Identification of small molecule regulators of the nuclear receptor HNF4α based on naphthofuran scaffolds

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Abstract

Nuclear receptors are ligand-activated transcription factors involved in all major physiological functions of complex organisms. In this respect, they are often described as drugable targets for a number of pathological states including hypercholesterolemia and atherosclerosis. HNF4α (NR2A1) is a recently ‘deorphanized’ nuclear receptor which is bound in vivo by linoleic acid, although this natural ligand does not seem to promote transcriptional activation. In mouse, HNF4α is a major regulator of liver development and hepatic lipid metabolism and mutations in human have been linked to diabetes. Here, we have used a yeast one-hybrid system to identify small molecule activators of HNF4α in a library of synthetic compounds and found one hit bearing a methoxy group branched on a nitronaphthofuran backbone. A collection of molecules deriving from the discovered hit was generated and tested for activity toward HNF4α in yeast one-hybrid system. It was found that both the nitro group and a complete naphthofuran backbone were required for full activity of the compounds. Furthermore, adding a hydroxy group at position 7 of the minimal backbone led to the most active compound of the collection. Accordingly, a direct interaction of the hydroxylated compound with the ligand binding domain of HNF4α was detected by NMR and thermal denaturation assays. When used in mammalian cell culture systems, these compounds proved to be highly toxic, except when methylated on the furan ring. One such compound was able to modulate HNF4α-driven transcription in transfected HepG2C3A cells. These data indicate that HNF4α activity can be modulated by small molecules and suggest new routes for targeting the receptor in humans.

Introduction

Orphan nuclear receptors are members of the nuclear receptor superfamily of ligand regulated transcription factors for which ligands have not been identified.1 Hepatocyte Nuclear Factor-4α, also known as HNF4α, is a highly conserved member of this nuclear receptor family that is expressed in liver, kidney, intestine, and pancreas, activates transcription likely as a homodimer2 and was considered as an orphan receptor until very recently. In the liver, HNF4α is known to up-regulate transcription of the cholesterol 7α-hydroxylase (Cyp7a1) gene encoding a major cholesterol catabolizing enzyme and is itself targeted by bile acids in a complex feed-back regulatory loop.3, 4 Thus, HNF4α is an attractive target for the design and development of a new generation of hypolididemic agents.5 Although fatty acyl-coenzyme A thioesters have been proposed to modulate HNF4α activity,6 it has been reported later7 that these molecules are not good candidates for being classical ligands for HNF4α. Fatty acids have also been suggested as endogeneous ligands of HNF4α on a structural basis. Indeed, they were found in the ligand binding pockets of crystallized HNF4α and HNF4γ.8, 9 However the crystallized HNF4α ligand binding domain (LBD) dimer is not in the fully active conformation since one monomer has helix 12 extended instead of the canonical ‘closed’ conformation observed in all activated receptors.10 Fatty acid fitting into a nuclear receptor ligand binding pocket is a recurrent observation that has been made for several other LBDs expressed in Escherichia coli and crystallized (ERα, RXRα, RORβ, PPARβ/δ). What is unusual however, in the case of HNF4α and γ LBDs produced in E. coli, is that the bound fatty acid cannot be exchanged.9, 11 These data suggested that fatty acids may be structural determinants of HNF4α rather than ligands regulating conformational transitions. A recent study highlighted linoleic acid as an endogenous HNF4α ligand in cultured mammalian cells as well as in mouse liver, but no function in the regulation of HNF4α transcriptional activity could be attributed to the newly discovered ligand.12 Interestingly, linoleic acid can be exchanged from the receptor ligand binding pocket in cells.12 Furthermore, in fasted animals, the ligand binding pocket was found to be devoid of ligand, indicating that, in certain physiological situations, HNF4α exists in the apo form. Hence, synthetic or natural molecules other than fatty acids might be used to manipulate HNF4α activity. If such synthetic compounds were to be obtained, they would prove to be extremely useful in precisely defining the physiological functions of HNF4α and would provide tools to intervene in physiological disorders like hypercholesterolemia.

In an attempt to identify HNF4α agonists, the initial screening of the National Center Institute (NCI, USA) diversity set of synthetic compounds (1990 molecules) was first undertaken in a yeast one-hybrid system. We discovered 4-methoxy-2-nitronaphtho[2,1-b]furan (1, Table 1) that modulates HNF4α activity in yeast, at the micromolar range. Through similarity searching of the complete library (250,000 compounds), we generated a first focused library containing 5 additional compounds (26) (Table 1) which were able to stimulate HNF4α-mediated transcription, compound 2 leading to the highest level of transcriptional induction. Synthesis of these molecules, were previously described by Royer and Buisson.13 Because we found that these compounds also appeared to reduce yeast growth, we chose to prepare a series of related derivatives and analogs for further investigation. Since substitution in position 7 seemed to give the most active compound such as 7-methoxy-2-nitronaphtho[2,1-b]furan (2), we decided to set up a new series with aromatic ring linked in this position. Thus, the application of a Suzuki–Miyaura cross-coupling approach14 to a range of phenyl C-7 substituted 2-nitronaphtho[2,1-b]furan from a 7-bromo-2-nitronaphtho[2,1-b]furan (21a) was undertaken.

Here, we report the identification of new activators of HNF4α using a random screening method through a yeast cell-based functional approach. We also report the chemical optimization study of the initial hit which led to the identification of a series of compounds that exhibit HNF4α agonist activities.

Section snippets

Chemistry

The strategy for hit modification is summarized in Scheme 1. To complete the series, starting from 2-nitronaphtho[2,1-b]furan, all the proposed synthetic modifications classified in two series are summarized in Table 2, Table 3, Table 4. The compound 7-methoxy-2-nitronaphtho[2,1-b]furan (2) was chosen as starting material and different modifications of this compound have been made such as the substitution (or modification) of methoxy group in position 7 or the substitution of nitro group in

Hit discovery

HNF4α agonist activities were evaluated using a one-hybrid system designed to express the full-length human nuclear receptor HNF4α fused in C-terminal of the DNA binding domain of the bacterial LexA protein in a yeast strain containing a β-galactosidase reporter gene controlled by a promoter harboring LexA binding sites. Screening of the National Center Institute (NCI, USA) diversity set of synthetic compounds with this system allowed us to discover that 4-methoxy-2-nitronaphtho[2,1-b]furan

Conclusion

Screening of a chemical compound library led to the discovery of a family of small molecules activating the nuclear receptor HNF4α in a yeast cell-based assay. We further demonstrated that these compounds can bind directly to the ligand binding domain of the nuclear receptor, most likely in the hydrophobic pocket known to be occupied in vivo by linoleic acid.12 Whereas the natural ligand (i.e., linoleic acid) does not seem to be able to regulate HNF4α transactivation function (our data and

General remarks

All reactions were performed under argon. Solvents were distilled from appropriate drying agent prior to use: Et2O and THF from sodium and benzophenone, toluene from sodium, CH2Cl2 from CaH2, CHCl3 from P2O5 and all other solvents were HPLC grade. Commercially available reagents were used without further purification unless otherwise stated. All reactions were monitored by TLC with Merck pre-coated aluminum foil sheets (Silica Gel 60 with fluorescent indicator UV254). Compounds were visualized

Acknowledgments

We thank the National Cancer Institute (USA) for providing compounds. We also thank Maud Bizot, Gaëlle Palierne and Catherine Martin for technical support. We are grateful to Dr. Eeckhoute for critical reading of the manuscript. This work was funded by the European Community (QLG1-CT-2001-01513 and LSHM-CT-2006-037498), the Région Bretagne (SIE 211-B3-11), the Centre National pour la Recherche Scientifique and the Ministère de l’enseignement supérieur et de la recherche.

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    Present address: U522 INSERM, Faculté de Médecine, Campus de Villejean, 35033 Rennes, France.

    These two authors contributed equally to this study.

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