Evaluation of Receptor Affinity, Analgesic Activity and Cytotoxicity of a Hybrid Peptide, AWL3020

Abstract

In the present contribution we report design, synthesis and evaluation of receptor affinity, analgesic activity and cytotoxicity of a hybrid peptide, AWL3020. The peptide includes two pharmacophores, one of δ-opioid receptor (δOR) agonists and one of neurokinin-1 receptor (NK1R) antagonists. The design was motivated by the desire to obtain a compound with strong analgesic action and potential additional antiproliferative action. The compound displays high δOR affinity (IC 50 = 29.5 nM). On the other hand, it has only poor affinity for the NK1R (IC 50 = 70.28 μM). The substance shows good analgesic action which is however weaker than that of morphine. Regarding the effect on proliferation, the compound exhibits no pro-proliferative action in the assayed range. In higher concentrations, it has also cytotoxic activity. This effect is however not selective. The strongest effect of AWL3020 was found for melanoma MeW164 cell line (EC 50 = 46.27 μM in reduction of cell numbers after a few days of incubation; EC 50 = 37.78 μM in MTT assay).

In Vivo, In Vitro and In Silico Studies of the Hybrid Compound AA3266, an Opioid Agonist/NK1R Antagonist with Selective Cytotoxicity

“…Finally, we determined the impact of AA3266 on the Ki-67 protein expression in three cell lines (2 cancer and 1 normal one, Figure 5 D). The Ki-67 protein is a marker of proliferating cells, since it is expressed during all phases of the cell cycle (G 1 , S, G 2 , M), but it is undetectable in the resting state (G 0 phase) [ 44 ]. The Ki-67 proliferation index is the percentage of cells that are found to express this protein (by immunostaining).…”

“…So far, the idea to combine opioids and NK1R antagonists to achieve auxiliary anticancer activity has been considered only a few times in the literature. These include our previous contribution on AA3266 [ 29 ] and the above mentioned AWL3020 [ 44 ]. Recently Dyniewicz et al reported a series of hybrid compounds in which various opioid sequences were appended (at the C-terminus) with the 3,5-bis(trifluoromethyl)phenyl moiety characteristic for neurokinin-1 antagonists [ 17 ].…”

“…The rats (adult male Wistar, weighing 200–250 g, 10 animals for each group) were prepared for the intrathecal administration (i.t.) by a method described previously in detail [ 44 ]. All housing and experiments were conducted in accordance with the Polish Act of 21 January 2005 on Experiments on Animals (Journal of Laws No 33 of the Republic of Poland, item 289 as amended).…”

“…The antinociceptive activity of AA3266 and morphine (MF) was measured by a tail-flick test, as described previously [ 44 ]. In brief, the compounds were dissolved in a small amount of DMSO and the saline was added to obtain a required stock concentration.…”

“…Data related to yet another endpoint, that is influence on the number of cells after 4 or 7 days incubation, come from the previous report [ 29 ]. The tests were performed following the procedures previously described [ 44 ]. In brief, the cells were incubated in the presence of three concentrations of AA3266 (25 µM, 50 µM and 100 µM, in separate wells/dishes).…”