A fluorescent analogue of tauroursodeoxycholic acid reduces ER stress and is cytoprotective.

Tauroursodeoxycholic acid (TUDCA) is a cytoprotective ER stress inhibitor and chemical chaperone. It has therapeutic potential in a wide array of diseases but a specific macromolecular target or molecular mechanism of action remains obscure. This Letter describes an effective new synthetic approach to taurine conjugation of bile acids which we used to prepare 3α-dansyl TUDCA (4) as a probe for TUDCA actions. As a model of ER stress we used the hepatocarcinoma cell line HUH7 and stimulation with either deoxycholic acid (DCA, 200µM) or tunicamycin (5µg/ml) and measured levels of Bip/GRP78, ATF4, CHOP and XBP1s/XBP1u. Compound 4 was more effective than UDCA at inhibiting ER stress markers and had similar effects to TUDCA. In a model of cholestasis using the cytotoxic DCA to induce apoptosis, pretreatment with 4 prevented cell death similarly to TUDCA whereas the unconjugated clinically used UDCA had no effect. 3α-Dansyl TUDCA (4) appears to be a suitable reporter for TUDCA effects on ER stress and related cytoprotective activity.

New highly toxic bile acids derived from deoxycholic acid, chenodeoxycholic acid and lithocholic acid.

We have prepared a new panel of 23 BA derivatives of DCA, chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) in order to study the effect of dual substitution with 3-azido and 24-amidation, features individually associated with cytotoxicity in our previous work. The effect of the compounds on cell viability of HT-1080 and Caco-2 was studied using the 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds with high potency towards reduction of cell viability were further studied using flow cytometry in order to understand the mechanism of cell death. Several compounds were identified with low micromolar IC50 values for reducing cell viability in the Caco-2 and HT1080 cell lines, making them among the most potent BA apoptotic agents reported to date. There was no evidence of relationship between overall hydrophobicity and cytotoxicity supporting the idea that cell death induction by BAs may be structure-specific. Compounds derived from DCA caused cell death through apoptosis. There was some evidence of selectivity between the two cell lines studied which may be due to differing expression of CD95/FAS. The more toxic compounds increased ROS production in Caco-2 cells, and co-incubation with the antioxidant N-acetyl cysteine blunted pro-apoptotic effects. The properties these compounds suggest that there may be specific mechanism(s) mediating BA induced cell death. Compound 8 could be useful for investigating this phenomenon.

Synthesis and biological evaluation of Foscan® bile acid conjugates to target esophageal cancer cells.

Porphyrins and chlorins such as Foscan® have a natural proclivity to accumulate in cancer cells. This trait has made them good candidates for photosensitizers and as imaging agents in phototherapy. In order to improve on cellular selectivity to lower post-treatment photosensitivity bile acid porphyrin bioconjugates have been prepared and investigated in esophageal cancer cells. Bile acids which are known to selectively bind to, or be readily taken up by cancer cells were chosen as targeting moieties. Synthesis of the conjugates was achieved via selective nucleophilic monofunctionalization of 5,10,15,20-tetrahydroxyphenylporphyrins with propargyl bromide followed by Cu(I) mediated cycloaddition with bile acid azides in good yields. The compounds were readily taken up by esophageal cancer cells but showed no PDT activity.

New fluorescent bile acids: synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid.

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3ß-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the ß-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 µM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 µM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes.

Ursodeoxycholic acid amides as novel glucocorticoid receptor modulators.

Ursodeoxycholic acid (UDCA) is used for the treatment of hepatic inflammatory diseases. Recent studies have shown that UDCA's biological effects are partly glucocorticoid receptor (GR) mediated. UDCA derivatives were synthesized and screened for ability to induce GR translocation in a high content analysis assay using the esophageal cancer SKGT-4 cell line. UDCA derivatives induced GR translocation in a time dependent manner with equal efficacy to that of dexamethasone (Dex) and with greatly increased potency relative to UDCA. The cyclopropylamide 1a suppressed TNF-α induced NF-κB activity and it induced GRE transactivation. 1a was unable to displace Dex from the GR ligand binding domain (LBD) in a competition experiment but was capable of coactivator recruitment in a time-resolved fluorescence energy transfer assay (TR-FRET). This represents a novel mechanism of action for a GR modulator. These derivatives could result in a new class of GR modulators.

Bile acid toxicity structure-activity relationships: correlations between cell viability and lipophilicity in a panel of new and known bile acids using an oesophageal cell line (HET-1A).

The molecular mechanisms and interactions underlying bile acid cytotoxicity are important to understand for intestinal and hepatic disease treatment and prevention and the design of bile acid-based therapeutics. Bile acid lipophilicity is believed to be an important cytotoxicity determinant but the relationship is not well characterized. In this study we prepared new azido and other lipophilic BAs and altogether assembled a panel of 37 BAs with good dispersion in lipophilicity as reflected in RPTLC RMw. The MTT cell viability assay was used to assess cytotoxicity over 24 h in the HET-1A cell line (oesophageal). RMw values inversely correlated with cell viability for the whole set (r2=0.6) but this became more significant when non-acid compounds were excluded (r2=0.82, n=29). The association in more homologous subgroups was stronger still (r2>0.96). None of the polar compounds were cytotoxic at 500 microM, however, not all lipophilic BAs were cytotoxic. Notably, apart from the UDCA primary amide, lipophilic neutral derivatives of UDCA were not cytotoxic. Finally, CDCA, DCA and LagoDCA were prominent outliers being more toxic than predicted by RMw. In a hepatic carcinoma line, lipophilicity did not correlate with toxicity except for the common naturally occurring bile acids and their conjugates. There were other significant differences in toxicity between the two cell lines that suggest a possible basis for selective cytotoxicity. The study shows: (i) azido substitution in BAs imparts lipophilicity and toxicity depending on orientation and ionizability; (ii) there is an inverse correlation between RMw and toxicity that has good predictive value in homologous sets; (iii) lipophilicity is a necessary but apparently not sufficient characteristic for BA cytocidal activity to which it appears to be indirectly related.