Opiate Aromatic Pharmacophore Structure−Activity Relationships in CTAP Analogues Determined by Topographical Bias, Two-Dimensional NMR, and Biological Activity Assays

Abstract

Topographically constrained analogues of the highly mu-opioid-receptor-selective antagonist CTAP (H-D-Phe-c[Cys-Tyr-D-Trp-Arg-Thr-Pen]-Thr-NH(2), 1) were prepared by solid-phase peptide synthesis. Replacement of the D-Phe residue with conformationally biased beta-methyl derivatives of phenylalanine or tryptophan (2R,3R; 2R,3S; 2S,3R; 2S,3S) yielded peptides that displayed widely varying types of biological activities. In an effort to correlate the observed biological activities of these analogues with their structures, two-dimensional (1)H NMR and molecular modeling was performed. Unlike the parent (1), which is essentially a pure mu antagonist with weak delta agonist activities in the MVD bioassay, the diastereomeric beta-MePhe(1)-containing peptides exhibited simultaneous delta agonism and mu antagonism by the (2R,3R)-containing isomer 2; mu antagonism by the (2R,3S)-containing isomer 3; weak mu agonism by the (2S,3R)-containing isomer 4; and delta agonism by the (2S,3S)-containing isomer 5. Incorporation of beta-MeTrp isomers into position 1 led to peptides that were mu antagonists (2R,3R), 8; (2R,3S), 9, or essentially inactive (<10%) in the MVD and GPI assays (2S,3R), 10; (2S,3S), 11. Interestingly, in vivo antinociceptive activity was predicted by neither MVD nor GPI bioactivity. When D-Trp was incorporated in position 1, the result (7) is a partial, yet relatively potent mu agonist which also displayed weak delta agonist activity. Molecular modeling based on 2D NMR revealed that low energy conformers of peptides with similar biological activities had similar aromatic pharmacophore orientations and interaromatic distances. Peptides that exhibit mu antagonism have interaromatic distances of 7.0-7.9 A and have their amino terminal aromatic moiety pointing in a direction opposite to the direction that the amino terminus points. Peptides with delta opioid activity displayed an interaromatic distance of <7 A and had their amino terminal aromatic moiety pointing in the same direction as the amino terminus.

The effect of β‐methylation on the conformation of α, β‐dehydrophenylalanine: a DFT study

“…Among the known dehydroamino acids, dehydrophenylalanine is the most commonly used to modify bioactive peptides because it is the most stable and relatively easy to obtain. On the other hand, among topographically constrained amino acids, β‐methylphenylalanine is very often used as the phenyl ring commonly serves as a pharmacophore45–51. This is an important feature as the advances in structure–activity relationships have shown that restrictions not only of the conformations (ϕ,ψ torsion angles) but also of topographies (χ torsion angles) of a single amino acid residues can affect the bioactivities of peptides52.…”

Somatostatin analogs

“…The elements of opioid peptides hypothesized to be responsible for their activities are a pair of aromatic amino acid residues, one in the N‐terminal position and another in either position 3 or 4 (110). Replacement of the d ‐Phe residue in CTAP with conformationally biased β‐methyl derivatives of Phe or Trp resulted in topographically constrained analogs that displayed widely varying types of biological activities (111). On the basis of the low‐energy conformations of these analogs, aromatic pharmacophore arrangements were discovered for opioid receptor activity.…”

Preparative separation and identification of derivatized β-methylphenylalanine enantiomers by chiral SFC, HPLC and NMR for development of new peptide ligand mimetics in drug discovery

“…Unnatural amino acids are often employed to affect these constraints. In particular, stereospecific ␤-methyl substitution is a widely used technique for constraining aromatic side-chains [4][5][6]. Additionally, it is most advantageous to examine only stereochemically pure substances, thereby precluding ambiguous physicochemical and biological data that may arise from the testing of enantiomeric/diastereomeric mixtures.…”

“…Nuclear magnetic resonance (NMR) Spectroscopy was performed on a Bruker Avance 300 MHz spectrometer (Billerica, MA, USA) equipped with a 5 mm auto-switchable quad nucleus (QNP) probe at 25 • C. NMR data were collected in DMSO-d 6 and standard Bruker zg30 and noesyst NMR pulse programs were used for the 1 H and NOESY experiments, respectively. Chemical shifts are referenced to tetramethylsilane (TMS).…”

“…Amino acids, in particular, unnatural amino acids, have been recognized as major tools for the preparation of peptide ligand mimetics with both enhanced biological activity and proteolytic resistance. These molecules represent a nearly infinite array of diverse building blocks for the development of new pharmacological leads [1][2][3][4][5][6]. However, amino acids are also chiral molecules.…”