Separation of α-glucosidase-inhibitory and liver X receptor-antagonistic activities of phenethylphenyl phthalimide analogs and generation of LXRα-selective antagonists
Introduction
Liver X receptors (LXRα and LXRβ) are members of the nuclear receptor superfamily.1 LXRα is most highly expressed in liver and is abundant in other tissues involved in lipid metabolism. LXRβ has a more widespread pattern of expression, being almost ubiquitous. Their physiological ligands are considered to be oxysterols, including 24(S),25-epoxycholesterol and 22(R)-hydroxycholesterol. Upon ligand binding, LXRs form heterodimers with the retinoid X receptor (RXR). The LXR/RXR heterodimer binds to LXR response elements in promoter regions of specific genes,2, 3 modulating gene transcription by recruiting coactivators.4, 5 LXRs are considered to function as cholesterol sensors, and to regulate the expression of genes associated with cholesterol efflux, absorption and transport.6, 7 The activation of LXRs leads to an increase in plasma HDL levels and net cholesterol efflux via up-regulation of gene expression of ATP-binding cassette (ABC) membrane transporters, including ABCA1,8, 9 ABCG5, ABCG810 and ABCG1.11, 12 ABCA1 functions in transporting cholesterol and phospholipids to ApoA-I, which is a critical step in cholesterol efflux via HDL. In animal studies, the activation of LXR led to an increase of HDL level and a decrease of atherosclerotic lesions through the induction of peripheral cholesterol efflux.9, 13 There is increasing evidence suggesting that the therapeutic effects of LXR activation on atherosclerosis are associated with stimulation of cholesterol efflux, rather than simply an increase of serum HDL.13, 14
Currently available typical synthetic LXR agonists T0901317 (1)15 and GW3965 (2)16 (Fig. 1) non-selectively activate both LXRα and LXRβ. They also activate triglyceride (TG) synthesis in the liver by the up-regulation of sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FAS), and this activity limits the clinical utility of these LXR agonists.15, 17 There is increasing evidence that the undesirable stimulation of lipogenesis by LXR ligands is largely attributable to LXRα. It has been demonstrated that administration of an LXRα/β dual agonist to LXRα-null mice results in increased HDL levels without significant hepatic TG accumulation.18 In these genetically modified mice, hepatic mRNA levels of many lipogenic genes, including SREBP1c, FAS, and lipoprotein lipase genes, were markedly reduced by LXR agonists, as compared to wild-type mice. More importantly, LXRα-deficient macrophages from LXRα-null mice retained the ability to increase ABCA1,9, 19 implying that LXRβ is capable of inducing cholesterol efflux in macrophages without inducing significant hepatic fatty acid synthesis.
These considerations led researchers to focus on LXRβ-selective ligands. LXRα and LXRβ are highly related and share 77% amino acid sequence identity in both the DNA-binding and ligand-binding domains (LBD).1 Therefore, the design of LXR subtype-selective ligands is challenging. To date, only one series of ligands, including 3, appear to be LXRβ-selective agonists based on binding assay, although they act as LXRα/β dual agonists in reporter gene assay.20 As an alternative approach, it seems plausible to speculate that usage of an LXRα-selective antagonist in conjunction with an LXR α/β dual agonist may circumvent the undesirable effect of increased lipogenesis mediated by LXRα while maintaining the enhanced cholesterol efflux mediated by LXRβ. There is some evidence in support of this idea. Herbal extracts, which have LXRα-selective antagonistic activity, have been reported to suppress the expression of LXRα-responsive genes, such as FAS and SREBP1c.21 These extracts also significantly reduce lipogenesis and adipocyte differentiation. However, the active ingredients have not been identified. As another example, an LXR α/β dual antagonist, fenofibrate (4), was reported to repress LXR agonist-induced transcription of hepatic lipogenic genes.22 Surprisingly, however, fenofibrate (4) did not repress LXR-induced transcription of ABCA1 in liver or in macrophages. Concerning LXR antagonists, those reported so far include riccardin C (5)23 (LXRα partial agonist/LXRβ antagonist), riccardin F (6)23 (LXRα antagonist), riccardin C analogs24 [LXRα/β dual antagonists and an antagonist with slight LXRα selectivity (7)], and 22(S)-hydroxycholesterol (8)25 (LXRα/β dual antagonists) (Fig. 1).
We have focused on the creation of LXR antagonists using a multi-template approach based on thalidomide. The multi-template approach is based on the reports that the number of three-dimensional spatial structures (fold structures) of human proteins is much smaller (more than 50 times smaller) than the number of human proteins (50,000–70,000).26, 27, 28, 29 Therefore, ignoring physical/chemical interactions, a template/scaffold structure which is spatially complementary to one fold structure might serve as a multi-template for structural development of ligands that would interact specifically with more than 50 different human proteins. In other words, the structures of ligands that bind to a protein having a certain fold structure may be useful for the development of novel ligands for other proteins possessing the same fold structure. One of the multi-templates that we have adopted is thalidomide, which is a drug first launched as a sedative/hypnotic agent, but withdrawn from the market because of its severe teratogenicity. Focusing on the potential of the thalidomide template for the treatment of a wide range of diseases, including cancers, diabetes, and rheumatoid arthritis, we have created compounds thalidomide analogs with a range of biological activities.30, 31, 32, 33 During our studies, we found that several thalidomide-related phthalimide derivatives, including PP2P (9) (Fig. 1), possess LXR-antagonistic activity as well as α-glucosidase-inhibitory activity.24, 34, 35 The co-existence of LXR-modulating activity and α-glucosidase-inhibitory activity may be general, because typical LXR ligands, including T0901317 (1), GW3965 (2), 22-(R)-hydroxycholesterol and riccardin C (5) were found to possess potent α-glucosidase-inhibitory activity.24 These results suggested that our previously reported thalidomide-related α-glucosidase inhibitors24, 34, 35, 36, 37, 38 might represent a superior scaffold structure for the development of LXR antagonists. The issues to be addressed were therefore the separation of α-glucosidase-inhibitory and LXR-antagonistic activities and the creation of LXRα-selective antagonists. Here, we describe the design, synthesis, and structure–activity relationship studies of phenethylphenyl phthalimide derivatives to generate an LXRα-selective antagonist.
Section snippets
Choice of the phenethylphthalimide skeleton as a scaffold
Initially, we selected our previously reported PP2P (9) as a prototype.24, 34, 35 We also noted the similarity of the biological activities (i.e., LXR-antagonistic activity and α-glucosidase-inhibitory activity) elicited by PP2P (9) and riccardin C (5).24 Although the binding site of PP2P (9)/riccardin C (5) on α-glucosidase, the crystal structure of α-glucosidase, and the precise binding mode of PP2P (9)/riccardin C (5) to LXR are all unknown, we expected that PP2P (9) and riccardin C (5)
Conclusion
Structural development studies of PP2P (9), which had been derived from thalidomide and found to possess LXRα/β-antagonistic and α-glucosidase-inhibitory activities, were performed. We were able to separate the LXR-antagonistic and α-glucosidase-inhibitory activities, at least under the experimental conditions used here. A potent LXRα-selective antagonist 23f with very low LXRβ-antagonistic activity (IC50 values of 0.2 μM for LXRα and >30 μM for LXRβ) and no α-glucosidase-inhibitory activity was
α-Glucosidase inhibition assay
α-Glucosidase (Saccharomyces sp., Wako) 0.2 mU/mL in 10 mM phosphate buffer (pH 7.0) was treated with DMSO solution of various compounds (final DMSO concentration 1% v/v) in a 96-well plate (final volume 90 μL). After 10 min incubation at 37 °C, 10 μL pNPG solution (final concentration 0.2 mM) was added. The mixture was incubated at 37 °C for 10 min, then basified by adding 100 μL of 0.5 M Na2CO3 solution. The amount of released p-nitrophenol was measured based on the absorbance at 405 nm. The experiment
Acknowledgments
The work described in this paper was partially supported by Grants-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Japan Society for the Promotion of Science.
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