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. 2021 Dec 28;27(1):151.
doi: 10.3390/molecules27010151.

Synthesis, Pharmacological Evaluation, and Computational Studies of Cyclic Opioid Peptidomimetics Containing β3-Lysine

Karol Wtorek  1 , Piotr F J Lipiński  2 , Anna Adamska-Bartłomiejczyk  1 , Justyna Piekielna-Ciesielska  1 , Jarosław Sukiennik  3 , Alicja Kluczyk  4 , Anna Janecka  1
Affiliations

Synthesis, Pharmacological Evaluation, and Computational Studies of Cyclic Opioid Peptidomimetics Containing β3-Lysine

Karol Wtorek et al. Molecules. .

Abstract

Our formerly described pentapeptide opioid analog Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (designated RP-170), showing high affinity for the mu (MOR) and kappa (KOR) opioid receptors, was much more stable than endomorphine-2 (EM-2) in the rat brain homogenate and displayed remarkable antinociceptive activity after central (intracerebroventricular) and peripheral (intravenous ) administration. In this report, we describe the further modification of this analog, which includes the incorporation of a β3-amino acid, (R)- and (S)-β3-Lys, instead of D-Lys in position 2. The influence of such replacement on the biological properties of the obtained analogs, Tyr-c[(R)-β3-Lys-Phe-Phe-Asp]NH2 (RP-171) and Tyr-c[(S)-β3-Lys-Phe-Phe-Asp]NH2, (RP-172), was investigated in vitro. Receptor radiolabeled displacement and functional calcium mobilization assays were performed to measure binding affinity and receptor activation of the new analogs. The obtained data revealed that only one of the diastereoisomeric peptides, RP-171, was able to selectively bind and activate MOR. Molecular modeling (docking and molecular dynamics (MD) simulations) suggests that both compounds should be accommodated in the MOR binding site. However, in the case of the inactive isomer RP-172, fewer hydrogen bonds, as well as instability of the canonical ionic interaction to Asp147, could explain its very low MOR affinity.

Keywords: functional assay; opioid receptors; peptide synthesis; receptor binding studies; β-amino acids.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Scheme 1
Scheme 1
Synthesis of Fmoc-(R)- and (S)-β3-Lys(Mtt).
Figure 1
Figure 1
Chemical structure of RP-171 and RP-172.
Figure 2
Figure 2
LC-MSn analysis of peptides RP-171 and RP-172. Extracted ion chromatogram (XIC) showing two peaks for the mixture of peptides (top panel): MS panels, all spectra recorded for the retention time of indicated peaks; MS spectra, MS/MS spectra for the precursor ions m/z 700.34, and MS/MS/MS spectra for the precursor ions m/z 586.30.
Figure 3
Figure 3
The lowest-lying conformers of RP-171 and RP-172 (at the B3LYP/6-31G(d,p) in the gas phase. Green dots show intramolecular hydrogen bonding.
Figure 4
Figure 4
(A) Binding mode of RP-171 (white sticks) in the MOR binding site, as found at t = 100.0 ns of the MD production run. The receptor is shown in a simplified manner, with only selected helices (ribbons) and side chains (thin sticks) shown. Display of nonpolar hydrogens is suppressed for clarity. (B) Diagram showing interactions between RP-171 and the MOR binding site (interaction types colored according to the legend). (C–F) Time evolutions of selected distances associated with protein–ligand interactions during the MD simulations.
Figure 5
Figure 5
(A) Binding mode of RP-172 (white sticks) in the MOR binding site, as found at t = 100.0 ns of the MD production run. The receptor is shown in a simplified manner, with only selected helices (ribbons) and side chains (thin sticks) shown. Display of nonpolar hydrogens is suppressed for clarity. (B) Diagram showing interactions between RP-172 and the MOR binding site (interaction types colored according to the legend). (C–F) Time evolutions of selected distances associated with protein–ligand interactions during the MD simulations.
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