siRNA-Mediated Timp1 Silencing Inhibited the Inflammatory Phenotype during Acute Lung Injury.

Acute lung injury is a complex cascade process that develops in response to various damaging factors, which can lead to acute respiratory distress syndrome. Within this study, based on bioinformatics reanalysis of available full-transcriptome data of acute lung injury induced in mice and humans by various factors, we selected a set of genes that could serve as good targets for suppressing inflammation in the lung tissue, evaluated their expression in the cells of different origins during LPS-induced inflammation, and chose the tissue inhibitor of metalloproteinase Timp1 as a promising target for suppressing inflammation. We designed an effective chemically modified anti-TIMP1 siRNA and showed that Timp1 silencing correlates with a decrease in the pro-inflammatory cytokine IL6 secretion in cultured macrophage cells and reduces the severity of LPS-induced acute lung injury in a mouse model.

Dual Effect of Soloxolone Methyl on LPS-Induced Inflammation In Vitro and In Vivo.

Plant-extracted triterpenoids belong to a class of bioactive compounds with pleotropic functions, including antioxidant, anti-cancer, and anti-inflammatory effects. In this work, we investigated the anti-inflammatory and anti-oxidative activities of a semisynthetic derivative of 18ßH-glycyrrhetinic acid (18ßH-GA), soloxolone methyl (methyl 2-cyano-3,12-dioxo-18ßH-olean-9(11),1(2)-dien-30-oate, or SM) in vitro on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and in vivo in models of acute inflammation: LPS-induced endotoxemia and carrageenan-induced peritonitis. SM used at non-cytotoxic concentrations was found to attenuate the production of reactive oxygen species and nitric oxide (II) and increase the level of reduced glutathione production by LPS-stimulated RAW264.7 cells. Moreover, SM strongly suppressed the phagocytic and migration activity of activated macrophages. These effects were found to be associated with the stimulation of heme oxigenase-1 (HO-1) expression, as well as with the inhibition of nuclear factor-κB (NF-κB) and Akt phosphorylation. Surprisingly, it was found that SM significantly enhanced LPS-induced expression of the pro-inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in RAW264.7 cells via activation of the c-Jun/Toll-like receptor 4 (TLR4) signaling axis. In vivo pre-exposure treatment with SM effectively inhibited the development of carrageenan-induced acute inflammation in the peritoneal cavity, but it did not improve LPS-induced inflammation in the endotoxemia model.

Novel 3'-Substituted-1',2',4'-Oxadiazole Derivatives of 18ßH-Glycyrrhetinic Acid and Their O-Acylated Amidoximes: Synthesis and Evaluation of Antitumor and Anti-Inflammatory Potential In Vitro and In Vivo.

A series of novel 18ßH-glycyrrhetinic acid (GA) derivatives containing 3'-(alkyl/phenyl/pyridin(-2″, -3″, and -4″)-yl)-1',2',4'-oxadiazole moieties at the C-30 position were synthesized by condensation of triterpenoid's carboxyl group with corresponding amidoximes and further cyclization. Screening of the cytotoxicity of novel GA derivatives on a panel of tumor cell lines showed that the 3-acetoxy triterpenoid intermediates-O-acylated amidoxime 3a-h-display better solubility under bioassay conditions and more pronounced cytotoxicity compared to their 1',2',4'-oxadiazole analogs 4f-h (median IC50 = 7.0 and 49.7 µM, respectively). Subsequent replacement of the 3-acetoxy group by the hydroxyl group of pyridin(-2″, 3″, and -4″)-yl-1',2',4'-oxadiazole-bearing GA derivatives produced compounds 5f-h, showing the most pronounced selective toxicity toward tumor cells (median selectivity index (SI) > 12.1). Further detailed analysis of the antitumor activity of hit derivative 5f revealed its marked proapoptotic activity and inhibitory effects on clonogenicity and motility of HeLa cervical carcinoma cells in vitro, and the metastatic growth of B16 melanoma in vivo. Additionally, the comprehensive in silico study revealed intermediate 3d, bearing the tert-butyl moiety in O-acylated amidoxime, as a potent anti-inflammatory candidate, which was able to effectively inhibit inflammatory response induced by IFNγ in macrophages in vitro and carrageenan in murine models in vivo, probably by primary interactions with active sites of MMP9, neutrophil elastase, and thrombin. Taken together, our findings provide a basis for a better understanding of the structure-activity relationship of 1',2',4'-oxadiazole-containing triterpenoids and reveal two hit molecules with pronounced antitumor (5f) and anti-inflammatory (3d) activities.

Trioxolone Methyl, a Novel Cyano Enone-Bearing 18ßH-Glycyrrhetinic Acid Derivative, Ameliorates Dextran Sulphate Sodium-Induced Colitis in Mice.

Semi-synthetic triterpenoids, bearing cyano enone functionality in ring A, are considered to be novel promising therapeutic agents with complex inhibitory effects on tissue damage, inflammation and tumor growth. Previously, we showed that the cyano enone-containing 18ßH-glycyrrhetinic acid derivative soloxolone methyl (SM) effectively suppressed the inflammatory response of macrophages in vitro and the development of influenza A-induced pneumonia and phlogogen-stimulated paw edema in vivo. In this work, we reported the synthesis of a novel 18ßH-glycyrrhetinic acid derivative trioxolone methyl (TM), bearing a 2-cyano-3-oxo-1(2)-en moiety in ring A and a 12,19-dioxo-9(11),13(18)-dien moiety in rings C, D, and E. TM exhibited a high inhibitory effect on nitric oxide (II) production by lipopolysaccharide-stimulated J774 macrophages in vitro and dextran sulfate sodium (DSS)-induced colitis in mice, displaying higher anti-inflammatory activity in comparison with SM. TM effectively suppressed the DSS-induced epithelial damage and inflammatory infiltration of colon tissue, the hyperproduction of colonic neutral mucin and TNFα and increased glutathione synthesis. Our in silico analysis showed that Akt1, STAT3 and dopamine receptor D2 can be considered as mediators of the anti-colitic activity of TM. Our findings provided valuable information for a better understanding of the anti-inflammatory activity of cyano enone-bearing triterpenoids and revealed TM as a promising anti-inflammatory candidate.

Deep insights into the response of human cervical carcinoma cells to a new cyano enone-bearing triterpenoid soloxolone methyl: a transcriptome analysis.

Semisynthetic triterpenoids, bearing cyano enone functionality in ring A, are considered now as novel promising anti-tumor agents. However, despite the large-scale studies, their effects on cervical carcinoma cells and, moreover, mechanisms underlying cell death activation by such compounds in this cell type have not been fully elucidated. In this work, we attempted to reconstitute the key pathways and master regulators involved in the response of human cervical carcinoma KB-3-1 cells to the novel glycyrrhetinic acid derivative soloxolone methyl (SM) by a transcriptomic approach. Functional annotation of differentially expressed genes, analysis of their cis- regulatory sequences and protein-protein interaction network clearly indicated that stress of endoplasmic reticulum (ER) is the central event triggered by SM in the cells. A range of key ER stress sensors and transcription factor AP-1 were identified as upstream transcriptional regulators, controlling the response of the cells to SM. Additionally, by using Gene Expression Omnibus data, we showed the ability of SM to modulate the expression of key genes involved in regulation of the high proliferative rate of cervical carcinoma cells. Further Connectivity Map analysis revealed similarity of SM's effects with known ER stress inducers thapsigargin and geldanamycin, targeting SERCA and Grp94, respectively. According to the molecular docking study, SM could snugly fit into the active sites of these proteins in the positions very close to that of both inhibitors. Taken together, our findings provide a basis for the better understanding of the intracellular processes in tumor cells switched on in response to cyano enone-bearing triterpenoids.

Novel Derivatives of Deoxycholic Acid Bearing Linear Aliphatic Diamine and Aminoalcohol Moieties and their Cyclic Analogs at the C3 Position: Synthesis and Evaluation of Their In Vitro Antitumor Potential.

A series of novel deoxycholic acid (DCA) derivatives containing aliphatic diamine and aminoalcohol or morpholine moieties at the C3 position were synthesized by 3,26-epoxide ring-opening reactions. These compounds were investigated for their cytotoxicity in four human tumor cell lines and murine macrophages and for inhibitory activity against macrophage-mediated NO synthesis in vitro. Obtained data revealed that: (i) all amine-containing substituents significantly increased the cytotoxicity of the novel compounds (IC502-10 = 1.0-36.0 µM) in comparison with DCA (IC50DCA ≥ 82.9 µM); (ii) aminoalcohol moieties were more preferable than diamine moieties due to the fact they imparted better selectivity for tumor cells of the novel derivatives; (iii) the susceptibility of tested cell lines to derivatives diminished in the following order: HuTu-80 (duodenal carcinoma) ≈ HepG2 (hepatocarcinoma) > KB-3-1 (cervical carcinoma) > RAW264.7 (macrophages) > A549 (lung carcinoma); (iv) compounds 8 and 9, bearing aminoethanol and aminopropanol moieties, respectively, exhibited high cytotoxic selectivity indexes (SIHuTu-80 = 7.9 and 8.3, respectively) and good drug-likeness parameters; (v) the novel compounds do not display anti-NO activity. Mechanistic study revealed that compound 9 induces ROS-dependent cell death by activation of intrinsic caspase-dependent apoptosis and cytodestructive autophagy in HuTu-80 cells and vitamin D receptor can be considered as its primary target.

Novel 5'-Norcarbocyclic Derivatives of Bicyclic Pyrrolo- and Furano[2,3-d]Pyrimidine Nucleosides.

Here we report the synthesis and biological activity of new 5'-norcarbocyclic derivatives of bicyclic pyrrolo- and furano[2,3-d]pyrimidines with different substituents in the heterocyclic ring. Lead compound 3i, containing 6-pentylphenyl substituent, displays inhibitory activity with respect to a number of tumor cells with a moderate selectivity index value. Compound 3i induces cell death by the apoptosis pathway with the dissipation of mitochondrial potential.

Soloxolone methyl inhibits influenza virus replication and reduces virus-induced lung inflammation.

Highly pathogenic influenza viruses pose a serious public health threat to humans. Although vaccines are available, new antivirals are needed to efficiently control disease progression and virus transmission due to the emergence of drug-resistant viral strains. In this study, we describe the anti-viral properties of Soloxolone methyl (SM) (methyl 2-cyano-3,12-dioxo-18ßH-olean-9(11),1(2)-dien-30-oate, a chemical derivative of glycyrrhetinic acid) against the flu virus. Anti-flu efficacy studies revealed that SM exhibits antiviral activity against the H1N1 influenza A virus in a dose-dependent manner causing a more than 10-fold decrease in virus titer and a reduction in the expression of NP and M2 viral proteins. In a time-of-addition study, SM was found to act at an early stage of infection to exhibit an inhibitory effect on both the attachment step and virus uptake into cells. Also, in infected cells SM downregulates the expression of the inflammatory cytokines IL-6 and TNF-α. In infected mice, SM administered intranasally prior to and after infection significantly decreases virus titers in the lung and prevents post-challenge pneumonia. Together, these results suggest that Soloxolone methyl might serve as an effective therapeutic agent to manage influenza outbreaks and virus-associated complications, and further preclinical and clinical investigation may be warranted.

Synthesis and evaluation of antitumor, anti-inflammatory and analgesic activity of novel deoxycholic acid derivatives bearing aryl- or hetarylsulfanyl moieties at the C-3 position.

Novel deoxycholic acid (DCA) derivatives were stereoselectively synthesised with -OH and -CH2SR moieties at the C-3 position, where R was a substituted aryl [2-aminophenyl (8) or 4-chlorophenyl (9)] or hetaryl [1-methylimidazolyl (5), 1,2,4-triazolyl (6), 5-amino-1,3,4-thiadiazolyl (7), pyridinyl (10) or pyrimidinyl (11)]. These compounds were prepared in good yields from the C-3ß-epoxy derivative 2 in the epoxide ring-opening reaction by S-nucleophiles. These derivatives were evaluated for their in vitro anti-proliferation activity in a panel of tumor cell lines. Data showed that: (i) heterocycle-containing derivatives displayed higher cytotoxicity profiles than the parent molecule; (ii) heterocyclic substituents were more preferable than aryl moieties for enhancing anti-proliferation activity; (iii) the sensitivity of tumor cell lines to analysed compounds decreased in the following order: HuTu-80 (duodenal carcinoma)>KB-3-1 (cervical carcinoma)>HepG2 (hepatocellular carcinoma)>MH-22a (hepatoma); (iv) compounds 5, 6 and 11 exhibited a high cytotoxic selectivity index (HuTu-80: SI>7.7, 38.5 and 12.0, respectively). Compounds 2 and 6-8 markedly inhibited NO synthesis by interferon γ-induced macrophages. Screening for anti-inflammatory activity of these derivatives in vivo showed their high potency on histamine- (5, 10) and formalin- (2, 10, 11) induced paw edema models.

Novel derivatives of deoxycholic acid bearing aliphatic or cyclic diamine moieties at the C-3 position: Synthesis and evaluation of anti-proliferative activity.

A new library of deoxycholic acid derivatives bearing nitrogen-containing moieties at the C-3 position was synthesised from epoxy derivative 1 via an epoxide ring-opening reaction promoted by aliphatic or cyclic diamines and fully characterised by NMR and mass-spectroscopy. The synthesised compounds were screened for cytotoxicity against four human tumour cell lines. The results showed that some of the novel diamine-bearing derivatives displayed improved anti-proliferative activities over the parent compound DCA. Among them, a 1-methylpiperazine containing compound (6) showed promising activity and the highest selectivity against tumour cells of enterohepatic origin (HepG2: IC50=3.6µM, SI=9.0; HuTu-80: IC50=4.6µM, SI=6.9) and was identified as a lead molecule.

Modulation of Tumour-Related Signaling Pathways by Natural Pentacyclic Triterpenoids and their Semisynthetic Derivatives.

Pentacyclic triterpenoids are a large class of natural isoprenoids that are widely biosynthesized in higher plants. These compounds are potent anticancer agents that exhibit antiproliferative, antiangiogenic, antiinflammatory and proapoptotic activities. Although their effects on multiple pathways have been reported, unifying mechanisms of action have not yet been established. To date, a huge number of semisynthetic derivatives have been synthesized in different laboratories on the basis of triterpenoid scaffolds, and many have been assayed for their biological activities. The present review focuses on natural triterpenoids of the oleanane-, ursane- and lupane-types and their semisynthetic derivatives. Here, we summarize the diverse cellular and molecular targets of these compounds and the signal pathways involved in the performance of their antitumour actions. Among the most relevant mechanisms involved are cell cycle arrest, apoptosis and autophagy triggered by the effect of triterpenoids on TGF-ß and HER cell surface receptors and the downstream PI3KAkt- mTOR and IKK/NF-kB signaling axis, STAT3 pathway and MAPK cascades.

Synthesis and biological activity of novel deoxycholic acid derivatives.

We report the synthesis and biological activity of new semi-synthetic derivatives of naturally occurring deoxycholic acid (DCA) bearing 2-cyano-3-oxo-1-ene, 3-oxo-1(2)-ene or 3-oxo-4(5)-ene moieties in ring A and 12-oxo or 12-oxo-9(11)-ene moieties in ring C. Bioassays using murine macrophage-like cells and tumour cells show that the presence of the 9(11)-double bond associated with the increased polarity of ring A or with isoxazole ring joined to ring A, improves the ability of the compounds to inhibit cancer cell growth.

Synthesis of novel 2-cyano substituted glycyrrhetinic acid derivatives as inhibitors of cancer cells growth and NO production in LPS-activated J-774 cells.

Here we report the synthesis and biological activity of new semi-synthetic derivatives of naturally occurring glycyrrhetinic acid bearing a 2-cyano-3-oxo-1-en moiety in the A-ring and double bonds and carbonyl groups in the C, D and E rings. Bioassays using murine macrophage-like and tumor cells show that compound 4, which differs from Soloxolone methyl by the absence of a 9(11)-double bond in the C-ring, displays anti-inflammatory and inhibitory activities with respect to tumor cells with a high selectivity index value.

Synthesis and pro-apoptotic activity of novel glycyrrhetinic acid derivatives.

Triterpenoids are used for medicinal purposes in many countries. Some, such as oleanolic and glycyrrhetinic acids, are known to be anti-inflammatory and anticarcinogenic. However, the biological activities of these naturally occurring molecules against their particular targets are weak, so the synthesis of new synthetic analogues with enhanced potency is needed. By combining modifications to both the A and C rings of 18ßH-glycyrrhetinic acid, the novel synthetic derivative methyl 2-cyano-3,12-dioxo-18ßH-olean-9(11),1(2)-dien-30-oate was obtained. This derivative displays high antiproliferative activity in cancer cells, including a cell line with a multidrug-resistance phenotype. It causes cell death by inducing the intrinsic caspase-dependent apoptotic pathway.