The receptor binding affinity of monocyclic [Ala 3, Xaa 11]endothelin-1 analogs correlates with inducible helix length

Abstract

Endothelin-1, a bicyclic 21-amino acid peptide with disulfide bridges between cysteines 1 and 15 as well as between cysteines 3 and 11, has been reported to be partially helical based on both CD and NMR data. However, this remains an area of controversy with some claims that CD data indicate no α-helical structure (Calas, B.; Harrican, M.-C.; Gulmard, L.; Heitz, F.; Mendre, C.; Chabrier, P. E.; Bennes, R. Peptide Res. 1992, 5, 97) and a recent X-ray crystal structure placing the helix at a different locus (Janes, R. W.; Peapus, D. H.; Wallace, B. A. Structural Biology 1994, 1, 311). The CD studies reported herein indicate that the helical structures reported in NMR studies (e.g. Andersen, N. H.; Chen, C.; Marschner, T. M.; Krystek, Jr. S. R.; Bassolino, D. A. Biochemistry 1992, 31, 1280) apply to pure aqueous media as well. The helix located from Lys 9 to the Cys 15/His 16 juncture is ca 75% populated in pH 4 aqueous buffer. Titration difference CDs reveal that the helix extent increases by one to two residues and that the ‘helical conformation’ is more completely populated upon addition of TFE to 50+ volume-%. Comparison with a more helical analog suggests that the helix propagates towards (but not to the end of) the C-terminus upon fluoroalcohol addition. A variety of monocyclic derivatives of [Nle 7] ET-1 lacking the 3,11-disulfide were evaluated for biological activity and examined by TFE titration difference CD. The series included an Aib 11 and a Pro 11 analog. The helix promoting Aib analog was the most active while the Pro analog exhibited significantly lower vasoconstrictor activity and binding affinity for the ET A receptor. All of the monocyclic analogs become significantly more helical upon addition of fluoroalcohols. The inclusion of a proline residue at position 11 does not preclude helix formation upon addition of fluoroalcohols. Rather, helix formation is relatively easily induced but limited to a 5 residue span. Apparently this is insufficient to orient required side chains optimally for interaction with the ET A receptor. For the 1,15-monocyclic analogs differing only at position 11, ET A binding affinity and vasoconstrictor potency correlate with the facility with which a 7–8 residue long helix can be induced. This presumably includes the segment Glu 10 → Cys 15 in all cases and may represent the full sequence from Lys 9 → His 16. CD studies also reveal that the C-terminal fragment of endothelins is not a fully disordered ‘random coil’ either alone or attached to the endothelin core. A variety of monocyclic derivatives of [Nle 7] ET-1 lacking the 3,11-disulfide were evaluated for biological activity and examined by TFE titration difference CD. For monocyclic analogs differing only at position 11, ET A binding affinity and vasoconstrictor potency correlate with the facility with which a 7–8 residue long helix can be induced. In the least active Pro 11 analog, helix formation is relatively easily induced but limited to a 5 residue span (presumably Glu 10 → Cys 15 without Asp 8 as the N-capping residue.