Discovery of novel modulators for the PPARα (peroxisome proliferator activated receptor α): Potential therapies for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a severe liver disease causing serious liver complications, including nonalcoholic steatohepatitis (NASH). Nuclear receptor PPARα (peroxisome proliferator-activated receptor α) has drawn special attention recently as a potential developmental drug target to treat type-2 diabetes and related diseases due to its unique functions in regulating lipid metabolism, promoting triglyceride oxidation, and suppressing hepatic inflammation, raising interest in PPARα agonists as potential therapies for NAFLD. However, how PPARα coordinates potential treatment of NAFLD and NASH between various metabolic pathways is still obscure. Here, we show that the DY series of novel selective PPARα modulators activate PPARα by up-regulating PPARα target genes directly involved in NAFLD and NASH. The design, synthesis, docking studies, and in vitro and in vivo evaluation of the novel DY series of PPARα agonists are described.

Identification of novel inverse agonists of estrogen-related receptors ERRγ and ERRß.

Estrogen-related receptors (ERRs, α, ß, and γ) are orphan nuclear receptors most closely related in sequence to estrogen receptors (ERα and ERß). Much attention has been paid recently to the functions of ERRs for their potential roles as new therapeutic targets implicated in the etiology of metabolic disorders. While no endogenous ligand has been identified for any of the ERR isoforms to date, the potential for using synthetic small molecules to modulate their activity has been demonstrated. In the present study, a series of novel inverse agonists of ERRγ and ERRß were synthesized using regio- and stereo-specific direct substitution of triarylethylenes. These compounds were evaluated for their ability to modulate the activities of ERRs. The rational directed substitution approach and extensive SAR studies resulted in the discovery of compound 4a (DY40) as the most potent ERRγ inverse agonist described to date with mixed ERRγ/ERRß functional activities, which potently suppressed the transcriptional functions of ERRγ with IC50=0.01µM in a cell-based reporter gene assay and antagonized ERRγ with a potency approximately 60 times greater than its analog Z-4-OHT (Z-4-hydroxytamoxifen). In addition, compound 3h (DY181) was identified as the most potent synthetic inverse agonist for the ERRß that exhibited excellent selectivity over ERRα/γ in functional assays. This selectivity was also supported by computational docking models that suggest DY181 forms more extensive hydrogen bound network with ERRß which should result in higher binding affinity on ERRß over ERRγ.

Novel FXR (farnesoid X receptor) modulators: Potential therapies for cholesterol gallstone disease.

Metabolic disorders such as diabetes are known risk factors for developing cholesterol gallstone disease (CGD). Cholesterol gallstone disease is one of the most prevalent digestive diseases, leading to considerable financial and social burden worldwide. Ursodeoxycholic acid (UDCA) is the only bile acid drug approved by FDA for the non-surgical treatment of gallstones. However, the molecular link between UDCA and CGD is unclear. Previous data suggest that the farnesoid X receptor (FXR), a bile acid nuclear receptor, may protect against the development of CGD. In studies aimed at identifying the role of FXR, we recently identify a novel chemical tool, 6EUDCA (6-αethyl-ursodeoxycholic acid), a synthetic derivative of UDCA, for studying FXR. We found that 6EUDCA binds FXR stronger than UDCA in a TR-FRET binding assay. This result was supported by computational docking models that suggest 6EUDCA forms a more extensive hydrogen bound network with FXR. Interestingly, neither compound could activate FXR target genes in human nor mouse liver cells, suggesting UDCA and 6EUDCA activate non-genomic signals in an FXR-dependent manner. Overall these studies may lead to the identification of a novel mechanism by which bile acids regulate cell function, and 6EUDCA may be an effective targeted CGD therapeutic.

Stereoselective synthesis, biological evaluation, and modeling of novel bile acid-derived G-protein coupled bile acid receptor 1 (GP-BAR1, TGR5) agonists.

GP-BAR1 (also known as TGR5), a novel G-protein coupled receptor regulating various non-genomic functions via bile acid signaling, has emerged as a promising target for metabolic disorders, including obesity and type II diabetes. However, given that many bile acids (BAs) are poorly tolerated for systemic therapeutic use, there is significant need to develop GP-BAR1 agonists with improved potency and specificity and there also is significant impetus to develop a stereoselective synthetic methodology for GP-BAR1 agonists. Here, we report the development of highly stereo-controlled strategies to investigate a series of naturally occurring bile acid derivatives with markedly enhanced GP-BAR1 activity. These novel GP-BAR1 agonists are evaluated in vitro using luciferase-based reporter and cAMP assays to elucidate their biological properties. In vivo studies revealed that the GP-BAR1 agonist 23(S)-m-LCA increased intestinal GLP-1 transcripts by 26-fold. Additionally, computational modeling studies of selected ligands that exhibit enhanced potency and specificity for GP-BAR1 provide information on potential binding sites for these ligands in GP-BAR1.

Identification of trisubstituted-pyrazol carboxamide analogs as novel and potent antagonists of farnesoid X receptor.

Farnesoid X receptor (FXR, NRIH4) plays a major role in the control of cholesterol metabolism. This suggests that antagonizing the transcriptional activity of FXR is a potential means to treat cholestasis and related metabolic disorders. Here we describe the synthesis, biological evaluation, and structure-activity relationship (SAR) studies of trisubstituted-pyrazol carboxamides as novel and potent FXR antagonists. One of these novel FXR antagonists, 4j has an IC50 of 7.5 nM in an FXR binding assay and 468.5 nM in a cell-based FXR antagonistic assay. Compound 4j has no detectable FXR agonistic activity or cytotoxicity. Notably, 4j is the most potent FXR antagonist identified to date; it has a promising in vitro profile and could serve as an excellent chemical tool to elucidate the biological function of FXR.

Development of time resolved fluorescence resonance energy transfer-based assay for FXR antagonist discovery.

FXR (farnesoid X receptor, NRIH4), a nuclear receptor, plays a major role in the control of cholesterol metabolism. FXR ligands have been investigated in preclinical studies for targeted therapy against metabolic diseases, but have shown limitations. Therefore, there is a need for new agonist or antagonist ligands of FXR, both for potential clinical applications, as well as to further elucidate its biological functions. Here we describe the use of the X-ray crystal structure of FXR complexed with the potent small molecule agonist GW4064 to design and synthesize a novel fluorescent, high-affinity probe (DY246) for time resolved fluorescence resonance energy transfer (TR-FRET) assays. We then used the TR-FRET assay for high throughput screening of a library of over 5000 bioactive compounds. From this library, we identified 13 compounds that act as putative FXR transcriptional antagonists.

Identification of an agonist ligand for estrogen-related receptors ERRbeta/gamma.

In order to develop agonist ligands that are specific for the estrogen-related receptors ERRbeta/gamma, a hydrazone with a 4-hydroxy group at one phenyl ring and a 4-diethylamino moiety at the other phenyl ring was synthesized. We demonstrate that compound 3 (DY131; N'-{(1E)-[4-(diethylamino)phenyl]methylene}-4-hydroxybenzohydrazide) effectively and selectively activates ERRbeta/gamma. DY131 had no effect on the structurally related receptors ERRalpha or the estrogen receptors alpha and beta (ERalpha/beta). This work defines a convenient synthesis for a novel and selective pharmacologic tool that can be used to elucidate the biological activities of ERRbeta/gamma.

Simple and efficient production of (Z)-4-hydroxytamoxifen, a potent estrogen receptor modulator.

A McMurry coupling reaction and selective crystallization were used to develop a simple and efficient two-step synthesis of (Z)-4-hydroxytamoxifen (2a). This compound is an active metabolite of tamoxifen, a selective estrogen receptor (ER) modulator widely used to treat breast cancer. The synthesis employed 1,1-bis(4-hydroxyphenyl)-2-phenylbut-1-ene (1) as a useful building block.