Computational exploration of Eucommia ulmoides flavonoids as potential RANKL inhibitors via molecular docking and dynamics simulations

Abstract

Osteoporosis, characterized by excessive osteoclast activation, is mediated through the RANKL/RANK/OPG signaling axis. While flavonoids from Eucommia ulmoides (EU) have demonstrated anti-osteoclastogenic activity, their atomic-level mechanisms remain elusive. Here, we investigated six EU-derived flavonoids (cyrtominetin, quercetin, syringetin, genistein, ombuin, and kaempferol) targeting RANKL using integrated computational approaches. Molecular docking revealed strong binding affinities (Total_Score > 4.0) for all compounds, with cyrtominetin exhibiting the highest affinity (-50.205 kJ/mol via MM-PBSA), primarily through hydrogen bonds with Gly178, His180, Lys181, and Asn295. Moreover, most flavonoids interacted with RANKL by forming strong hydrogen bonds with Gly178 and Asn295, exhibiting higher binding affinity that was identified as essential for the activity. All-atom molecular dynamics simulations (100 ns) confirmed complex stability, demonstrating: low RMSD fluctuations (< 4.0 Å) and compact Rg values (16.0-17.0 Å). Notably, binding free energy decomposition identified both electrostatic and van der Waals contributions as critical for stabilization. These results identify cyrtominetin as a promising lead compound for RANKL inhibition, providing structural insights for designing flavonoid-based therapeutics against osteoporosis.

Keywords: Flavonoids; Molecular docking; Molecular dynamics; Osteoporosis; RANKL.

Fig. 1

(A) Overall structure of RANKL/OPG complex. (B) The Ramachandran plot of RANKL. Red, yellow, light yellow, and white are the most favorable, additional allowed, generously allowed, and disallowed regions in conformation, respectively.

Fig. 2

Overall view of RANKL/OPG binding interface. (A) Sites I and II in the binding interface. The OPG is shown in surface colored with violet. The RANKL is colored with blue. (B) An open-book view of the contact residues in binding sites I and II. The contact residues in binding sites I and II are colored with grey and orange, respectively.

Fig. 3

Binding mode of flavonoids in the active site of RANKL. (A) Cyrtominetin (orange), (B) Quercetin (blue), (C) Syringetin (cyan), (D) Genistein (purple), (E) Ombuin (yellow), (F) Kaempferol (green), and (G) control group (pink). Protein residues are shown in pale gold sticks.

Fig. 4

Time evolution RMSD trajectories of the RANKL-flavonoids complexes over 100 ns all-atom MD simulation. (A) The RMSD of backbone Cα-atoms. (B) The relative frequency distribution of RMSD.

Fig. 5

Analysis of RMSF trajectories versus residue number of the RANKL-flavonoids complexes over 100 ns all-atom MD simulation.

Fig. 6

Time evolution Rg trajectories of the RANKL-flavonoids complexes over 100 ns all-atom MD simulation. (A) The Rg of the RANKL-flavonoids complexes. (B) The relative frequency distribution of Rg.

Fig. 7

Time evolution SASA trajectories of the RANKL-flavonoids complexes over 100 ns all-atom MD simulation. (A) The SASA of the RANKL-flavonoids complexes. (B) The relative frequency distribution of SASA.

Fig. 8

The sidechain-sidechain contact maps for RANKL monomer of all flavonoids simulation systems. (A) Cyrtominetin, (B) Quercetin, (C) Syringetin, (D) Genistein (E) Ombuin (F) Kaempferol (G) control group and (H) apo. The distance is given in nm and indicated by the color code.

Fig. 9

Time evolution plot of hydrogen bond interaction between the RANKL and flavonoids over 100 ns all-atom MD simulation.

Fig. 10

FEL of RANKL-flavonoids complexes. (A) Cyrtominetin, (B) Quercetin, (C) Syringetin, (D) Genistein, (E) Ombuin, (F) Kaempferol, (G) control group, and (H) apo. The free energy is given in kcal/mol and indicated by the color code, from lower to higher energy in the right panel.

Fig. 11

Time evolution secondary structures of the RANKL-flavonoids complexes over 100 ns all-atom MD simulation. (A) Cyrtominetin, (B) Quercetin, (C) Syringetin, (D) Genistein, (E) Ombuin, (F) Kaempferol, (G) control group, and (H) apo.

Fig. 12

The residue decomposition plot (MM-PBSA) representing the binding energy contribution of the residues energetically stabilizing the flavonoids at binding pocket. (A) Cyrtominetin, (B) Quercetin, (C) Syringetin, (D) Genistein, (E) Ombuin, (F) Kaempferol and (G) control group.