Molecular determinants of magnolol targeting both RXRα and PPARγ

Abstract

Nuclear receptors retinoic X receptor α (RXRα) and peroxisome proliferator activated receptor γ (PPARγ) function potently in metabolic diseases, and are both important targets for anti-diabetic drugs. Coactivation of RXRα and PPARγ is believed to synergize their effects on glucose and lipid metabolism. Here we identify the natural product magnolol as a dual agonist targeting both RXRα and PPARγ. Magnolol was previously reported to enhance adipocyte differentiation and glucose uptake, ameliorate blood glucose level and prevent development of diabetic nephropathy. Although magnolol can bind and activate both of these two nuclear receptors, the transactivation assays indicate that magnolol exhibits biased agonism on the transcription of PPAR-response element (PPRE) mediated by RXRα:PPARγ heterodimer, instead of RXR-response element (RXRE) mediated by RXRα:RXRα homodimer. To further elucidate the molecular basis for magnolol agonism, we determine both the co-crystal structures of RXRα and PPARγ ligand-binding domains (LBDs) with magnolol. Structural analyses reveal that magnolol adopts its two 5-allyl-2-hydroxyphenyl moieties occupying the acidic and hydrophobic cavities of RXRα L-shaped ligand-binding pocket, respectively. While, two magnolol molecules cooperatively accommodate into PPARγ Y-shaped ligand-binding pocket. Based on these two complex structures, the key interactions for magnolol activating RXRα and PPARγ are determined. As the first report on the dual agonist targeting RXRα and PPARγ with receptor-ligand complex structures, our results are thus expected to help inspect the potential pharmacological mechanism for magnolol functions, and supply useful hits for nuclear receptor multi-target ligand design.

Figure 1. Magnolol as a dual agonist of RXRα and PPARγ.

(A) Chemical structure of magnolol. (B–C) Magnolol dose-dependently activated the transcription of GAL4DBD-RXRαLBD (B) and GAL4DBD-PPARγLBD (C) in HEK-293T cells, which could be suppressed by RXRα antagonist HX531 and PPARγ antagonist GW9662, respectively. RXRα agonist 9-cis-retinoic acid (9cRA) and PPARγ agonist Rosiglitazone were used as positive controls. (D–E) Magnolol dose-dependently bound to RXRαLBD (D) and PPARγLBD (E) in SPR technology based assays. (F–G) Magnolol dose-dependently enhanced SRC1 recruitment to RXRαLBD (F), instead of PPARγLBD (G) in SPR technology based assays.

Figure 2. Magnolol as a biased agonist on PPRE transcription.

(A–B) Magnolol could not activate the transcription of RXRE mediated by RXRα:RXRα homodimer (A), while activating the transcription of PPRE mediated by RXRα:PPARγ heterodimer in a dose-dependent manner (B). RXRα agonist 9cRA, RXRα antagonist HX531, PPARγ agonist Rosiglitazone, and PPARγ antagonist GW9662 were used as controls. (C) Activating curves of magnolol and Rosiglitazone on PPRE transcription indicated that magnolol was a PPARγ full agonist, although magnolol exhibited lower activities in their lower concentrations.

Figure 3. Crystal structures of RXRαLBD-magnolol-SRC1 and PPARγLBD-magnolol.

(A) Electron density of magnolol bound into RXRα ligand-binding pocket in stereo view (contoured at 1.0σ level). (B) Comparison of receptor-ligand interactions between 9cRA-bound and magnolol-bound RXRαLBDs. 9cRA (in blue sticks) formed hydrogen bonds with Arg316 (in magenta sticks) in the C-terminus of helix 5 (in magenta ribbon), while magnolol (in yellow sticks) formed hydrogen bonds with Asn306 (in cyan sticks) in the N-terminus of helix 5. Density map around Asn306 was shown in the right to indicate its conformational changes. All other hydrophobic residues involving 9cRA (in magenta lines) or magnolol interactions were the same (shown in cyan lines). (C) Electron density map of magnolol bound into PPARγ ligand-binding pocket in stereo view (contoured at 1.0σ level). (D) The two magnolol molecules formed hydrogen bonds with Ser342 in β-sheet, Tyr473 in AF-2 motif, and Ser289 in helix 3 of PPARγ, as well as water-mediated hydrogen bonds.

Figure 4. Key interactions for magnolol function on RXRα and PPARγ.

(A) Magnolol exhibited three different conformations upon binding into RXRα and PPARγ ligand-binding pockets. Magnolol in RXRα ligand-binding pocket was shown in yellow, while the two magnolol molecules in PPARγ ligand-binding pocket were shown in green and cyan, respectively. (B–C) Secondary structures with which magnolol interacted were shown in both RXRα (B) and PPARγ (C) ligand-binding pockets. The functions of these secondary structures in the coactivator recruitment, homo-/heterodimerization and DNA-binding domain (DBD) interactions were indicated.