In Vitro and In Silico Studies of Neolignans from Magnolia grandiflora L. Seeds against Human Cannabinoids and Opioid Receptors

Abstract

Magnolia grandiflora L. (Magnoliaceae) is a plant of considerable medicinal significance; its flowers and seeds have been used in various traditional remedies. Radioligand binding assays of n-hexane seeds extract showed displacement of radioligand for cannabinoid (CB1 and CB2) and opioid δ (delta), κ (kappa), and µ (mu) receptors. Bioactivity-guided fractionation afforded 4-O-methylhonokiol (1), magnolol (2), and honokiol (3), which showed higher binding to cannabinoid rather than opioid receptors in radioligand binding assays. Compounds 1-3, together with the dihydro analog of 2 (4), displayed selective affinity towards CB2R (K_i values of 0.29, 1.4, 1.94, and 0.99 μM, respectively), compared to CB1R (K_i 3.85, 17.82, 14.55, and 19.08 μM, respectively). An equal mixture of 2 and 3 (1:1 ratio) showed additive displacement activity towards the tested receptors compared to either 2 or 3 alone, which in turn provides an explanation for the strong displacement activity of the n-hexane extract. Due to the unavailability of an NMR or X-ray crystal structure of bound neolignans with the CB1 and CB2 receptors, a docking study was performed to predict ligand-protein interactions at a molecular level and to delineate structure-activity relationships (SAR) of the neolignan analogs with the CB1 and CB2 receptors. The putative binding modes of neolignans 1-3 and previously reported related analogs (4, 4a, 5, 5a, 6, 6a, and 6b) into the active site of the CB1 and CB2 receptors were assessed for the first time via molecular docking and binding free-energy (∆G) calculations. The docking and ∆G results revealed the importance of a hydroxyl moiety in the molecules that forms strong H-bonding with Ser383 and Ser285 within CB1R and CB2R, respectively. The impact of a shift from a hydroxyl to the methoxy group on experimental binding affinity to CB1R versus CB2R was explained through ∆G data and the orientation of the alkyl chain within the CB1R. This comprehensive SAR, influenced by the computational study and the observed in vitro displacement binding affinities, has indicated the potential of magnolia neolignans for developing new CB agonists for potential use as analgesics, anti-inflammatory agents, or anxiolytics.

Keywords: 4-O-methylhonokiol; Magnolia grandiflora; cannabinoid; honokiol; magnolol; molecular docking; opioid; tetrahydromagnolol.

Figure 1

Compounds isolated/identified (1–4) from M. grandiflora seed and related (4a–6b) compounds [29] were used for molecular docking studies.

Figure 2

The radioligand displacement curves were obtained for compounds (1–4) for the CB1R. CP55,940 was used as a positive control.

Figure 3

The radioligand displacement curves were obtained for compounds (1–4) for the CB2R. CP55,940 was used as a positive control.

Figure 4

The radioligand displacement curves were obtained for compounds (2 and 3) for the µ opioid receptor. Naloxone hydrochloride was used as a positive control.

Figure 5

2D interaction diagrams of (A) 4 and (B) 4a along with the 3D overlaid representations of (C) 4 (carbon in orange) with 4a (carbon in plum) against the CB1R and (D) 4 (carbon in orange) with 4a (carbon in plum) against the CB2R. The key residues are shown in the ball and stick model (carbon in grey).

Figure 6

2D interaction diagrams of (A) 5 and (B) 5a along with the 3D overlaid representations of (C) 5 (carbon in yellow) with 5a (carbon in magenta) against the CB1R and (D) 5 (carbon in yellow) with 5a (carbon in magenta) against the CB2R. The key residues are shown in the ball and stick model (carbon in grey).

Figure 7

2D interaction diagrams of (A) 6 and (B) 6a, along with the 3D overlaid representations of (C) 6 (carbon in orange) with 6a (carbon in plum) against the CB1R and (D) 6 (carbon in orange) with 6a (carbon in plum) and 6b (carbon in yellow) against the CB2R. The key residues are shown in the ball and stick model (carbon in grey).

Figure 8

2D interaction diagrams of (A) 1 and (B) 3 along with the 3D overlaid representations of (C) 1 (carbon in blue) with 3 (carbon in orange) against the CB1R and (D) 1 (carbon in blue) with 3 (carbon in orange) against the CB2R. The key residues are shown in the ball and stick model (carbon in grey).