Evidences for the action mechanism of angiotensin II and its analogs on Plasmodium sporozoite membranes.

Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d-amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so-called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all-D-AII and all-D-VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro-AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a ß-turn conformation in different solutions. In the presence of membrane-mimetic micelles, AII had also a ß-turn conformation, while retro-AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro-AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro-AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation.

Effects of the angiotensin II Ala-scan analogs in erythrocytic cycle of Plasmodium falciparum (in vitro) and Plasmodium gallinaceum (ex vivo).

The anti-plasmodium activity of angiotensin II and its analogs have been described in different plasmodium species. Here we synthesized angiotensin II Ala-scan analogs to verify peptide-parasite invasion preservation with residue replacements. The analogs were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (t-Boc) solid phase methods, purified by liquid chromatography and characterized by mass spectrometry. The results obtained in Plasmodium falciparum assays indicated that all analogs presented some influence in parasite invasion, except [Ala(4)]-Ang II (18% of anti-plasmodium activity) that was not statistically different from control. Although [Ala(8)]-Ang II presented a lower biological activity (20%), it was statistically different from control. The most relevant finding was that [Ala(5)]-Ang II preserved activity (45%) relative to Ang II (47%). In the results of Plasmodium gallinaceum assays all analogs were not statistically different from control, except [Ala(6)]-Ang II, which was able to reduce the parasitemia about 49%. This approach provides insight for understanding the importance of each amino acid on the native Ang II sequence and provides a new direction for the design of potential chemotherapeutic agents without pressor activity.

A mild removal of Fmoc group using sodium azide.

A mild method for effectively removing the fluorenylmethoxycarbonyl (Fmoc) group using sodium azide was developed. Without base, sodium azide completely deprotected N (α)-Fmoc-amino acids in hours. The solvent-dependent conditions were carefully studied and then optimized by screening different sodium azide amounts and reaction temperatures. A variety of Fmoc-protected amino acids containing residues masked with different protecting groups were efficiently and selectively deprotected by the optimized reaction. Finally, a biologically significant hexapeptide, angiotensin IV, was successfully synthesized by solid phase peptide synthesis using the developed sodium azide method for all Fmoc removals. The base-free condition provides a complement method for Fmoc deprotection in peptide chemistry and modern organic synthesis.

Antiplasmodial activity study of angiotensin II via Ala scan analogs.

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid-phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide-capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile(5) or the His(6) residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala(6) ]-AII (79%), and [Ala(5) ]-AII (75%). Analogs [Ala(3) ]-AII, [Ala(1) ]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala(8) ]-AII, [Ala(7) ]-AII, [Ala(4) ]-AII, and [Ala(2) ]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a ß-fold conformation in different solutions. All AII analogs, except [Ala(4) ]-AII and [Ala(8) ]-AII, show contractile responses and interact with the AT1 receptor, [Ala(5) ]-AII and [Ala(6) ]-AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds.

Beta-homo-amino acid scan of angiotensin IV.

Angiotensin IV, a metabolite of angiotensin II, inhibits the enzyme insulin regulated aminopeptidase or IRAP and also, although with lower potency, aminopeptidase-N (AP-N). When both beta (2)-homo amino acid- and beta (3)-homo amino acid substitutions were used, allowed the identification of H-( R)beta (2)hVal-Tyr-Ile-His-Pro-beta (3)hPhe-OH as a potent and stable Ang IV analog with high selectivity for IRAP versus AP-N and the AT1 receptor.

Angiotensin Peptides as AT2 Receptor Agonists.

In 2004, the first nonpeptide selective angiotensin II type 2 receptor (AT2R) agonist was reported. This nonpeptide (C21), which, exerts anti-inflammatory and antifibrotic actions in vivo, has been extensively explored and is currently in clinical trials. Subsequently, a large number of related drug-like AT2R agonists have been disclosed. Reviews that summarize known structure-activity relationships (SAR) of nonpeptide AT2R agonists have recently appeared in the literature; however, very few reviews discuss the role of angiotensin peptides as AT2R agonists. Furthermore, to date, there have been no reports focusing on the medicinal chemistry perspective of peptide AT2R agonists. In the present review, reports on linear and conformationally constrained Ang II analogues, with a focus on AT2R selective ligands that are proven to act as agonists at the AT2 receptor are summarized. The impact of truncations and macrocyclizations of Ang II analogues and of incorporation of scaffolds that mimic secondary structures into Ang II related peptides is highlighted. A survey of the efforts to transform the nonselective octapeptide Ang II to more drug-like selective AT2R agonists is presented. The relationship between the structures of the AT2R agonists and their affinity to the AT2R is briefly discussed and common pharmacophore elements of AT2R selective Ang II peptide analogues and selective nonpeptide AT2R agonists are compared.

Stereospecific synthesis of a carbene-generating angiotensin II analogue for comparative photoaffinity labeling: improved incorporation and absence of methionine selectivity.

A stereospecific convergent synthesis of N-[(9-fluorenyl)methoxycarbonyl]-p-[3-(trifluoromethyl)-3H-diazirin-3-yl]-l-phenylalanine (Fmoc-12, Fmoc-Tdf) and its incorporation into the C-terminal position of the angiotensin II (AngII) peptide to form (125)I[Sar(1),Tdf(8)]AngII ((125)I-13) is presented. This amino acid photoprobe is a highly reactive carbene-generating diazirine phenylalanine derivative that can be used for photoaffinity labeling. Using model receptors, we compared the reactivity and the Met selectivity of 12 to that of the widely used and reputedly Met-selective p-benzoyl-l-phenylalanine (Bpa) photoprobe. Wild-type and mutant AngII type 2 receptors, a G protein-coupled receptors, were photolabeled with (125)I-13 as well as with (125)I[Sar(1),Bpa(8)]AngII ((125)I-14), and the respective incorporation yields were assessed. The carbene-generating 12 was more reactive toward inert residues and was not Met-selective compared to the biradical ketone-generating Bpa, allowing for more precise determination of ligand contact points in peptidergic receptors.

Angiotensin II analogues with N-terminal lactam bridge cyclization: an overview on AT1 receptor activation and tachyphylaxis.

Angiotensin II (AngII) is the final active product of the renin enzymatic cascade, which is responsible for sustaining blood pressure. To investigate the effect of N-terminal cyclization on AT1 activation and tachyphylaxis, we designed conformationally constrained analogues with an i-(i + 1) lactam bridge. All analogues presented the same binding coefficient and tachyphylactic index, but some of them such as Cyclo (0-1a) [Glu0 , endo-(Lys1a )]-AngII and Cyclo (0-1a) [Asp0 , endo-(Orn1a )]-AngII showed higher potency. The same tachyphylactic index presented by AngII and cyclic analogues was surprising. We expected a variation after the modification of AngII N-terminal region.

Angiotensin II pseudopeptides containing 1,3,5-trisubstituted benzene scaffolds with high AT2 receptor affinity.

Two 1,3,5-trisubstituted aromatic scaffolds intended to serve as gamma-turn mimetics have been synthesized and incorporated in five pseudopeptide analogues of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), replacing Val-Tyr-Ile, Val-Tyr, or Tyr-Ile. All the tested compounds exhibited nanomolar affinity for the AT2 receptor with the best compound (3) having a K(i) of 1.85 nM. Four pseudopeptides were AT2 selective, while one (5) also exhibited good affinity for the AT1 receptor (K(i) = 30.3 nM). This pseudopeptide exerted full agonistic activity in an AT2 receptor induced neurite outgrowth assay but displayed no agonistic effect in an AT1 receptor functional assay. Molecular modeling, using the program DISCOtech, showed that the high-affinity ligands could interact similarly with the AT2 receptor as other ligands with high affinity for this receptor. A tentative agonist model is proposed for AT2 receptor activation by angiotensin II analogues. We conclude that the 1,3,5-trisubstituted benzene rings can be conveniently prepared and are suitable as gamma-turn mimics.

Structure-activity relationships for the competitive angiotensin antagonist [sarcosine,O-methyltyrosine4]angiotensin II (sarmesin).

Analogues of the competitive angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (sarmesin) in which the sarcosine-1, O-methyltyrosine-4, and phenylalanine-8 residues were modified have been synthesized by the solid-phase method. The agonist and antagonist potencies of the 23 peptides synthesized were determined in the rat isolated uterus assay. At position 1, replacement of Sar with Asp, Ala, or Pro gave inactive analogues, and deletion of the N-terminal amino acid produced inactive heptapeptides for all analogues investigated. At position 4, substitution of Tyr with Tyr(Et), D-Tyr, D-Phe, Ile, Thr, or Hyp resulted in inactive analogues, whereas substitution of Phe gave a potent competitive antagonist (pA2 = 7.9), which retained significant agonist activity (22%). For position 8, [Sar1,Tyr(Me)4,Ile8]ANG II and [Sar1,Phe4,Ile8]ANG II were weaker antagonists (pA2 = 6.6 and 6.7, respectively) than [Sar1,Ile8]ANG II (pA2 apparent = 8.1) and, moreover, were reversible competitive antagonists. These findings demonstrate that the structural requirements for receptor blockade by sarmesin are remarkably stringent--modifications at positions 1, 4, and 8 markedly reduce the antagonist activity of this peptide.

The role of position 4 in angiotensin II antagonism: a structure-activity study.

A number of [Sar1,(pX)Phe4]-ANG II and [Sar1,(pX)Phe4,Ile8]-ANG II analogues were prepared. A good correlation between pX structure in [Sar1,(pX)Phe4]-ANG II and antagonist activity could not be found. However, the data suggest a general trend: Position 4 para substituents that are hydrophilic and capable of donating a hydrogen atom in a hydrogen bond promote agonist activity, while para substituents that are hydrophobic and incapable of donating a hydrogen atom promote antagonist activity. These properties were found to be optimal in the p-chloro substituent. The resulting analogue [Sar1,(pCl)Phe4]-ANG II is a potent ANG II antagonist in vivo. The pX substituents that promote antagonist activity in the [Sar1,(pX)Phe4]-ANG II series were unfavorable in [Sar1,(pX)Phe4,Ile8]-ANG II analogues. ANG II analogues that are antagonists by virtue of an alteration in position 8 require a position 4 agonist side chain. Concurrent modifications of positions 4 and 8 do not give rise to potent antagonists with reduced partial agonist activity.

Role of the C-terminal carboxylate in angiotensin II activity: alcohol, ketone, and ester analogues of angiotensin II.

[Ac-Asn1, Val5]angiotensin II analogues containing a C-terminal alcohol (Phe-ol), methyl ketone (Pmk), methyl ester (Phe-OMe), or alpha-methyl methyl ester (Phe(alpha Me)-OMe) were prepared in order to examine the relative importance of COOH-mediated ionic vs. hydrogen bonding interactions in angiotensin activities. Based on the observation that only [Ac-Asn1,Phe-OMe8]AII (AII, angiotensin II) had significant activities (20% oxytocic and 13% pressor) in the rat, with all other analogues having negligible agonistic and antagonistic effects, it is concluded that ionic interaction of the C-terminal carboxylate with the receptor is necessary for angiotensin binding and that hydrogen bonding has little effect. Thus, the different potencies observed for the AII methyl ester and for various C-terminal analogues previously reported may simply reflect their relative abilities to generate the active carboxylate species in situ.

Synthesis and specific pressor activity of [1-aspartic acid,5-valine,9-serine]angiotensin I ("fowl angiotensin I").

[Asp1, Val5, Ser9]angiotensin I was synthesized by Merrifield's solid-phase procedure. The dansylated derivative of this angiotensin was cochromatographed on the TLC with the dansylated angiotensin decapeptide isolated from white leghorn fowl. Either angiotensin showed identical behavior. The per mole pressor activity of the synthetic decapeptide (in rats anesthetized with pentobarbital and treated with pentolinium) as compared to mammalian angiotensins, namely, [Ile5]angiotensin I, [Val5]angiotensin I, [Ile5]angiotensin II, and [Val5]angiotensin II, was 157, 181, 114, and 85%, respectively.

Synthesis of fluorine-containing peptides. Analogues of angiotensin II containing hexafluorovaline.

Gamma, gammma, gammma, gammma', gamma', gammma'-Hexafluorovaline and derivatives have been prepared and incorporated into angiotensin II by fragment condensation and solid-phase peptide synthesis. Hexafluorovaline derivatives showed general resistance toward various enzymatic digestions and the tendency to racemize extensively upon carboxyl activation. When the angiotensin II analogues were assayed on rat uterus, [Hfv5]AII had 133% activity, [D-Hfv5]AII was inactive, and [Ac-Asn1,DL-Hfv8]AII was a potent inhibitor of angiotensin II in vitro and in vivo.

A selective AT2 receptor ligand with a gamma-turn-like mimetic replacing the amino acid residues 4-5 of angiotensin II.

Three angiotensin II (Ang II) analogues encompassing a benzodiazepine-based gamma-turn-like scaffold have been synthesized. Evaluation of the compounds in a radioligand binding assay showed that they had no affinity to the rat liver AT(1) receptor. However, one of the compounds displayed considerable affinity to the pig uterus AT(2) receptor (K(i) = 3.0 nM) while the other two lacked affinity to this receptor. It was hypothesized that the reason for the inactivity of one of these analogues to the AT(2) receptor was that the guanidino group of the Arg(2) residue and/or the N-terminal end of the pseudopeptide could not interact optimally with the receptor. To investigate this hypothesis, a conformational analysis was performed and a comparison was carried out with the monocyclic methylenedithioether analogue cyclo(S-CH(2)-S)[Cys(3,5)]Ang II which is known to bind with high affinity to the AT(2) receptor (K(i) = 0.62 nM). This comparison showed that, in the compounds with high AT(2) receptor affinity, the guanidino group of the Arg(2) residue and the N-terminal end could access common regions of space that were not accessible to the inactive compound. To examine the importance of the guanidino group for binding, the Arg side chain was removed by substituting Arg(2) for Ala(2) in the analogue having the high affinity. This analogue lacked affinity to AT(2) receptors, which supports the role of the guanidino group in receptor binding.

Novel synthesis of cyclic amide-linked analogues of angiotensins II and III.

Cyclic amide-linked angiotension II (ANGII) analogues have been synthesized by novel strategies, in an attempt to test the ring clustering and the charge relay bioactive conformation recently suggested. These analogues were synthesized by connecting side chain amino and carboxyl groups at positions 1 and 8, 2 and 8, 3 and 8, and 3 and 5, N-terminal amino and C-terminal carboxyl groups at positions 1 and 8, 2 and 8, and 4 and 8, and side chain amino to C-terminal carboxyl group at positions 1 and 8. All these analogues were biologically inactive, except for cyclic [Sar1, Asp3, Lys5]ANGII (analogue 10) which had high contractile activity in the rat uterus assay (30% of ANGII) and [Lys1, Tyr(Me)4, Glu8]ANGII (analogue 7) which had weak antagonist activity (PA2 approximately 6). Precyclic linear peptides synthesized using 2-chlorotrityl chloride resin and N alpha-Fmoc-amino acids with suitable side chain protection were obtained in high yield and purity and were readily cyclized with benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate as coupling reagent. Molecular modeling suggests that the ring structure of the potent analogue can be accommodated in the charge relay conformation proposed for ANGII.

Synthesis of angiotensin II analogues by incorporating beta-homotyrosine or beta-homoisoleucine residues.

[1-Sarcosine,4-beta-homotyrosine]-(I), [5-beta-homoisoleucine]-(II), and [1-sarcosine,5-beta-homoisoleucine]angiotensin II (III) were synthesized by Merrifield's solid-phase procedure to study the effect of pressor activity and duration of action. The analogues I--III possessed, respectively, 1.98, 2.82, and 29.2% pressor activity of angiotensin II (vagotomized, ganglion-blocked rats by single-injection procedure) and duration of action of 5.5, 6.7, and 4.7 min; the comparative duration of action of an equipressor dose of angiotensin II was 5.2, 6.3, and 5.3 min, respectively. When incubated with leucine aminopeptidase, degradation of II was as fast as that of angiotensin II; this degradation became considerably slower when position 1 was replaced with sarcosine. Incubation of all these analogues with chymotrypsin showed very little or no degradation up to 3 h. The results indicate that an increase in the chain length by one carbon atom in position 4 or 5 of angiotensin II increased resistance to degradation by chymotrypsin without any increase in in vivo duration of action. Further, all analogues showed low pressor activity.

Synthesis of angiotensin II antagonists containing sarcosine in position 7.

Analogues of the type [1-sarcosine,7-sarcosine, 8-X]angiotensin II, where X = isoleucine, leucine, alanine, methionine, O-methylthreonine, or DL-alloisoleucine, were synthesized by the solid-phase method and purified by partition chromatography, cation-exchange chromatography, and high-pressure liquid chromatography. In the isolated rat uterus, these analogues had activities of less than 0.1, less than 0.1, less than 0.1, less than 0.1, less than 0.1, and 0.7%, respectively, of the hyotropic activity of angiotensin II and inhibited the contractile response to angiotensin II with pA2 values of 8.1, 7.2, 6.7, 7.7, 7.4, and 8.4, respectively. In the vagotomized ganglion blocked rat, the analogues had 0.7, 0.21, 0.06, 0.72, 0.13, and 12.5%, respectively, of the pressor activity of angiotensin II.

Synthesis and evaluation of (Des-Asp1)angiotensin I as a precursor for (Des-Asp1)angiotensin II ("Angiotensin III").

The nonapeptide [des-Asp1]angiotensin I (IV), synthesized by Merrifield's solid-phase procedure, was tested as a possible substrate for the converting enzymes from porcine lung and plasma. IV, [des-Asp1]angiotensin II (III), [des-(Asp1,Arg2)]angiotensin II (V), [des-(Asp1,Arg2,Val3)]angiotensin II (VI), [Sar1,Ile8]angiotensin II (VII), and [des-Asp1,Ile8]angiotensin II (VIII) possessed 0.5, 20, 2, 0 less than 0.1, and less than 0.01% of the inotropic activity (rabbit atria), 1, 15, 5, 0, 3, and 0% secretory activity of the cat adrenal medulla, and 0.0, 150, 0.5, 3, and 10% of the adrenal steroidogenic activity of angiotensin II, respectively. When tested for their antagonistic activity in the above tissues, only VII and VIII were found to inhibit responses to angiotensin II. The pA2 values for VII and VIII were 8.31 and 10.0 in the adrenal cortex and 9.31 and 9.16 in the adrenal medulla, respectively. All these peptides were also tested as product inhibitors for the plasma and lung converting enzymes. With the plasma enzyme, the ID50 values were II, 1.6 X 10(-4) M; III, 5 X 10(-5) M; V, 1.2 X 10(-4) M; VI 5 X 10(-4) M; VII 5 X 10(-5) M; VIII, 5 X 10(-4) M. Thus, IV is a good substrate for converting enzymes from lung and plasma while all other compounds were inhibitors of these enzymes. The most potent inhibitors of converting enzyme were III followed by VII and VIII. With the exception of II and III, all the other analogs had very low intrinsic activities, per se. These results suggest (a) an alternate pathway for the formation of heptapeptide III, viz., by the action of converting enzyme on the nonapeptide IV, and (b) that III may also be acting as inhibitor of the converting enzyme by the feedback mechanism.

Synthesis of biologically active retroenantiomers of angiotensin peptides.

The effect of the reversal of the direction of amide bonds in the peptide chain of angiotensin was determined by the synthesis and study of retroenantiomers of the following peptides: 1 [Val5]angiotensin II (angiotensin); 2, [Suc1]angiotensin (desamino-angiotensin); 3, [Ala7]desamino-angiotensin; 4, [beta-Ala7]desamino-angiotensin. In all of these retroenantiomers, the N-terminal Phe residue was replaced by a benzylmalonyl moiety in order to maintain the topological features of angiotensin's C terminus which are important for biological activity. The separation of the diasteromeric peptides containing D- or L-benzylmalonyl residues was possible in the cases of the retroenantiomers of 1 and 2 but not in those of 3 and 4. The retroenantiomers of 1 and 2 were devoid of smooth muscle contracting activities, while those of 3 and 4 contracted the isolated guinea-pig ileum and rat uterus with activities ranging from 8 to 24%, when compared with the respective parent compounds. The results indicate that (a) the sense of the peptide bonds in angiotensin's backbone is not essential for activity, and (b) the Pro7 residue in angiotensin is important for maintaining an "active" conformation of the molecule. The compounds reported in this paper are the first retroenantiomers of linear peptide hormones that have been shown to retain the biological activities of the parent compounds.