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

Abstract

AA3266 is a hybrid compound consisting of opioid receptor agonist and neurokinin-1 receptor (NK1R) antagonist pharmacophores. It was designed with the desire to have an analgesic molecule with improved properties and auxiliary anticancer activity. Previously, the compound was found to exhibit high affinity for μ- and δ-opioid receptors, while moderate binding to NK1R. In the presented contribution, we report on a deeper investigation of this hybrid. In vivo, we have established that AA3266 has potent antinociceptive activity in acute pain model, comparable to that of morphine. Desirably, with prolonged administration, our hybrid induces less tolerance than morphine does. AA3266, contrary to morphine, does not cause development of constipation, which is one of the main undesirable effects of opioid use. In vitro, we have confirmed relatively strong cytotoxic activity on a few selected cancer cell lines, similar to or greater than that of a reference NK1R antagonist, aprepitant. Importantly, our compound affects normal cells to smaller extent what makes our compound more selective against cancer cells. In silico methods, including molecular docking, molecular dynamics simulations and fragment molecular orbital calculations, have been used to investigate the interactions of AA3266 with MOR and NK1R. Insights from these will guide structural optimization of opioid/antitachykinin hybrid compounds.

Keywords: NK1 receptor antagonists; cytotoxicity; fragment molecular orbitals; melanoma; molecular dynamics; morphine; multitarget ligands; opioid; pain; tolerance.

Figure 1

Structure of AA3266.

Figure 2

(A) Time and dose-dependence of antinociceptive effect (i.t. administration, tail-flick test) of AA3266 compared to positive control (morphine 12 nmol/kg) and negative control (NaCl). Thin bars show standard error of the mean. The asterisks denote statistical significance of the difference between the value found for a particular AA3266 concentration and the positive control (* p  ≤  0.05, ** p  ≤  0.01). The statistical analysis used is the Fisher’s Least Significant Difference test (planned comparisons; not corrected for multiple comparisons) at significance level α  =  0.05. (B) Area under the antinociceptive response curve.

Figure 3

(A) Time and dose-dependence of antinociceptive effect (i.t. administration, tail-flick test) of AA3266 (30 nmol/kg) compared to morphine (20 nmol/kg) on Day 1 and on Day 6 after prolonged administration. The results for the negative control (NaCl) were no different than zero in all the time points and so they are omitted from the plot for clarity. Thin bars show standard error of the mean. The results of statistical testing are given in a tabular form below the plots. The asterisks denote statistical significance of the difference between the compared values (* p  ≤  0.05, ** p  ≤  0.01, *** p  ≤  0.001, ns—not significant). The statistical analysis used is the Fisher’s Least Significant Difference test (planned comparisons; not corrected for multiple comparisons) at significance level α  =  0.05. (B) Area under the antinociceptive response curve (prolonged administration).

Figure 4

The influence of the prolonged administration of AA3266, MF and NaCl on (A) the cumulative faecal index, (B) food intake, (C) water consumption and (D) faecal water content. The line in the (A) subplot is a curve of linear relationship between the index and the experiment day. The dotted lines represent 95% confidence intervals of the linear curve. Whether the regression slopes are different for the groups, was tested with the extra sum-of-squares F test. In the subplots (B–D), the bars represent the mean over the 6 days with the standard error of the mean. For these data (food intake, water consumption and faecal water content), according to the one-way analysis of variance (ANOVA), there is no significant difference between the means (α = 0.05).

Figure 5

The cellular pharmacological effects of AA3266 in selected cell lines. (A) Number of cells counted in haemocytometer (data taken from Ref. [29]) (B) Results of the MTT assay. (C) Effect on the extent of the colony formation. (D) Effect on the expression of the Ki67 protein. The data are expressed as percentage of the values found for the control. Bar colouring corresponds to the concentration of AA3266 (red—100 µM; orange—50 µM; yellow—25 µM; blue—0 µM, control). Cell lines designations given in text. Blue thin bar shows standard deviation. The data come from two independent experiments done in triplicate. The asterisks denote statistical significance of the difference between the given value found for the given concentration and the control (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p < 0.0001). The statistical analysis used is the one-way ANOVA with post-hoc Dunnett test at significance level α = 0.05.

Figure 6

Representative structures for the clusters from MD simulations of AA3266 in the µOR binding site. Focus on the opioid part. (A) MOR-1 cluster, (B) MOR-2 cluster, (C) MOR-3 cluster, (D) MOR-4 cluster. Only a few receptor (yellow) side chains and helices are shown for clarity. The surface covers a few side chains from TM3, ECL2, ECL3 and TM5 important for binding. The red number is the cluster population.

Figure 7

Representative structures for the clusters from MD simulations of AA3266 in the MOR binding site. Focus on the antitachykinin part. (A) MOR-a cluster, (B) MOR-b cluster, (C) MOR-c cluster, (D) MOR-d cluster, (E) MOR-e cluster, (F) MOR-f cluster, (G) MOR-g cluster, (H) MOR-h cluster, (I) MOR-i cluster. Receptor is represented as yellow cylinders (transmembrane helices, TM). The ligand is shown as green sticks. The red number is the cluster population.

Figure 8

Representative structures for the clusters from MD simulations of AA3266 in the NK1R binding site. Focus on the -NH-NH<-Z-D-Trp part. (A) NK1-a cluster, (B) NK1-b cluster, (C) NK1-c cluster, (D) NK1-d cluster. Only a few receptor (orange) side chains and helices are shown for clarity. The blue number is the cluster population.

Figure 9

Representative structures for the clusters from MD simulations of AA3266 in the NK1R binding site. Focus on the Tyr-d-Ala-Gly-Phe-fragment. (A) NK1-1 cluster, (B) NK1-2 cluster, (C) NK1-3 cluster, (D) NK1-4 cluster, (E) NK1-5 cluster, (F) NK1-6 cluster, (G) NK1-7 cluster, (H) NK1-8 cluster, (I) NK1-9 cluster, (J) NK1-10 cluster. Receptor is represented as orange cylinders (transmembrane helices, TM). The ligand is shown as green sticks. The blue number is the cluster population.

Figure 10

Superposition of the AA3266 conformations found in simulations with MOR and NK1R (A) Focus on the Tyr-d-Ala-Gly-Phe-fragment, (B) Focus on the -NH-NH-Z-d-Trp fragment. Green sticks represent the structures from simulations with MOR, and pink sticks represent the structures from simulations with NK1R. The reference structures are MOR-1 for (A) and NK1-a for (B) as the most populated clusters at the respective receptors.