In this study, we explored the candidate components which were expected to play a role in analgesic activity of PAT. From our data we identified ignavine and found this candidate molecule to affect MOR activity in a unique manner and to exert analgesic activity in mice.

A pharmacokinetic study of GJG showed that ignavine and benzoylmesaconine could be detected in the plasma 1 hour after administration (Fig. 1A). Benzoylmesaconine is known to transit to the systemic blood circulation after oral administration of either benzoylmesaconine alone or of PAT-containing prescriptions20,21. The result shown in this study is in line with previous reports. Although benzoylmesaconine has analgesic activity itself15, it does not interact with opioid receptors according to the results of our binding assay (Fig. 2). Therefore, benzoylmesaconine must act to suppress pain independent of opioid signaling. On the other hand, ignavine was detected at the highest concentration among the measured components. This is the first report to show that ignavine could be absorbed into the systemic blood. Ignavine is a diterpene alkaloid, with a different structure from the aconitine alkaloids and is a component of Aconitum tuber22 and PAT23. Ignavine has been reported to be one of the low-toxicity components of Aconitum tuber and to have anti-inflammatory activity in the acetic acid-induced writhing and carrageenan paw edema test without any adverse effects24. However, no target molecules of ignavine have been identified to date. In this study, using the receptor binding assay, we found that ignavine specifically interacts with MOR.

An in vitro functional assay revealed that ignavine exhibited bidirectional activity in a concentration-dependent manner. At high concentrations (>10 μM), ignavine inhibited MOR activity both in a receptor internalization assay and in an intracellular cAMP assay (Figs 3 and 4). It has been reported that high doses of PAT inhibit the antinociception induced by morphine in mice25. The inhibitory activity of ignavine might therefore contribute to this phenotype. In contrast, enhancement of MOR activity was observed at low concentrations of ignavine (~1 μM). In the tail-flick test, 0.1 mg/kg ignavine showed the maximum response, while higher concentrations of ignavine weakened analgesic activity (Fig. 6A). The tail pressure test also showed the same tendency towards dose dependence. These bell-shaped dose-dependency curves observed in vivo were in good accord with the in vitro profiles of ignavine. Since ignavine was shown to enhance the activity of the endogenous MOR ligand, endomorphin-1 (Fig. 4D), it is possible that it may exert analgesic activity by single administration in vivo. Therefore, it can be presumed that the interaction of ignavine with the MOR might be involved in analgesia in these mice.

Ignavine positively modulated the response of DAMGO at a low concentration (~1 μM). In the present study, ignavine accelerated the receptor internalization triggered by DAMGO (Fig. 3) and enhanced the potency of several MOR agonists in the intracellular cAMP assay (Fig. 4) without affecting MOR activity when used alone. These features are comparable to those of the positive allosteric modulator, BMS-986122, reported by Burford et al.26. Although the binding site of BMS-986122 is different from that of ignavine (ignavine binds the MOR orthosterically (see below)), BMS-986122 showed positive modulation of endomorphin-I both in β-arrestin recruitment and in intracellular cAMP assay. Since positive modulators of MOR are expected to maintain the temporal and spatial fidelity of native signaling, they are proposed to avoid on-target side effects of exogenous MOR agonists such as respiratory suppression, constipation, allodynia, tolerance, dependence and withdrawal symptoms27. Our results showed that a single administration of ignavine exerted an analgesic effect in mice. Therefore, ignavine is also likely to be a promising agent for analgesia. A long-term dosing study should be performed to investigate any adverse effects of ignavine.

Regarding the binding site of ignavine, the receptor binding assay showed that ignavine inhibited the binding of [3H]-diprenorphine, which has the same structure as morphine and β-FNA. The docking simulation supports the result of the binding assay. Like morphine and β-FNA, ignavine has a quaternary amine structure. This structure has been reported to interact with Asp147 of the rat MOR (Asp149 in the human MOR as shown in Fig. 7) and this interaction is important for the pharmacological properties of morphine19,28. Taken together with these results, these data strongly suggest that ignavine directly interacts with the MOR at the orthosteric binding site.

Additionally, MD simulation of an ignavine-MOR complex model indicates that orthosteric binding of ignavine induces structural change in a unique manner (Fig. 8). PCA analysis of MD simulation clearly distinguishes the trajectory of conformation change induced by ignavine from those brought about by a representative MOR agonist and antagonist. Since the PC-1 axis divides the three compounds, the difference might reflect the unique character of ignavine. On the PC-2 axis, the plot area of ignavine overlapped that of β-FNA and was separate from that of morphine, indicating that the PC-2 axis might differentiate between agonist and antagonist.

Docking simulation indicates that a single MOR binding pocket does not have enough capacity to accommodate both ignavine and an agonist simultaneously. Since opioid receptors including MOR can form homo- and hetero-dimers in recombinant-expressing cells29, these dimeric opioid receptors would have two binding pockets. The binding assay clearly showed that ignavine is an orthosteric ligand for MOR. Therefore, the excess amount of ignavine should occupy both binding pockets in an MOR dimer as suggested in Fig. 2B (high concentrations of ignavine completely inhibited the binding of [3H]-diprenorphine). However, if the concentration of ignavine was not sufficient, ignavine would not occupy both pockets, leaving one of the pockets vacant. Because the low and high concentrations of ignavine increased and decreased the activity of agonists, respectively, we propose the following hypothesis: when ignavine and another agonistic ligand (such as DAMGO, morphine, or endogenous endomorphin-1) occupy each binding pocket in a homodimer, ignavine might exert positive modulatory activity. On the other hand, ignavine at higher concentration would bind to both binding pockets in a homodimer, exerting antagonistic activity. It has been reported that DOR ligands can enhance DAMGO binding capacity in a μ-δ heteromer30. It is hypothesized that DOR ligands bind the DOR of the heteromer and “allosterically” modulate the activity or binding capacity of the partner receptor. Ignavine is also expected to exert an enhancing effect on activity by a similar mechanism. Smith et al. named the allosteric modulation mentioned above as “off-target allosterism”, meaning allosteric modulation of another binding site (orthosteric or allosteric) on a distinct protein such as a dimeric partner31. Therefore ignavine could be defined as an “off-target allosteric modulator”.

Since PAT can produce analgesia directly or indirectly through the KOR and/or induction of dynorphin release2,10, it is possible that ignavine might contribute to the analgesic activity of PAT by modulating μ-κ heteromer signaling. To test this hypothesis, it will be necessary to evaluate the effect of ignavine on μ-κ heteromer signaling. We plan to carry out the characterization of ignavine’s activity with heteromers including μ-κ heteromer.

In conclusion, we identified ignavine and demonstrated the possibility that ignavine produces analgesia via positive modulation of MOR signaling. Pain management using a positive modulator of opioid receptors is an attractive approach to avoid the serious adverse effects of conventional opioid drugs. It is expected that ignavine could be a candidate compound to evaluate this promising strategy.