraction which is related to the release of nitric oxide from vascular endothelium in aorta rings of rats and rabbits. The reduced (CH2NH) amide bond is a useful peptide bond surrogate in the design of opioid mimetics because it could enhance conformational flexibility and metabolic stability. The present work was designed to investigate the vascular activities of interrelated endomorphin analogues: endomorphin-1[psi] (Tyr[psi(CH2NH)]Pro-Trp-Phe-NH2, EM1[psi]) and endomorphin-2[psi] (Tyr[psi(CH2NH)]Pro-Phe-Phe-NH2, EM2[psi]). The effect of EM1[psi] (1, 2, 3, 4, 5 microM) and EM2[psi] (0.001, 0.01, 0.1, 1, 5 microM) were evaluated on rat thoracic aortic rings pre-contracted with PE (0.1 microM). EM1[psi] and EM2[psi] both caused a concentration-dependent relaxation. The IC50 of EM1[psi] and EM2[psi] was 3.332 microM and 1.226 microM, respectively. The vasorelaxant effect of EMs[psi] is 216.9 and 237.1 fold more potent than EMs. Moreover, the vasorelaxant effect of EMs[psi] was blocked by naloxone (NaCl, 1 microM) and was reduced by N(omega)-nitro-L-arginine (L-NNA, 1 microM) and removal of endothelium. The present study demonstrated that EMs[psi] had more potent vasorelaxant effects and their activities were naloxone-sensitive and endothelium-dependent and partially NO-dependent, similar to the mechanism of parent EMs.     

Potent and selective antagonists of the antidiuretic responses to arginine-vasopressin based on modifications of [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-isoleucine,4- valine]arginine-vasopressin at position 4

As part of a program in which we are attempting (a) to obtain more potent and/or more selective antagonists of the antidiuretic responses to arginine-vasopressin (AVP) and (b) to delineate the structural features at positions 1-9 required for antidiuretic antagonism, we have synthesized 13 new analogues of the antidiuretic antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-isoleucine,4- valine]arginine-vasopressin [d(CH2)5[D-Ile2]VAVP] in which the valine residue at position 4 has been replaced by the L-amino acids Abu, Ile, Thr, Ala, Ser, Nva, Gln, Leu, Lys, Cha, Asn, Orn, and Phe and two new analogues of the antidiuretic antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-phenylalanine,4- valine]arginine-vasopressin [d(CH2)5[D-Phe2]VAVP] with the Val4 residue replaced by Ser and Orn. These analogues are 1, d(CH2)5[D-Ile2,Abu4]AVP; 2, d(CH2)5[D-Ile2,Ile4]AVP; 3, d(CH2)5[D-Ile2,Thr4]AVP; 4, d(CH2)5[D-Ile2,Ala4]AVP; 5, d(CH2)5[D-Ile2,Ser4]AVP; 6, d(CH2)5[D-Ile2,Nva4]AVP; 7, d(CH2)5[D-Ile2]AVP; 8, d(CH2)5[D-Ile2,Leu4]AVP; 9, d(CH2)5[D-Ile2,Lys4]AVP; 10, d(CH2)5[D-Ile2,Cha4]AVP; 11, d(CH2)5[D-Ile2,Asn4]AVP; 12, d(CH2)5[D-Ile2,Orn4]AVP; 13, d(CH2)5[D-Ile2,Phe4]AVP; 14, d(CH2)5[D-Phe2,Ser4]AVP; and 15, d(CH2)5[D-Phe2,Orn4]AVP. The protected peptide precursors for these peptides were prepared by the solid-phase method, followed by ammonolytic cleavage. The free peptides 1-15 were obtained by deblocking with Na in NH3, oxidation of the resultant disulfhydryl compounds with dilute K3[Fe(CN)6], and purification on Sephadex G-15 in a two-step procedure with 50% HOAc and 0.2 M HOAc as eluants. Analogues 1-15 were tested in rats for agonistic and antagonistic activities by antidiuretic, vasopressor, and oxytocic assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and initial characterization of a putative neuromedin B-type receptor from rat urinary bladder membranes.

Receptor binding site(s) on the rat urinary bladder membranes were characterized using a biologically active analog of bombesin, [Tyr4,Leu14]bombesin, and a 50,000 x g total particulate preparation. The binding was specific, reversible, saturable, time- and concentration-dependent. A dissociation curve showed that both bombesin and neuromedin B equally displaced the radioligand in the first 10 min after saturation. From the rate constant of association K + 1 = 7.60 x 10(9) M-1 min-1, and the rate constant of dissociation k-1 = 0.050 min-1, the apparent equilibrium dissociation constant Kd = 6.57 +/- 1.09 pM was determined. A linear Scatchard plot of the specific binding of 125I-[Tyr4,Leu14]bombesin to the membranes revealed that the radioligand bound with high affinity, Kd = 6.38 +/- 0.86 pM, to a single class of sites (Bmax = 2.3 fmol/mg protein). The Hill coefficient of the same binding data was 1.05 +/- 0.21, indicating that the radioligand was binding to a single population of noninteracting binding sites. Both bombesin and neuromedin B displaced the radioligand dose dependently (IC50 = 0.3 nM). Neurokinin A and neurokinin B were less potent (IC50 = 20 and 110 nM, respectively). Substance P, or the specific bombesin receptor antagonists [D-Phe6]bombesin-(6-13) methyl ester, [D-F5Phe6,D-Ala11]bombesin-(6-11) methyl ester, [D-Phe6]bombesin-(6-13) propylamide, [D-Phe6,Leu13psi(CH2NH)Leu14]bombesin or [D-Cpa6,Phe14(psi13-14)]bombesin-(6-14) had an IC50 greater than 1 microM. The results presented suggest the presence of neuromedin B receptor sites on the rat urinary bladder membranes that can be occupied also by some other peptides, notably bombesin, neurokinin A and neurokinin B.(ABSTRACT TRUNCATED AT 250 WORDS)