1a,25-Dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], a vitamin D receptor (VDR) ligand, regulates calcium homeostasis and also exhibits noncalcemic actions on immunity and cell differentiation. In addition to disorders of bone and calcium metabolism, VDR ligands are potential therapeutic agents in the treatment of immune disorders, microbial infections, and malignancies. Hypercalcemia, the major adverse effect of vitamin D 3 derivatives, limits their clinical application. The secondary bile acid lithocholic acid (LCA) is an additional physiological ligand for VDR, and its synthetic derivative, LCA acetate, is a potent VDR agonist. In this study, we found that an additional derivative, LCA propionate, is a more selective VDR activator than LCA acetate. LCA acetate and LCA propionate induced the expression of the calcium channel transient receptor potential vanilloid type 6 (TRPV6) as effectively as that of 1a,25-dihydroxyvitamin D 3 24-hydroxylase (CYP24A1), whereas 1,25(OH) 2 D 3 was more effective on TRPV6 than on CYP24A1 in intestinal cells. In vivo experiments showed that LCA acetate and LCA propionate effectively induced tissue VDR activation without causing hypercalcemia.These bile acid derivatives have the ability to function as selective VDR modulators. , and regulates calcium and bone homeostasis, immunity, and cellular growth and differentiation (1-3). 1,25(OH) 2 D 3 has been demonstrated to inhibit the proliferation and/or to induce the differentiation of various types of malignant cells, including breast, prostate, and colon cancers, as well as myeloid leukemia cells in vitro (1). The administration of 1,25(OH) 2 D 3 and its analogs has therapeutic effects in mouse models of malignancies such as myeloid leukemia (4). 1,25(OH) 2 D 3 was also demonstrated to exert immunomodulatory and antimicrobial functions (5). VDR activation by 1,25(OH) 2 D 3 induces the cathelicidin antimicrobial peptide (CAMP) and kills Mycobacterium tuberculosis in monocytes (6). Although they have been used successfully in the treatment of bone and skin disorders, adverse effects, especially hypercalcemia, limit the clinical application of vitamin D and its synthetic analogs in the management of diseases other than bone and mineral disorders (5). Combined dosing of 1,25(OH) 2 D 3 with other drugs is one approach to overcome its adverse effects (7,8). The development of synthetic vitamin D analogs that retain VDR transactivation but have low calcemic activity provides another approach (9). With an improved understanding of the mechanisms of VDR signaling, the possibility of identifying VDR ligands with selective action is emerging (10).

These differences in ligand-receptor interaction may contribute to the differential recruitment of coactivators to VDR and selective biological actions (7).Nuclear receptors are ligand-inducible transcription factors that are involved in many biological processes, including cell growth and differentiation, embryonic development, and metabolic homeostasis (8). Recently, nuclear receptors belonging to the NR1H and NR1I subfamilies, including VDR, have been shown to control cholesterol Abbreviations: BSEP, bile salt export pump; CAR, constitutive androstane receptor; ER, estrogen receptor; FXR, farnesoid X receptor; IBABP, ileal bile acid binding protein;

The vitamin D receptor (VDR), initially identified as a nuclear receptor for 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], regulates calcium metabolism, cellular proliferation and differentiation, immune responses, and other physiological processes. Recently, secondary bile acids such as lithocholic acid (LCA) were identified as endogenous VDR agonists. To identify structural determinants required for VDR activation by 1alpha,25(OH)2D3 and LCA, we generated VDR mutants predicted to modulate ligand response based on sequence homology to pregnane X receptor, another bile acid-responsive nuclear receptor. In both vitamin D response element activation and mammalian two-hybrid assays, we found that VDR-S278V is activated by 1alpha,25(OH)2D3 but not by LCA, whereas VDR-S237M can respond to LCA but not to 1alpha,25(OH)2D3. Competitive ligand binding analysis reveals that LCA, but not 1alpha,25(OH)2D3, effectively binds to VDR-S237M and both 1alpha,25(OH)2D3 and LCA bind to VDR-S278V. We propose a docking model for LCA binding to VDR that is supported by mutagenesis data. Comparative analysis of the VDR-LCA and VDR-1alpha,25(OH)2D3 structure-activity relationships should be useful in the development of bile acid-derived synthetic VDR ligands that selectively target VDR function in cancer and immune disorders without inducing adverse hypercalcemic effects.

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Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders.

Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has attracted interest as a novel target for the treatment of obesity and metabolic diseases. Starting from N-phenylbenzenesulfonamide derivative 1 with moderate potency for MGAT2 inhibition, we explored an effective location of the hydrophobic group at the 1-position to enhance MGAT2 inhibitory activity. Shifting the hydrophobic group to the adjacent position followed by introduction of a bicyclic central core to restrict the substituent orientation produced N-phenylindoline-5-sulfonamide derivative 10b, which displayed much improved potency, with an IC50 value of 1.0 nM. This compound also exhibited excellent selectivity (greater than 30,000-fold) against related acyltransferases (MGAT3, DGAT1, DGAT2, and ACAT1). Subsequent optimization efforts were directed toward improving pharmacokinetic profiles, which resulted in the identification of 5-[(2,4-difluorophenyl)sulfamoyl]-7-(2-oxopyrrolidin-1-yl)-N-[4-(trifluoromethyl)phenyl]-2,3-dihydro-1H-indole-1-carboxamide (24d) endowed with potent MGAT2 inhibitory activity (IC50 = 3.4 nM) and high oral bioavailability (F = 52%, mouse). In a mouse oral fat tolerance test, oral administration of this compound effectively suppressed the elevation of plasma triacylglycerol levels.

Scleroderma has clinical characteristics including skin and other tissue fibrosis, but there is an unmet need for anti-fibrotic therapy. Halofuginone (HF) is a well-known anti-fibrosis agent in preclinical and clinical studies which exerts its effect via inhibition of TGF-β/Smad3 signaling pathway. Recently, prolyl-tRNA synthetase (PRS) was elucidated as a target protein for HF that binds to the proline binding site of the catalytic domain of PRS. Here, we characterized a new class of PRS inhibitor (T-3833261) that is carefully designed in a way that binds to the ATP site of the catalytic domain and does not disrupt binding of proline. The anti-fibrotic activity and the mechanism of action for T-3833261 on TGF-β-induced fibrotic assay were compared with those of HF in primary human skin fibroblast. We evaluated in vivo effect of topical application of T-3833261 and HF on TGF-β-induced fibrotic genes expression in mice. We found that T-3833261 suppressed TGF-β-induced α-smooth muscle actin (α-SMA) and type I collagen α1 (COL1A1) expression through the Smad3 axis in a similar fashion to HF. In vivo topical application of T-3833261 reduced the increase of fibrotic genes expression such as α-Sma, Col1a1 and Col1a2 by TGF-β intradermal injection to the ear of a mouse. We revealed that T-3833261 is more effective than HF under the conditions of high proline concentration, as reported in fibrotic tissues. These results suggest the potential of ATP competitive PRS inhibitors for the treatment of fibrotic diseases such as scleroderma.