New peptidic angiotensin II analogues their synthesis and pharmacological characterization
In this study we present the synthesis and pharmacological characterization of different classes of peptidic Angiotensin II (AngII) analogues modified principally at position 8. These analogues can be used to explain the conformational changes, which AngII receptors undergo upon ligand binding. The first class of AngII analogues synthesized were Tyr[superscript 4] -palmitoylated analogues of general structure [Sar[superscript 1] ,Y(O-Pal)[superscript 4] ,X]AngII, where X presented either Gly, Ala, Ile, Leu, Br[subscript 5]Phe (pentabromophenylalanine), D-[bêta]Nal (beta-naphtylalanine) or Phe. Binding studies on bovine adrenocortical membranes showed that these analogues keep their affinity for the AT[subscript 1] receptor despite palmitoylation having pK[subscript i] values around 8. Pharmacologically these analogues resemble their non-palmitoylated equivalents. All the analogues were antagonists of AT[subscript 1] , except [Sar[superscript 1] ,Y(O-Pal)[superscript 4] ]AngII, which exerted full agonistic properties. Furthermore, this study showed that palmitoylated antagonists exerted long-lasting antagonistic effect in vitro , which persisted for several hours, and that these analogues acted specifically and all the observed effects were driven via the AT[subscript 1] receptor. The second class of analogues contained a benzoylphenylalanine-like (Bpa) residue at the position 8 of AngII. Four new ligands, which were potentially photoactive were synthesized, three of which could be directly iodinated within the Bpa-moiety. Photolabeling studies revealed that two of the four tested analogues, [superscript 125]I-[Sar1 ,p ' -I-Bpa8 ]AngII and [superscript 125] I-[Sar[superscript 1] ,p' -NO[subscript 2] -Bpa[superscript 8]]AngII photolabeled the human AT[subscript 2] receptor (hAT[subscript 2] ) but did not photolabel the human AT[subscript 1] receptor (hAT[subscript 1] ). CNBr chemical digestion of the photolabeled hAT[subscript 2] complexes confirmed that the site of attachment of p' -I-Bpa[superscript 8] and p' -NO[subscript 2] -Bpa[superscript 8] corresponded to that of Bpa[superscript 8] previously reported to be situated in the 3rd TMD within M[superscript 128] -M[superscript 138] . Site-directed mutagenesis of hAT[subscript 1] and hAT[subscript 2] , where different mutations were introduced close to the previously reported contact points of Bpa[superscript 8] with the receptor, have shown that selective photolabeling of hAT[subscript 2] by [superscript 125]I-[Sar[superscript 1] ,p' -NO[subscript 2] -Bpa[superscript 8] ]AngII resulted from an increased selectivity of Bpa and p' -NO[subscript 2] -Bpa for M presented in the site of attachment of hAT[subscript 2] . Furthermore, these results indicated that M[superscript 128] and M[superscript 138] from the 3rd TMD of hAT[subscript 2] equally contribute to the binding of position 8 of AngII.