Synthesis and biological evaluation of novel hydrogen sulfide releasing glycyrrhetic acid derivatives

<p>A series of hybrids, which are composed of glycyrrhetic acid (GA) and slowly hydrogen sulfide-releasing donor ADT-OH, were designed and synthesized to develop anticancer and anti-inflammatory agents. Most of the compounds, whose inhibitory rates were comparable to or higher than those of GA and aspirin, respectively, significantly inhibited xylene-induced ear edema in mice. Especially, compound V<b>₄</b>exhibited the most potent inhibitory rate of 60.7%. Furthermore, preliminary structure–activity relationship studies demonstrated that 3-substituted GA derivatives had stronger anti-inflammatory activities than the corresponding 3-unsubstituted GA derivatives. In addition, anti-proliferative activities of compounds <b>V₁₋₉</b> were evaluated in three different human cancer cell lines. Compound <b>V₄</b> showed the most high potency against all three tumor cell lines with IC₅₀ values ranging from 10.01 μM in Hep G2 cells to 17.8 μM in MDA-MB-231 cells, which were superior to positive GA.</p

Iron-Catalyzed 1,2-Selective Hydroboration of <i>N</i>‑Heteroarenes

A N₂-bridged diiron complex [Cp*­(Ph₂PC₆­H₄S)­Fe]₂­(μ-N₂) (<b>1</b>) has been found to catalyze the hydroboration of <i>N</i>-heteroarenes with pinacolborane, giving <i>N</i>-borylated 1,2-reduced products with high regioselectivity. The catalysis is initiated by coordination of <i>N</i>-heteroarenes to the iron center, while the B–H bond cleavage is the rate-determining step

Cysteine Oxidation Reactions Catalyzed by a Mononuclear Non-heme Iron Enzyme (OvoA) in Ovothiol Biosynthesis

OvoA in ovothiol biosynthesis is a mononuclear non-heme iron enzyme catalyzing the oxidative coupling between histidine and cysteine. It can also catalyze the oxidative coupling between hercynine and cysteine, yet with a different regio-selectivity. Due to the potential application of this reaction for industrial ergothioneine production, in this study, we systematically characterized OvoA by a combination of three different assays. Our studies revealed that OvoA can also catalyze the oxidation of cysteine to either cysteine sulfinic acid or cystine. Remarkably, these OvoA-catalyzed reactions can be systematically modulated by a slight modification of one of its substrates, histidine

Activation of Epoxides by a Cooperative Iron–Thiolate Catalyst: Intermediacy of Ferrous Alkoxides in Catalytic Hydroboration

This paper describes a cooperative iron–thiolate catalyst Cp*Fe­(1,2-Ph₂PC₆H₄S)­(NCMe) (Cp*^– = C₅Me₅^–, [<b>1</b>(NCMe)]) for regioselective hydroboration of aryl epoxide by pinacolborane (HBpin). The critical catalytic step involves the direct addition of epoxide to the catalyst rather than activation of the B–H bond of HBpin. Through iron–thiolate cooperation, [<b>1</b>(NCMe)] opens the aryl epoxide rings affording ferrous–alkoxide compounds. Notably, the ferrous–alkoxide intermediate (<b>4</b>) was structurally characterized after its isolation from the reaction of [<b>1</b>(NCMe)] with <i>trans</i>-2,3-diphenyloxirane. The more Lewis acidic hydroboranes such as H₃B·THF and 9-BBN (BBN = bora­bicyclo­nonane) can also be captured by [<b>1</b>(NCMe)]. The resulting iron–borane adducts [<b>1</b>H­(BH₂)] and [<b>1</b>H­(BBN)] feature an agnostic Fe···B–H interaction. DFT calculations indicate that the addition of HBpin across the iron–thiolate sites is endergonic by 12.9 kcal/mol, whereas it is exergonic by 20.2 kcal/mol with BH₃ and 4.6 kcal/mol with 9-BBN. Combining the experimental data with theoretical studies, a mechanism of the substrate activation by [<b>1</b>(NCMe)], followed by HBpin addition, is proposed for the catalysis

Use of a Tyrosine Analogue To Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C–S Bond Formation

Ovothiol is a histidine thiol derivative. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidation of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addition, by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3-(methylthio)­phenyl)­propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways

Bioinformatic and Biochemical Characterizations of C–S Bond Formation and Cleavage Enzymes in the Fungus <i>Neurospora crassa</i> Ergothioneine Biosynthetic Pathway

Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C–S bond formation and a PLP-mediated C–S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus <i>Neurospora crassa</i> has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the <i>N. crassa</i> C–S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible

FTY720 Induces Apoptosis of M2 Subtype Acute Myeloid Leukemia Cells by Targeting Sphingolipid Metabolism and Increasing Endogenous Ceramide Levels

<div><p>The M2 subtype Acute Myeloid Leukemia (AML-M2) with t(8;21) represents an unmet challenge because of poor clinical outcomes in a sizable portion of patients. In this study,we report that FTY720 (Fingolimod), a sphingosine analogue and an FDA approved drug for treating of multiple sclerosis, shows antitumorigenic activity against the Kasumi-1 cell line, xenograft mouse models and leukemic blasts isolated from AML-M2 patients with t(8;21) translocation. Primary investigation indicated that FTY720 caused cell apoptosis through caspases and protein phosphatase 2A (PP2A) activation. Transcriptomic profiling further revealed that FTY720 treatment could upregulate AML1 target genes and interfere with genes involved in ceramide synthesis. Treatment with FTY720 led to the elimination of AML1-ETO oncoprotein and caused cell cycle arrest. More importantly, FTY720 treatment resulted in rapid and significant increase of pro-apoptotic ceramide levels, determined by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry based lipidomic approaches. Structural simulation model had also indicated that the direct binding of ceramide to inhibitor 2 of PP2A (I2PP2A) could reactivate PP2A and cause cell death. This study demonstrates, for the first time, that accumulation of ceramide plays a central role in FTY720 induced cell death of AML-M2 with t(8;21). Targeting sphingolipid metabolism by using FTY720 may provide novel insight for the drug development of treatment for AML-M2 leukemia.</p></div

FTY720 induces Kasumi-1 cell apoptosis primarily through elevating ceramide levels.

<p>(A, B) Ceramide levels in Kasumi-1 cells treated FTY720 with indicated concentrations (A) and different times (B) were determined by HPLC-ESI-MS/MS assay, TC means total ceramide (C14-, C16-, C18-, C20-, and C22-Ceramide). *P<0.05. (C) Kasumi-1 cells were treated with FTY720 for indicated times, and mitochondria was separated. The contents of ceramide in mitochondria of FTY720 treated Kasumi-1 cells were determined by HPLC-ESI-MS/MS assay. * P<0.05. (D) Kasumi-1 cells treated with or without Ara-C (7.5 µM) for 24 h, the contents of sphingolipid were determined by HPLC-ESI-MS/MS assay. (E, F) Kasumi-1 cells treated with indicated concentrations of FTY720 for 24 h, the contents of S1P (E) or SPH (F) were determined by HPLC-ESI-MS/MS assay. *P<0.05. (G) Kasumi-1 cells treated with indicated concentrations of FTY720 for 24 h, the signals of sphingolipids detected by HPLC-ESI-MS/MS assay were shown. (H) Kasumi-1 cells were pretreated with 20 µM FB1 or GW4869 for 3 h, and treated with 7.5 µM FTY720 for further 21 h. The effects of FB1 or GW4869 on FTY720-induced apoptosis were evaluated by flow cytometry. *P<0.05. (I) Kasumi-1 cells were pretreated with amitriptyline for 3 h, and treated with 7.5 µM FTY720 for further 21 h. The effects of amitriptyline on FTY720-mediated apoptosis were determined by flow cytometry.</p

FTY720 induces elimination of AML1-ETO oncoprotein and causes cell cycle arrest.

<p>(A) Kasumi-1 cells were treated with indicated concentrations of FTY720 for 24 h, and cell lysate was subjected to western blotting analysis. Anti-ETO antibody was used to detect AML1-ETO fusion protein. (B) Kasumi-1 cells were treated with FTY720 for 72 hours, and cell cycle of Kasumi-1 cells treated FTY720 was analyzed by flow cytometry after propidium iodide (PI) staining, representative histograms were shown. (C) Cell cycle progression of Kasumi-1 treated with FTY720 was evaluated, nuclei stained with PI was used to DNA content analysis by flow cytometry. (D) Morphologic changes in Kasumi-1 treated for 3 days with indicated concentrations of FTY720. (E, F) Quantification of CD11b (E), CD11c (F), and CD117 expression in Kaumi-1 cells treated for 3 days with FTY720 was detected with flow cytometry.</p

<i>In vitro</i> efficacy of FTY720 on Kasumi-1 cells and fresh leukemic cells.

<p>(A) Kasumi-1 and SKNO-1 cells were treated with indicated concentration of FTY720 for 48 h, and the cell viability was determined with a CCK-8 kit. (B) Kasumi-1 cells were grown in methylcellulose for 14 d in presence of FTY720. **P<0.01, student's <i>t</i> test compared with untreated. (C) PBMCs from leukemia patients or healthy donor were treated with or without FTY720 for 24 h, and then subjected to apoptosis analysis by flow cytometry. *P<0.05, **P<0.01. (D) Kasumi-1 (top panel) and fresh leukemic cells (bottom panel) from AML-M2 patient were treated or untreated with FTY720 (7.5 µM), and analyzed by wright staining. Partial apoptotic cells were indicated by red arrows, and blue arrow presented cells might undergo differentiation.</p