Structural basis for iloprost as a dual peroxisome proliferator-activated receptor alpha/delta agonist

Abstract

Iloprost is a prostacyclin analog that has been used to treat many vascular conditions. Peroxisome proliferator-activated receptors (PPARs) are ligand-regulated transcription factors with various important biological effects such as metabolic and cardiovascular physiology. Here, we report the crystal structures of the PPARα ligand-binding domain and PPARδ ligand-binding domain bound to iloprost, thus providing unambiguous evidence for the direct interaction between iloprost and PPARs and a structural basis for the recognition of PPARα/δ by this prostacyclin analog. In addition to conserved contacts for all PPARα ligands, iloprost also initiates several specific interactions with PPARs using its unique structural groups. Structural and functional studies of receptor-ligand interactions reveal strong functional correlations of the iloprost-PPARα/δ interactions as well as the molecular basis of PPAR subtype selectivity toward iloprost ligand. As such, the structural mechanism may provide a more rational template for designing novel compounds targeting PPARs with more favorable pharmacologic impact based on existing iloprost drugs.

FIGURE 1.

Iloprost is a dual PPARα/δ agonist. A, receptor-specific transactivation by 1 μm iloprost. The COS-7 cells were cotransfected with the plasmids encoding various full-length nuclear receptors and their cognate luciferase reporters (see “Experimental Procedures”). After transfection, cells were treated with dimethyl sulfoxide (DMSO) or 1 μm iloprost. B, dose responses of iloprost in transactivating PPAR (α, δ, and γ). COS-7 cells were cotransfected with plasmids encoding full-length PPARs and a PPRE luciferase reporter. After transfection, cells were treated with dimethyl sulfoxide or iloprost ligand with various concentrations.

FIGURE 2.

Iloprost promotes the interaction of coactivator LXXLL motifs with PPARα and PPARδ, respectively. Modulation of the interaction of PPARα, PPARδ, and PPARγ with various coactivator LXXLL motifs and corepressor motifs in response to 1 μm iloprost is shown by AlphaScreen assays. The peptide sequences are listed under “Experimental Procedures”.

FIGURE 3.

Recognition of iloprost by PPARα and PPARδ. A and B, structures of iloprost bound with PPARα (A) and PPARδ (B) in ribbon representation. PPARα and PPARδ are colored in green and blue, respectively. The bound iloprost is shown in stick representation with carbon and oxygen atoms depicted in yellow and red, respectively. C and D, 2F_o − F_c electron density map (1.0σ) showing bound iloprost to the PPARα LBD (C) and PPARδ LBD (D). The bound iloprost is shown in stick representation with carbon and oxygen atoms depicted in yellow and red, respectively.

FIGURE 4.

Schematic representation of key interactions between iloprost and PPARα (A) and PPARδ (B). Key hydrocarbon positions of iloprost molecule are indicated. Hydrophobic interactions are indicated by lines, and hydrogen bonds are indicated by arrows from proton donors to acceptors.

FIGURE 5.

Functional correlation of the iloprost-PPARs interactions. A–D, molecular determinants of the interactions between iloprost ligand with PPARα (A–C) and PPARδ (D). The bound iloprost is shown in stick representation with carbon and oxygen atoms depicted in yellow and red, respectively. The PPARα ligand GW735 resulted from superposition and is shown in stick representation with carbon, nitrogen, and oxygen atoms depicted in purple, blue, and red, respectively. The hydrophobic interactions are shown with lines, and the hydrogen bonds are indicated by arrows. E, effects of mutations of key PPARα/δ residues on its transcriptional activity in response to iloprost treatment in cell-based reporter gene assays. COS-7 cells were cotransfected with plasmids encoding full-length PPARα/δ or PPARα/δ mutants as indicated in the figures together with a PPRE luciferase reporter.

FIGURE 6.

Molecular determinants of PPAR subtype selectivity toward iloprost ligand. A–C, superpositions of the PPARα-iloprost structure (green) with the PPARγ (gold) structure with ligand iloprost shown in yellow. Hydrophobic interactions between PPARα and iloprost are indicated by lines. D, sequence alignment of a fragment from human PPARγ LBD with PPARα and PPARδ. The secondary structural elements are boxed and annotated below the sequences, and the residues that determine the iloprost binding selectivity are noted with arrows. E, effects of mutations of key PPARγ residues on its transcriptional activity in response to iloprost treatment in cell-based reporter gene assays. COS-7 cells were cotransfected with plasmids encoding full-length PPARγ or PPARγ mutants as indicated in the figure together with a PPRE luciferase reporter.