Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5ß-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile.

Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we identified N-phenyl-3α,7α,12α-trihydroxy-5ß-cholan-24-amide as an inhibitor of C. difficile strain R20291 with an IC50 of 1.8 µM. Studies of bile salts on spore germination have shown that chenodeoxycholate, ursodeoxycholate and lithocholate are more potent inhibitors of germination compared to cholate. Given this, we created amide analogs of chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids using amines identified from our previous studies. We found that chenodeoxy- and deoxycholate derivatives were active with potencies equivalent to those for cholanamides. This indicates that only 2 out of the 3 hydroxyl groups are needed for activity and that the alpha stereochemistry at position 7 is required for inhibition of spore germination.

The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory ß1 subunit.

Large conductance, voltage- and calcium-gated potassium (BK) channels regulate several physiological processes, including myogenic tone and thus, artery diameter. Nongenomic modulation of BK activity by steroids is increasingly recognized, but the precise location of steroid action remains unknown. We have shown that artery dilation by lithocholate (LC) and related cholane steroids is caused by a 2× increase in vascular myocyte BK activity (EC(50) = 45 µM), an action that requires ß1 but not other (ß2-ß4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK α- (cbv1) and ß1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK ß1 transmembrane domain 2 proposed by computational dynamics: the outer site includes L157, L158, and T165, whereas the inner site includes T169, L172, and L173. As expected from computational modeling, cbv1+rß1T165A,T169A channels were LC-unresponsive. However, cbv1 + rß1T165A and cbv1 + rß1T165A,L157A,L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + rß1T169A was LC-insensitive, with rß1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + rß1L172A, and cbv1 + rß1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid α-hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for LC action. Therefore, ß1 TM2 T169,L172,L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK ß1, our study provides a structural basis for advancing ß1 subunit-specific pharmacology of BK channels.

Cerebrovascular dilation via selective targeting of the cholane steroid-recognition site in the BK channel ß1-subunit by a novel nonsteroidal agent.

The Ca(2+)/voltage-gated K(+) large conductance (BK) channel ß1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK ß1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK ß1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate ß1-containing BK channels and evoke vasodilation remain unknown. We performed a chemical structure database similarity search using LCA as a template, along with a two-step reaction to generate sodium 3-hydroxyolean-12-en-30-oate (HENA). HENA activated the BK (cbv1 + ß1) channels cloned from rat cerebral artery myocytes with a potency (EC50 = 53 µM) similar to and an efficacy (×2.5 potentiation) significantly greater than that of LCA. This HENA action was replicated on native channels in rat cerebral artery myocytes. HENA failed to activate the channels made of cbv1 + ß2, ß3, ß4, or ß1T169A, indicating that this drug selectively targets ß1-containing BK channels via the BK ß1 steroid-sensing site. HENA (3-45 µM) dilated the rat and C57BL/6 mouse pressurized cerebral arteries. Consistent with the electrophysiologic results, this effect was larger than that of LCA. HENA failed to dilate the arteries from the KCNMB1 knockout mouse, underscoring BK ß1's role in HENA action. Finally, carotid artery-infusion of HENA (45 µM) dilated the pial cerebral arterioles via selective BK-channel targeting. In conclusion, we have identified for the first time a nonsteroidal agent that selectively activates ß1-containing BK channels by targeting the steroid-sensing site in BK ß1, rendering vasodilation.

Solomonsterol A, a marine pregnane-X-receptor agonist, attenuates inflammation and immune dysfunction in a mouse model of arthritis.

In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFα, IFNγ and IL-17 and chemokines MIP1α and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists.

Modification in the side chain of solomonsterol A: discovery of cholestan disulfate as a potent pregnane-X-receptor agonist.

Seven synthetic analogues of the PXR (pregnane-X-receptor) potent natural agonist solomonsterol A were prepared by total synthesis. Their activity toward PXR was assessed by transactivation and RT-PCR assays. The study discloses cholestan disulfate (8) as a new, simplified agonist of PXR. By in vitro studies on hepatic cells we have demonstrated that this compound is a potent PXR agonist and functional characterization in human macrophages and hepatic stellate cells provided evidence that cholestan disulfate (8) has the ability to modulate the immune response triggered by bacterial endotoxin as well as to counter-activate hepatic stellate cell activation induced by thrombin. Because inhibition of immune-driven circuits might have relevance in the treatment of inflammation and liver fibrosis, the present data support the development of cholestan disulfate (8) in preclinical models of inflammatory diseases.

A concise synthesis and antimicrobial activities of 3-polyamino-23,24-bisnorcholanes as steroid-polyamine conjugates.

A series of steroid-polyamine conjugates were synthesized and evaluated for their antimicrobial activity. This study was focused on the effect of stereochemistry at the C-3 and C-5 of steroids and types of polyamine at C-3 on activity against various human pathogens. All the conjugates exhibited strong antimicrobial activities against Gram-positive strains. Compound 18 was found to be the most potent in these series with a MIC value as low as 1 µg/mL against the bacterium Staphylococcus aureus ATCC6538P.

Transcriptome Analysis of Dual FXR and GPBAR1 Agonism in Rodent Model of NASH Reveals Modulation of Lipid Droplets Formation.

Non-alcoholic steatohepatitis (NASH) is a progressive, chronic, liver disease whose prevalence is growing worldwide. Despite several agents being under development for treating NASH, there are no drugs currently approved. The Farnesoid-x-receptor (FXR) and the G-protein coupled bile acid receptor 1 (GPBAR1), two bile acid activated receptors, have been investigated for their potential in treating NASH. Here we report that BAR502, a steroidal dual ligand for FXR/GPBAR1, attenuates development of clinical and liver histopathology features of NASH in mice fed a high fat diet (HFD) and fructose (F). By RNAseq analysis of liver transcriptome we found that BAR502 restores FXR signaling in the liver of mice feed HFD-F, and negatively regulates a cluster of genes including Srebf1 (Srepb1c) and its target genes-fatty acid synthase (Fasn) and Cell death-inducing DFF45-like effector (CIDE) genes, Cidea and Cidec-involved in lipid droplets formation and triglycerides storage in hepatocytes. Additionally, BAR502 increased the intestinal expression of Fgf15 and Glp1 and energy expenditure by white adipose tissues. Finally, exposure to BAR502 reshaped the intestinal microbiota by increasing the amount of Bacteroidaceae. In conclusion, we have shown that dual FXR/GPBAR1 agonism might have utility in treatment of NASH.

BAR502, a dual FXR and GPBAR1 agonist, promotes browning of white adipose tissue and reverses liver steatosis and fibrosis.

Non-alcoholic steatohepatitis (NASH) is a highly prevalent chronic liver disease. Here, we have investigated whether BAR502, a non-bile acid, steroidal dual ligand for FXR and GPBAR1, reverses steato-hepatitis in mice fed a high fat diet (HFD) and fructose. After 9 week, mice on HFD gained ≈30% of b.w (P < 0.01 versus naïve) and were insulin resistant. These overweighting and insulin resistant mice were randomized to receive HFD or HFD in combination with BAR502. After 18 weeks, HFD mice developed NASH like features with severe steato-hepatitis and fibrosis, increased hepatic content of triacylglycerol and cholesterol and expression of SREPB1c, FAS, ApoC2, PPARα and γ, α-SMA, α1 collagen and MCP1 mRNAs. Treatment with BAR502 caused a ≈10% reduction of b.w., increased insulin sensitivity and circulating levels of HDL, while reduced steatosis, inflammatory and fibrosis scores and liver expression of SREPB1c, FAS, PPARγ, CD36 and CYP7A1 mRNA. BAR502 increased the expression of SHP and ABCG5 in the liver and SHP, FGF15 and GLP1 in intestine. BAR502 promoted the browning of epWAT and reduced liver fibrosis induced by CCl4. In summary, BAR502, a dual FXR and GPBAR1 agonist, protects against liver damage caused by HFD by promoting the browning of adipose tissue.

Impaired Itching Perception in Murine Models of Cholestasis Is Supported by Dysregulation of GPBAR1 Signaling.

BACKGROUND & AIMS: In cholestatic syndromes, body accumulation of bile acids is thought to cause itching. However, the mechanisms supporting this effect remain elusive. Recently, GPBAR1 (TGR5) a G-protein coupled receptor has been shown to mediate itching caused by intradermal administration of DCA and LCA. 6α-ethyl-3α, 7α-dihydroxy-24-nor-5ß-cholan-23-ol (BAR502) is a non-bile acid dual ligand for FXR and GPBAR1. METHODS: Cholestasis was induced in wild type and GPBAR1-/- mice by administration of α-naphthyl-isothiocyanate (ANIT) or 17α-ethynylestradiol. RESULTS: In naïve mice skin application of DCA, TLCA, 6-ECDCA, oleanolic and betulinic acid induces a GPBAR1 dependent pruritogenic response that could be desensitized by re-challenging the mice with the same GPBAR1 agonist. In wild type and GPBAR1-/- mice cholestasis induced by ANIT fails to induce spontaneous itching and abrogates scratching behavior caused by intradermal administration of DCA. In this model, co-treatment with BAR502 increases survival, attenuates serum alkaline phosphatase levels and robustly modulates the liver expression of canonical FXR target genes including OSTα, BSEP, SHP and MDR1, without inducing pruritus. Betulinic acid, a selective GPBAR1 ligand, failed to rescue wild type and GPBAR1-/- mice from ANIT cholestasis but did not induced itching. In the 17α-ethynylestradiol model BAR502 attenuates cholestasis and reshapes bile acid pool without inducing itching. CONCLUSIONS: The itching response to intradermal injection of GPBAR1 agonists desensitizes rapidly and is deactivated in models of cholestasis, explain the lack of correlation between bile acids levels and itching severity in cholestatic syndromes. In models of non-obstructive cholestasis, BAR502 attenuates liver injury without causing itching.

Antibacterial properties of bile acid oxazoline compounds.

Chenooxazoline (50-100 microM) inhibited (greater than 50%) both 7 alpha and 7 beta-dehydroxylase activities in whole cells and cell extracts of Eubacterium sp. V.P.I. 12708. Chenooxazoline (greater than or equal to 50 microM) and methylchenooxazoline (greater than 25 microM) but not lithooxazoline (less than or equal to 100 microM) inhibited growing cultures of Eubacterium sp. V.P.I. 12708. Chenooxazoline (100 microM) also inhibited the growth of certain members of the genera Eubacterium, Clostridium, Bacteroides and Staphylococcus but not Pseudomonas, Escherichia, Salmonella or the eucaryotic microorganism, Saccharomyces cerevisiae (less than or equal to 400 microM).

Quantitative structure-antimicrobial activity relationship in 5 beta-cholanyl-24-benzylamine derivatives.

Some representative physicochemical properties of benzylamido and amino derivatives of common bile acids have been determined and correlated with their antimicrobial activity against gram-positive bacterial strains. Steroid hydroxyls do not affect the basicity of amino derivatives; they promote solubility in a parallel way to unconjugated bile acids and mainly control hydrophobicity of this class of compounds as measured by log P values. Activity was correlated to hydrophobicity; that is, the nature of the side chain modulated activity, affected basicity, and facilitated changes in partition ability. Benzylamino derivatives proved to be even more active than the corresponding amides when ionization is taken into account. Trihydroxy derivatives possess the lowest log P values and were practically inactive. Decreased activity was also observed in those cases where, due to the orientation of the hydroxy group in the 6 or 7 position, the back beta face of the molecule had a reduced hydrophobic surface area. Antimicrobial activity, in terms of -log MIC (minimal inhibitory concentration), was found to correlate linearly with log P values of uncharged species. This linear relationship is discussed with respect to the structure of the steroid moiety and the ability of these molecules to cross cellular membranes.

Effect of basic cholane derivatives on intestinal cholic acid metabolism: in vitro and in vivo activity.

A representative series of hydroxy-5 beta-cholanyl-24-amines were tested both in vitro and in vivo with respect to their activity against the intestinal bacteria responsible for bile acid metabolism. For the in vitro studies, radiolabeled [14C]cholic acid was incubated with human stools both in aerobic and anaerobic conditions in the presence of the title compounds at a dose of 10 micrograms/mL, and the biotransformation of cholic acid into radiolabeled deoxycholic acid and other metabolites was followed by TLC-radiochromatography. Of the compounds studied, 3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-N-methylamine showed the highest activity. This compound was used for the in vivo studies and was shown to inhibit the formation of endogenous secondary bile acids when chronically administered to rats at a dose of 60 micrograms/day for 15 days. The treated rats showed an increased ratio of taurocholic acid (primary bile acid) to taurodeoxycholic acid (secondary bile acid) in bile, a fact further suggesting a potent antibacterial activity of the compound toward bacteria responsible for bile acid metabolism.

Effects of bile acid oxazolines on gallstone formation in prairie dogs.

The effects of 2 bile acid analogs, chenodeoxy-oxazoline [2-(3 alpha, 7 alpha-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2-oxazoline] and ursodeoxy-oxazoline [2-(3 alpha, 7 beta-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2-oxazoline] were examined in the prairie dog model of cholesterol cholelithiasis. Gallstones and biliary cholesterol crystals were induced in 5 out of 6 male prairie dogs fed a semisynthetic diet containing 0.4% cholesterol for 8 weeks. Six animals maintained on a low cholesterol control diet (0.08% cholesterol) exhibited neither gallstones nor biliary cholesterol crystals. The addition of 0.06% chenodeoxy-oxazoline to the lithogenic diet did not prevent induced cholelithiasis or the appearance of cholesterol crystals in bile. In contrast, 0.06% dietary ursodeoxy-oxazoline prevented gallstones in 5 out of 6 prairie dogs (but cholesterol crystals were present in the bile of 4 of these animals). Histologically, most of the livers from the prairie dogs fed the cholesterol-supplemented semisynthetic diet showed bile duct proliferation, inflammatory infiltration and fibrosis along the portal tracts. These pathologic changes were generally not ameliorated by adding chenodeoxy-oxazoline or chenodeoxy-oxazoline plus chenodeoxycholic acid to the diet. Portal tract pathology was markedly reduced in most animals by adding ursodeoxy-oxazoline to the cholesterol-supplemented diet. The pathologic changes overall could best be correlated with the presence of gallstones, but not with the incidence of biliary cholesterol crystals.

Design, synthesis, and biological evaluation of potent dual agonists of nuclear and membrane bile acid receptors.

Bile acids exert genomic and nongenomic effects by interacting with membrane G-protein-coupled receptors, including the bile acid receptor GP-BAR1, and nuclear receptors, such as the farnesoid X receptor (FXR). These receptors regulate overlapping metabolic functions; thus, GP-BAR1/FXR dual agonists, by enhancing the biological response, represent an innovative strategy for the treatment of enteroendocrine disorders. Here, we report the design, total synthesis, and in vitro/in vivo pharmacological evaluation of a new generation of dual bile acid receptor agonists, with the most potent compound, 19, showing promising pharmacological profiles. We show that compound 19 activates GP-BAR1, FXR, and FXR regulated genes in the liver, increases the intracellular concentration of cAMP, and stimulates the release of the potent insulinotropic hormone GLP-1, resulting in a promising drug candidate for the treatment of metabolic disorders. We also elucidate the binding mode of the most potent dual agonists in the two receptors through a series of computations providing the molecular basis for dual GP-BAR1/FXR agonism.

Marine sponge steroids as nuclear receptor ligands.

Nuclear receptors (NRs) are a large family of evolutionarily conserved and ligand-regulated transcription factors. The farnesoid X receptor (FXR) and the pregnane X receptor (PXR) are two bile-acid-activated receptors highly expressed in enterohepatic tissues essential for bile acids and xenobiotic metabolism. More than 1600 new steroidal structures have been isolated from marine organisms. Chemical, structural, and pharmacological characterization of sponge steroid libraries has allowed the identification of steroids that regulate FXR and PXR: selective FXR antagonists, FXR modulators, FXR antagonists endowed with PXR agonism, and selective PXR agonists. Selective FXR antagonists (theonellasterol) have proven effective in protecting against liver injury in models of cholestasis. Selective PXR agonists (natural and synthetic solomonsterols) have been effective in reducing nuclear factor (NF)-κB activity and intestinal inflammation. Identification of marine steroids endowed with dual FXR and PXR agonism-antagonism probably reflects the common identity of the unique ancestral precursor of these NRs. These findings pave the way to the development of novel FXR and PXR agonists and antagonists to target human diseases.

Solomonsterols A and B from Theonella swinhoei. The first example of C-24 and C-23 sulfated sterols from a marine source endowed with a PXR agonistic activity.

The finding of new PXR modulators as potential leads for treatment of human disorders characterized by dysregulation of innate immunity and with inflammation is of wide interest. In this paper, we report the identification of the first example of natural marine PXR agonists, solomonsterols A and B, from a Theonella swinhoei sponge. The structures were determined by interpretation of NMR and ESIMS data, and the putative binding mode to PXR has been obtained through docking calculations.

Total synthesis and pharmacological characterization of solomonsterol A, a potent marine pregnane-X-receptor agonist endowed with anti-inflammatory activity.

Recently, we reported the identification of a novel class of pregnane-X-receptor (PXR) agonists, solomonsterols A and B, isolated from the marine sponge Theonella swinhoei. Preliminary pharmacological studies demonstrated that these natural compounds are potential leads for the treatment of human disorders characterized by dysregulation of innate immunity. In this article, we describe the first total synthesis of solomonsterol A and its in vivo characterization in animal models of colitis. Using transgenic mice expressing the human PXR, we found that administration of synthetic solomonsterol A effectively protects against development of clinical signs and symptoms of colitis and reduced the generation of TNFα, a signature cytokine for this disorder. In addition, we have provided the first evidence that solomonsterol A might act by triggering the expression of TGFß and IL-10, potent counter-regulatory cytokines in inflammatory bowel diseases (IBD). Finally, we have shown that solomonsterol A inhibits NF-κB activation by a PXR dependent mechanism. In summary, solomonsterol A is a marine PXR agonist that holds promise in the treatment of inflammation-driven immune dysfunction in clinical settings.