Interconversion of Functional Activity by Minor Structural Alterations in Nonpeptide AT2 Receptor Ligands.

Migration of the methylene imidazole side chain in the first reported selective drug-like AT2 receptor agonist C21/M024 (1) delivered the AT2 receptor antagonist C38/M132 (2). We now report that the AT2 receptor antagonist compound 4, a biphenyl derivative that is structurally related to 2, is transformed to the agonist 6 by migration of the isobutyl group. The importance of the relative position of the methylene imidazole and the isobutyl substituent is highlighted herein.

From the first selective non-peptide AT(2) receptor agonist to structurally related antagonists.

A para substitution pattern of the phenyl ring is a characteristic feature of the first reported selective AT(2) receptor agonist M024/C21 (1) and all the nonpeptidic AT(2) receptor agonists described so far. Two series of compounds structurally related to 1 but with a meta substitution pattern have now been synthesized and biologically evaluated for their affinity to the AT(1) and AT(2) receptors. A high AT(2)/AT(1) receptor selectivity was obtained with all 41 compounds synthesized, and the majority exhibited K(i) ranging from 2 to 100 nM. Five compounds were evaluated for their functional activity at the AT(2) receptor, applying a neurite outgrowth assay in NG108-15 cells. Notably, four of the five compounds, with representatives from both series, acted as potent AT(2) receptor antagonists. These compounds were found to be considerably more effective than PD 123,319, the standard AT(2) receptor antagonist used in most laboratories. No AT(2) receptor antagonists were previously reported among the derivatives with a para substitution pattern. Hence, by a minor modification of the agonist 1 it could be transformed into the antagonist, compound 38. These compounds should serve as valuable tools in the assessment of the role of the AT(2) receptor in more complex physiological models.

Selective angiotensin II AT(2) receptor agonists with reduced CYP 450 inhibition.

Structural alterations to the benzylic position of the first drug-like selective angiotensin II AT(2) receptor agonist (1) have been performed, with the emphasis to reduce the CYP 450 inhibitory property of the initial structure. The imidazole moiety, responsible for the CYP 450 inhibitory effect in 1, was replaced with various heterocycles. In addition, the modes of attachment of the heterocycles, that is, carbon versus nitrogen attachment, and introduction of carbonyl functionalities to the benzylic position have been evaluated. In all the three series, AT(2) receptor ligands with affinity in the lower nanomolar range were identified. None of the analogues, regardless of the substituents, exhibited any affinity for the AT(1) receptor. Compounds with substantially reduced inhibition of the CYP 450 enzymes were obtained. Among them the compound 60 was found to induce neurite elongation in NG 108-15 cells and served as potent AT(2) selective agonist.

Discovery of dipeptides with high affinity to the specific binding site for substance P1-7.

Substance P 1-7 (SP(1-7), H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is the major bioactive metabolite of substance P. The interest in this heptapeptide originates from the observation that it modulates, and in certain cases opposes the effects of the parent peptide, e.g., the nociceptive effect. The mu-opioid receptor agonist endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH(2)) has been found to also interact with the specific binding site of SP(1-7) with only a 10-fold lower affinity compared to the native peptide. Considering the smaller size of EM-2 compared to the target heptapeptide, it was selected as a lead compound in the development of low-molecular-weight ligands to the SP(1-7) binding site. An alanine scan and truncation study led to the unexpected discovery of the dipeptide H-Phe-Phe-NH(2) (K(i) = 1.5 nM), having equal affinity as the endogenous heptapeptide SP(1-7.) Moreover, the studies show that the C-terminal phenylalanine amide is crucial for the affinity of the dipeptide.

The C-terminal amidated analogue of the substance P (SP) fragment SP(1-7) attenuates the expression of naloxone-precipitated withdrawal in morphine dependent rats.

We previously demonstrated that intracerebroventricular (i.c.v.) administration of the substance P (SP) aminoterminal fragment SP(1-7) attenuates the expression of morphine withdrawal in the male rat. In this study we have used a synthetic analogue of this peptide, i.e. the SP(1-7) amide showing higher binding potency than the native heptapeptide, in a similar experimental set-up. Thus, Wistar male rats were made tolerant to morphine by daily injections of the opiate during 8 days. Following peptide administration (i.c.v.) and a subsequent naloxone challenge a variety of physical syndromes of withdrawal were recorded. We observed that the SP(1-7) amide potently and dose-dependently reduced several signs of reaction to morphine withdrawal. Interestingly, the effect of the peptide amide was significantly attenuated by the addition of the sigma agonist (+)-SKF-10047. We conclude that the SP(1-7) amide mimics the effect of the native SP fragment and that the mechanisms for its action involve a sigma receptor site.

Endomorphins interact with the substance P (SP) aminoterminal SP(1-7) binding in the ventral tegmental area of the rat brain.

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP(1-7) in the rat spinal cord. This site appeared very specific for SP(1-7) as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP(1-7) from this site. In the present work using a [(3)H]-labeled derivative of the heptapeptide we have identified and characterized [(3)H]-SP(1-7) binding in the rat ventral tegmental area (VTA). Similarly to the [(3)H]-SP(1-7) binding in the spinal cord the affinity of unlabeled SP(1-7) to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP(1-7) site was 4-5 times weaker than that for SP(1-7) but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP(1-7) binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.

Selective angiotensin II AT2 receptor agonists: Benzamide structure-activity relationships.

In the investigation of the structure-activity relationship of nonpeptide AT(2) receptor agonists, a series of substituted benzamide analogues of the selective nonpeptide AT(2) receptor agonist M024 have been synthesised. In a second series, the biphenyl scaffold was compared to the thienylphenyl scaffold and the impact of the isobutyl substituent and its position on AT(1)/AT(2) receptor selectivity was also investigated. Both series included several compounds with high affinity and selectivity for the AT(2) receptor. Three of the compounds were also proven to function as agonists at the AT(2) receptor, as deduced from a neurite outgrowth assay, conducted in NG108-15 cells.

Small peptides mimicking substance P (1-7) and encompassing a C-terminal amide functionality.

Some of the biological effects demonstrated after administration of substance P (SP) in vivo can indirectly be attributed to the fragmentation of the undecapeptide to its N-terminal bioactive fragment SP(1-7). This heptapeptide (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is a major bioactive metabolite from SP that frequently exerts similar biological effects as the parent peptide but also, in several cases, completely opposite actions. Specific binding sites for the heptapeptide SP(1-7) that are separate from the SP preferred NK receptors have been identified. In this study we demonstrate that (a) the C-terminal part of the SP metabolite SP(1-7) is most important for binding as deduced from an Ala scan and that a replacement of Phe(7) for Ala is deleterious, (b) truncation of the N-terminal amino acid residues of SP(1-7) delivers peptides with retained binding activity, although with somewhat lower binding affinities than SP(1-7) and (c) a C-terminal amide group as a replacement for the terminal carboxy group of SP(1-7) and for all of the truncated ligands synthesized affords approximately 5-10-fold improvements of the binding affinities.

Selective angiotensin II AT(2) receptor agonists devoid of the imidazole ring system.

A versatile parallel synthetic method to obtain three series of non-cyclic analogues of the first drug-like selective angiotensin II AT(2) receptor agonist (1) has been developed. In analogy with the transformation of losartan to valsartan it was demonstrated that a non-cyclic moiety could be employed as an imidazole replacement to obtain AT(2) selective compounds. In all the three series, AT(2) receptor ligands with affinities in the lower nanomolar range were found. None of the analogues exhibited any affinity for the AT(1) receptor. Four compounds, 17, 22, 39 and 51, were examined in a neurite outgrowth cell assay. All four compounds were found to exert a high agonistic effect as deduced from their capacity to induce neurite elongation in neuronal cells, as does angiotensin II.

Synthesis of a new class of druglike angiotensin II C-terminal mimics with affinity for the AT2 receptor.

Four tripeptides corresponding to the C-terminal region of angiotensin II were synthesized. One of these peptides (Ac-His-Pro-Ile) showed moderate binding affinity for the AT2 receptor. Two aromatic histidine-related scaffolds were synthesized and introduced in the tripeptides to give eight new peptidomimetic structures. Three of the new peptide-derived druglike molecules exhibited selective, nanomolar affinity for the AT2 receptor. These ligands may become lead compounds in the future development of novel classes of selective AT2 receptor agonists.

Selective angiotensin II AT2 receptor agonists: arylbenzylimidazole structure-activity relationships.

Structural alterations in the 2- and 5-positions of the first drug-like selective angiotensin II AT2 receptor agonist (1) have been performed. The imidazole ring system was proven to be a strong determinant for the AT2 selectivity, and with few exceptions all variations gave good AT2 receptor affinities and with retained high AT2/AT1 selectivities. On the contrary to the findings with AT1 receptor agonists, the impact of structural modifications in the 5-position of the AT2 selective compounds were less pronounced regarding activation of the AT2 receptor. The butyloxyphenyl (56) and the propylthienyl (50) derivatives were found to exert a high agonistic effect as deduced from their capacity to induce neurite elongation in neuronal cells, as does angiotensin II.

Design, synthesis, and incorporation of a beta-turn mimetic in angiotensin II forming novel pseudopeptides with affinity for AT1 and AT2 receptors.

A benzodiazepine-based beta-turn mimetic has been designed, synthesized, and incorporated into angiotensin II. Comparison of the mimetic with beta-turns in crystallized proteins showed that it most closely resembles a type II beta-turn. The compounds exhibited high to moderate binding affinity for the AT2 receptor, and one also displayed high affinity for the AT1 receptor. Molecular modeling showed that the high-affinity compounds could be incorporated into a previously derived model of AT2 receptor ligands.

Short pseudopeptides containing turn scaffolds with high AT2 receptor affinity.

Two pentapeptides, Ac-Tyr-Ile-His-Pro-Phe/Ile, were synthesized and shown to have angiotensin II AT2 receptor affinity and agonistic activity. Based on these peptides, a new series of 13 pseudopeptides was synthesized via introduction of five different turn scaffolds replacing the Tyr-Ile amino acid residues. Pharmacological evaluation disclosed subnanomolar affinities for some of these compounds at the AT2 receptor. Substitution of Phe by Ile in this series of ligands enhanced the AT2 receptor affinity of all compounds. These results suggest that the C-terminal amino acid residues can be elaborated on to enhance the AT2 receptor affinity in truncated Ang II analogues.

Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SP1-7 in the rat spinal cord.

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the mu-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP1-7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP1-7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP1-7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar9, Met(O2)11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP1-7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP1-7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP1-7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPgammaS assay. This suggests that the identified SP1-7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors.

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.

New selective AT2 receptor ligands encompassing a gamma-turn mimetic replacing the amino acid residues 4-5 of angiotensin II act as agonists.

New benzodiazepine-based gamma-turn mimetics with one or two amino acid side chains were synthesized. The gamma-turn mimetics were incorporated into angiotensin II (Ang II) replacing the Val(3)-Tyr(4)-Ile(5) or Tyr(4)-Ile(5) peptide segments. All of the resulting pseudopeptides displayed high AT(2)/AT(1) receptor selectivity and exhibited AT(2) receptor affinity in the low nanomolar range. Molecular modeling was used to investigate whether the compounds binding to the AT(2) receptor could position important structural elements in common areas. A previously described benzodiazepine-based gamma-turn mimetic with high affinity for the AT(2) receptor was also included in the modeling. It was found that the molecules, although being structurally quite different, could adopt the same binding mode/interaction pattern in agreement with the model hypothesis. The pseudopeptides selected for agonist studies were shown to act as AT(2) receptor agonists being able to induce outgrowth of neurite cells, stimulate p42/p44(mapk), and suppress proliferation of PC12 cells.

First reported nonpeptide AT1 receptor agonist (L-162,313) acts as an AT2 receptor agonist in vivo.

In this investigation, it is demonstrated that the first nonpeptide AT(1) receptor agonist L-162,313 (1), disclosed in 1994, also acts as an agonist at the AT(2) receptor. In anesthetized rats, administration of compound 1 intravenously or locally in the duodenum increased duodenal mucosal alkaline secretion, effects that were sensitive to the selective AT(2) receptor antagonist PD-123,319. The data strongly suggest that 1 is an AT(2) receptor agonist in vivo. To the best of our knowledge, this substance is the first nonpeptidic low-molecular weight compound with an agonistic effect mediated through the AT(2) receptor.

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.