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. 1999 Jan 5;96(1):266-71.
doi: 10.1073/pnas.96.1.266.

Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta

B A Janowski  1 , M J Grogan, S A Jones, G B Wisely, S A Kliewer, E J Corey, D J Mangelsdorf
Affiliations

Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta

B A Janowski et al. Proc Natl Acad Sci U S A. .

Abstract

LXRalpha and -beta are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXRalpha-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.

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Figures

Figure 1
Figure 1
Naturally occurring oxysterols bind LXRα and LXRβ. (A) Structure of [3H]-24(S),25-EC, showing the incorporation of tritium atoms (T). (B) Binding specificity for LXRs and RXR by their cognate ligands. Receptor protein (His6-hLXRαLBD, His6-hLXRβLBD, or His10-hRXRα) immobilized on SPA beads was incubated with [3H]-24(S),25-EC (shaded bars) or [3H]-9cRA (hatched bars) in the absence or presence of 1000-fold excess of nonradiolabeled 24(S),25-EC (open bars in left panel) or 9cRA (open bar in right panel). (C and D) Competition curves for 24(S),25-EC binding His6-hLXRαLBD or His6-hLXRβLBD. LXRLBD protein immobilized on SPA beads was incubated with 25 nM [3H]-24(S),25-EC and increasing concentrations (3 nM to 50 μM) of nonradiolabeled 24(S),25-EC. (E) Binding of naturally occurring oxysterols to LXRα. Competition of 25 nM [3H]-24(S),25-EC with either nonradiolabeled 24(S)-HC (•) or 22(R)-HC (□) at concentrations ranging from 3 nM to 50 μM. Values for cholesterol (▴) and 9cRA (▵) are shown at the highest tested concentration, 50 μM.
Figure 2
Figure 2
Structures of naturally occurring and synthetic oxysterols, with chemical substitutions on the side chain carbons 21–27 (R1) or the B-ring (R2) of the sterol scaffold.
Figure 3
Figure 3
Multiple oxidation decreases binding and gene activation, whereas single hydrogen bond acceptors are potent ligands and activators of LXR. (A–C) Transcriptional activation profile of LXRα with increasing concentrations of 22(R),24(S)-diHC (•), 22(R)-HC (○), or 24(S)-HC (■) (A); 7α- (○) or 7β- (•) -hydroxy-24(S),25-EC, 7-oxo-24(S),25-EC (□), or 24(S),25-EC (■) (B); dimethylhydroxycholenamide (○), 24(S),25-iminocholesterol (•), or 24(S),25-EC (■) (C). Compounds were tested for their ability to activate the LXR-responsive element in the Cyp7a gene in CV-1 cells. Activation is represented as RLU. (D) Competition binding profile of His6-hLXRαLBD with dimethylhydroxycholenamide (○), 24(S),25-iminocholesterol (•), or 24(S),25-EC (■). LXRLBD protein, SPA beads, and 25 nM [3H]-24(S),25-EC were incubated with increasing concentrations of nonradiolabeled competitors (as in Fig. 1). Similar results were observed with LXRβ.
Figure 4
Figure 4
Identification of a LXRα-selective ligand. (A) Structure of 5,6-α-24(S),25-diEC. (B) Binding profile of 5,6–24(S),25-diEC for His6-hLXRαLBD (•) or His6-hLXRβLBD (○). LXRα or LXRβ protein, SPA beads, and 25 nM [3H]-24(S),25-EC were incubated with increasing concentrations of nonradiolabeled 5,6–24(S),25-diEC. (C and D) Activation profiles with increasing concentrations (3 nM to 5 μM) of either 5,6–24(S),25-diEC (○) or 24(S),25-EC (■) for LXRα or LXRβ on the LXR-responsive element in the Cyp7a gene. Transcriptional activation is represented in RLU.
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