Design, Synthesis, and Characterization of the Macrocyclic Tetrapeptide cyclo[Pro-Sar-Phe-d-Phe]: A Mixed Opioid Receptor Agonist–Antagonist Following Oral Administration

Abstract

Substance abuse remains a serious public health crisis, affecting millions of people worldwide. Macrocyclic tetrapeptides like CJ-15,208 demonstrate opioid activity shown to attenuate the rewarding effects of cocaine in conditioned place preference assays in mice, making them promising lead compounds for treating substance abuse. In the present study, we report the rational design, synthesis, conformational analysis, and continued pharmacological evaluation of the novel macrocyclic tetrapeptide cyclo[Pro-Sar-Phe-D-Phe] to further explore this unique molecular scaffold. This peptide was rationally designed based on X-ray and NMR structures of related macrocyclic tetrapeptides. Following synthesis, its solution-phase conformations were determined by NMR and molecular modeling. The peptide adopted multiple conformations in polar solvents, but a single conformation in chloroform that is stabilized by intramolecular hydrogen bonding. The peptide is orally bioavailable, producing antinociception and antagonism of kappa opioid receptor (KOR) stimulation following oral administration in a mouse 55 °C warm-water tail-withdrawal assay. Notably, cyclo[Pro-Sar-Phe-D-Phe] blocked both stress-and drug-induced reinstatement of cocaine and morphine conditioned place preference in mice following oral administration, and displayed a decreased side-effect profile compared to morphine. Thus, cyclo[Pro-Sar-Phe-D-Phe] is a promising lead compound for the treatment of substance abuse.

Phenylalanine Stereoisomers of CJ-15,208 and [d-Trp]CJ-15,208 Exhibit Distinctly Different Opioid Activity Profiles

“…Unlike the parent compounds, all of the stereoisomers except 2 demonstrated antinociception also mediated in part by DOR. This is in contrast to the in vitro results, where the stereoisomers all lacked affinity for DOR in radioligand binding assays; such discrepancies between in vitro and in vivo activity have been found for other CJ-15,208 analogs [ 26 , 27 , 28 ]. d -Phenylalanine in position 1 appears to favor DOR agonist activity; of the four isomers where DOR contributes to the observed antinociception only 5 does not contain d -Phe 1 , and DOR appears to contribute less to the antinociception of this isomer than for the other isomers ( Figure 4 B).…”

Multifunctional opioid receptor agonism and antagonism by a novel macrocyclic tetrapeptide prevents reinstatement of morphine‐seeking behaviour

“…The present results provide evidence that cyclo [Pro‐Sar‐Phe‐ d ‐Phe] is functioning through both μ‐ and κ‐opioid receptors, but how this activity is produced remains uncertain. Initial studies found that cyclo [Pro‐Sar‐Phe‐ d ‐Phe] showed poor affinity for opioid receptors in traditional radioligand binding assays (Ferracane et al, 2020). Molecular modelling in that study suggested that cyclo [Pro‐Sar‐Phe‐ d ‐Phe] adopts a variety of conformations depending on the polarity of its environment, with different conformations potentially exhibiting distinct mechanisms of action that could possibly account for the observed range of activity.…”

“…CJ‐15,208 exhibits antinociception mediated by μ‐ and κ‐opioid receptor agonism, followed by κ‐opioid receptor antagonism of finite duration (lasting hours, rather than days) that prevented stress‐induced reinstatement of extinguished cocaine‐conditioned place preference (CPP) (Ross, Reilley, Murray, Aldrich, & McLaughlin, 2012). To explore structure–activity relationships of CJ‐15,208 and enhance opioid activity, the novel peptide analogue cyclo [Pro‐Sar‐Phe‐ d ‐Phe] (Figure 1) was designed and synthesized (Ferracane et al, 2020). We hypothesized that cyclo [Pro‐Sar‐Phe‐ d ‐Phe] would demonstrate similar multifunctional opioid receptor activity, producing antinociception and preventing reinstatement of extinguished morphine‐CPP.…”

Fluorescence-Based Sensing of Pesticides Using Supramolecular Chemistry

“…Synthetic macrocycles have significant advantages, including the ability to rationally design a structure for targeted binding of particular pesticide analytes (Ferracane et al, 2020). Drawbacks to such macrocycles include the general need for complex, multistep syntheses, which are often plagued by low yields, particularly in the cyclization step (Mortensen et al, 2019).…”