Novel Cyclic Biphalin Analogues by Ruthenium-Catalyzed Ring Closing Metathesis: in Vivo and in Vitro Biological Profile

Abstract

In this work we report the application of the ring-closing metathesis (RCM) to the preparation of two cyclic olefin-bridged analogues of biphalin (Tyr-D-Ala-Gly-Phe-NH-NH ← Phe ← Gly ← D-Ala ← Tyr), using the second generation Grubbs' catalyst. The resulting cis-and trans-cyclic isomers were identified, fully characterized, and tested in vitro at μ (ΜΟR), δ (DOR), and κ (KOR) opioid receptors and in vivo for antinociceptive activity. Both were shown to be full agonists at MOR and potential partial antagonists at DOR, with low potency KOR agonism. They also share a strong antinociceptive effect after intracerebroventricular (i.c.v.) and intravenous (i.v.) administration, higher than that of the cyclic biphalin analogues containing a disulfide bridge between the side chains of two D-Cys or D-Pen residues, previously described by our group.

Developing Cyclic Opioid Analogues: Fluorescently Labeled Bioconjugates of Biphalin

“…We next measured the functional activity of all compounds at the opioid receptors using the same CHO cell lines used above for the binding. We used 35 S-GTPγS coupling, which we’ve used extensively to characterize the functional activity of our compounds. , …”

“…We used 35 S-GTPγS coupling, which we've used extensively to characterize the functional activity of our compounds. 20,25 We first found that all 3 compounds were partial agonists at the DOR (E MAX 47−62%), with modest potencies (EC 50 40− 250 nM, Figure 2). These potencies were all about 2-fold worse than the binding affinities measured in Figure 1, suggesting that these ligands have somewhat poor intrinsic efficacy at the DOR.…”

“…The cells were grown and harvested as above; the assay protocol used was also reported previously in Stefanucci et al , 15 μg of cell membrane protein was combined with concentration curves of experimental or positive control agonist and 0.1 nM 35 S-GTPγS (PerkinElmer) in a 200 μL volume. Reactions were incubated at 30 °C for 1 h and then harvested and data collected as above.…”

Strategies towards safer opioid analgesics—A review of old and upcoming targets

“…This peptide then served as a scaffold for the creation of numerous cyclized and linear derivatives, mainly headed by Adriano Mollica's research group. These daughter compounds also showed potent anti-nociception and, in some cases, reduced side effect liability (e.g., Stefanucci et al, 2019).…”

Central residues of FSHβ (89–97) peptide are not critical for FSHR binding: Implications for peptidomimetic design

“…The peptidomimetic analogs, generated by modifying the parent compounds, are initially assessed through in vitro methods such as radio-receptor and cAMP assays and eventually tested for in vivo activity. Analogs that do not display a remarkable difference in potency as compared to the parent or template molecule through in vitro studies may prove to be significantly more potent based on in vivo analysis (Stefanucci et al, 2019).…”