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.
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.
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.
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.
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.
The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT2 receptor. A Gly scan was performed where each amino acid in Ang II was substituted one-by-one with glycine. The resulting set of peptides was tested for affinity to the AT2 receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT1 receptor (rat liver membranes). Only the substitution of Arg2 reduced affinity to the AT2 receptor considerably (92-fold when compared with Ang II). For the other Gly-substituted analogues the affinity to the AT2 receptor was only moderately affected. To further investigate the role of the Arg2 side chain for receptor binding, we synthesized some N-terminally modified Ang II analogues. According to these studies a positive charge in the N-terminal end of angiotensin III [Ang II (2-8)] is not required for high AT2 receptor affinity but seems to be more important in Ang II. With respect to the AT1 receptor, [Gly2]Ang II and [Gly8]Ang II lacked binding affinity (Ki > 10 microM). Replacement of the Val3 or Ile5 residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr4 or His6, which are known to be very important for AT1 receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.
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.
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