Thiostrepton Research Articles

Thiostrepton Hijacks Pyoverdine Receptors To Inhibit Growth of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and is intrinsically resistant to many antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered that the thiopeptide antibiotic thiostrepton (TS), which is considered to be inactive against Gram-negative bacteria, stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is characteristic of exposure to antimicrobial compounds at subinhibitory concentrations, suggesting that TS was active against P. aeruginosa Supporting this observation, TS inhibited the growth of a panel of 96 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low-micromolar concentrations. TS also had activity against Acinetobacter baumannii clinical isolates. The expression of Tsr, a 23S rRNA-modifying methyltransferase from TS producer Streptomyces azureus, in trans conferred TS resistance, confirming that the drug acted via its canonical mode of action, inhibition of ribosome function. The deletion of oligopeptide permease systems used by other peptide antibiotics for uptake failed to confer TS resistance. TS susceptibility was inversely proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A. baumannii was potentiated by the FDA-approved iron chelators deferiprone and deferasirox and by heat-inactivated serum. Screening of P. aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to cross the outer membrane. We show that the biofilm stimulation phenotype can reveal cryptic subinhibitory antibiotic activity, and that TS has activity against select multidrug-resistant Gram-negative pathogens under iron-limited growth conditions, similar to those encountered at sites of infection.

8-bromo-7-methoxychrysin targets NF-κB and FoxM1 to inhibit lung cancer stem cells induced by pro-inflammatory factors.

We have previously reported that 8-bromo-7-methoxychrysin (BrMC), a novel synthetic derivative of chrysin, was demonstrated anti-tumor activities against several human cancers, including lung cancer. Interaction between inflammation and cancer stem cell are recently increasingly recognized in tumorigenesis and progression. The purpose of this study was to investigate whether BrMC inhibits lung cancer stemness of H460 cells induced by inflammatory factors (TGF-β combined with TNF-α) and its potential mechanism. Our results showed that BrMC inhibited lung cancer stemness, as validated by enhanced self-renewal ability, higher in vitro tumorigenicity, and increased expression of CD133, CD44, Bmi1 and Oct4 in H460 cells administered TNF-α after prolonged induction by TGF-β, in a concentration-dependent manner. Both NF-κB inhibition by SN50 and FoxM1 suppression by thiostrepton (THI) prompted the inhibition of BrMC on lung CSCs. Conversely, overexpression of NF-κBp65 significantly antagonized the above effects of BrMC. Meanwhile, overexpression of FoxM1 also significantly compromised BrMC function on suppression of FoxM1 and NF-κBp65 as well as stemness of lung CSCs. Our results suggest that activation of NF-κB and FoxM1 by cytokines facilitate the acquisition CSCs phenotype, and compromise the chemical inhibition, which may represent an effective therapeutic target for treatment of human lung cancer. Moreover, BrMC may be a potential promising candidate for targeting NF-κB/ FoxM1 to prevent the tumorigenesis under inflammatory microenvironment.

Development of co-solvent freeze-drying method for the encapsulation of water-insoluble thiostrepton in sterically stabilized micelles

The purpose of this work was to develop a practical and scalable method to encapsulate the hydrophobic antibiotic thiostrepton (TST) in sterically stabilized micelles (SSM). Using the conventional method of thin-film hydration, we encapsulated up to 5 drug molecules per SSM (diameter ∼ 16 nm). However, since this method is not suitable for large-scale production – a limiting factor for clinical translation – we applied the co-solvent freeze-drying method using tert-butanol (TBA): water co-solvent system. We found that the presence of phosphate-buffered saline (PBS) salts in the lyophilized cake accelerated the reconstitution time and allowed efficient drug encapsulation without the formation of larger drug particles. In addition, TBA proportion of 50% (v/v) was sufficient to maintain both phospholipid and drug in solution prior to the freeze-drying. The increase of drug and phospholipid concentrations in the formulation extended the reconstitution time and led to drug precipitation. Therefore, to increase the strength of the formulation, we prepared lyophilized cakes with lower phospholipid content (5 mM) and reconstituted them in one-third of the fill volume. In conclusion, we found optimum conditions to prepare TST-SSM using the co-solvent freeze-drying method. This scalable production method can facilitate the further clinical development and industrial production of TST-SSM nanomedicine.

Psychromicrobium lacuslunae sp. nov., isolated from a high altitude lake.

The novel strain IHBB 11108T was a psychrotolerant and alkaliphilic bacterium isolated from the subsurface water of Chandra Tal Lake in the Lahaul-Spiti valley located in the Indian trans-Himalayas. Cells were Gram-stain-positive, aerobic, non-motile, catalase-positive and oxidase-negative. The strain grew at 5-37 °C (optimum 28 °C), pH 5.0-12.0 (optimum pH 7.0) and with up to 8 % NaCl (optimum 1 %). The 16S rRNA gene sequence analysis revealed the highest relatedness of strain IHBB 11108T with Psychromicrobium silvestre DSM 102047T (97.5 %), Arthrobacter russicus DSM 14555T (97.4 %) and Renibacterium salmoninarum ATCC 33209T (97.4 %). The strain contained a quinone system with 57.2 % MK-9(H2), 39.1 % MK-10(H2), 3.0 % MK-8(H2) and 0.7 % MK-7(H2). The polar lipids detected were diphosphatidylglycerol, dimannosylglyceride, phosphatidylinositol, phosphatidylglycerol, monogalactosyldiacylglycerol, one unidentified glycolipid and four unidentified lipids. The cell-wall peptidoglycan structure type was A3α l-Lys-l-Thr-l-Ala with substitution of the α-carboxyl group of d-Glu by alanine amide. Anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0 were the predominant fatty acids. The genomic DNA G+C content was 59.0 mol%. The DNA-DNA relatedness of strain IHBB 11108T was 46.7±2.2, 43.1±2.5 and 19.1±2.4 % with P. silvestre DSM 102047T, A. russicus DSM 14555T and R. salmoninarum ATCC 33209T, respectively. On the basis of the results of the phenotypic, chemotaxonomic and phylogenetic analyses, IHBB 11108T is considered to represent a novel species of the genus Psychromicrobium for which the name Psychromicrobium lacuslunae sp. nov. is proposed. The type strain is IHBB 11108T (=MTCC 12460T=MCC 2780T=JCM 31143T=KACC 19070T).

Isolation and characterization of racemase from Ensifer sp. 23-3 that acts on α-aminolactams and α-amino acid amides.

Limited information is available on α-amino-ε-caprolactam (ACL) racemase (ACLR), a pyridoxal 5'-phosphate-dependent enzyme that acts on ACL and α-amino acid amides. In the present study, eight bacterial strains with the ability to racemize α-amino-ε-caprolactam were isolated and one of them was identified as Ensifer sp. strain 23-3. The gene for ACLR from Ensifer sp. 23-3 was cloned and expressed in Escherichia coli. The recombinant ACLR was then purified to homogeneity from the E. coli transformant harboring the ACLR gene from Ensifer sp. 23-3, and its properties were characterized. This enzyme acted not only on ACL but also on α-amino-δ-valerolactam, α-amino-ω-octalactam, α-aminobutyric acid amide, and alanine amide.

Processing 2-Methyl-l-Tryptophan through Tandem Transamination and Selective Oxygenation Initiates Indole Ring Expansion in the Biosynthesis of Thiostrepton.

Thiostrepton (TSR), an archetypal member of the family of ribosomally synthesized and post-translationally modified thiopeptide antibiotics, possesses a biologically important quinaldic acid (QA) moiety within the side-ring system of its characteristic thiopeptide framework. QA is derived from an independent l-Trp residue; however, its associated transformation process remains poorly understood. We here report that during the formation of QA, the key expansion of an indole to a quinoline relies on the activities of the pyridoxal-5'-phosphate-dependent protein TsrA and the flavoprotein TsrE. These proteins act in tandem to process the precursor 2-methyl- l-Trp through reversible transamination and selective oxygenation, thereby initiating a highly reactive rearrangement in which selective C2-N1 bond cleavage via hydrolysis for indole ring-opening is closely coupled with C2'-N1 bond formation via condensation for recyclization and ring expansion in the production of a quinoline ketone intermediate. This indole ring-expansion mechanism is unusual, and represents a new strategy found in nature for l-Trp-based functionalization.

Peroxiredoxins prevent oxidative stress during human sperm capacitation.

Do peroxiredoxins (PRDXs) control reactive oxygen species (ROS) levels during human sperm capacitation? PRDXs are necessary to control the levels of ROS generated during capacitation allowing spermatozoa to achieve fertilizing ability. Sperm capacitation is an oxidative event that requires low and controlled amounts of ROS to trigger phosphorylation events. PRDXs are antioxidant enzymes that not only act as scavengers but also control ROS action in somatic cells. Spermatozoa from infertile men have lower levels of PRDXs (particularly of PRDX6), which are thiol-oxidized and therefore inactive. Semen samples were obtained from a cohort of 20 healthy nonsmoker volunteers aged 22-30 years old over a period of 1 year. Sperm from healthy donors was capacitated with fetal cord serum ultrafiltrate (FCSu) in the absence or presence of thiostrepton (TSP), inhibitor of 2-Cys PRDXs or 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium (MJ33), inhibitor of calcium independent-phospholipase A2 (Ca2+-iPLA2) activity of PRDX6, added at different times of incubation. Capacitation was also induced by the dibutyryl cAMP+3-isobuty1-1-methylxanthine system. Sperm viability and motility were determined by the hypo-osmotic swelling test and computer-assisted semen analysis system, respectively. Capacitation was determined by the ability of spermatozoa to undergo the acrosome reaction triggered by lysophosphatidylcholine. Percentages of acrosome reaction were obtained using the FITC-conjugated Pisum sativum agglutinin assay. Phosphorylation of tyrosine residues and of protein kinase A (PKA) substrates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblotting with specific antibodies. Actin polymerization was determined by phalloidin labeling. TSP and MJ33 prevented sperm capacitation and its associated actin polymerization in spermatozoa incubated with 10% FCSu (capacitation inducer) compared to non-capacitated controls (P < 0.05) without altering sperm viability. PKA substrates and tyrosine phosphorylations were prevented in FCSu-treated spermatozoa in a differential fashion depending on the type and the time of addition of the inhibitor used compared to non-capacitated controls (P < 0.05). TSP and MJ33 promoted an increase of lipid peroxidation in spermatozoa (P < 0.01) and these levels were higher in those spermatozoa incubated with the inhibitors and FCSu compared to those capacitated spermatozoa incubated without the inhibitors (P < 0.0001). Inhibition of 2-Cys PRDXs by TSP generated an oxidative stress in spermatozoa, affecting their viability compared to controls (P < 0.05). This oxidative stress was prevented by nuclephile D-penicillamine (PEN). MJ33 also promoted an increase of lipid peroxidation and impaired sperm viability compared to non-treated controls (P < 0.05) but its effect was not circumvented by PEN, suggesting that not only peroxidase but also Ca2+-iPLA2 activity of PRDX6 are necessary to guarantee viability in human spermatozoa. Not applicable. We focused on the global effect of PRDXs inhibitors on human sperm capacitation and in two of its associated phosphorylation events. Thus, other phosphorylation events and mechanisms necessary for capacitation may also be affected. PRDXs are the major antioxidant system in ejaculated spermatozoa and are necessary to allow spermatozoon to achieve fertilizing ability (capacitation and acrosome reaction). This research was supported by Canadian Institutes of Health Research (MOP 133661) and the Fonds de Recherché en Santé Quebec (FRSQS #22151) to C.O. The authors have nothing to disclose.

An α/β-hydrolase fold protein in the biosynthesis of thiostrepton exhibits a dual activity for endopeptidyl hydrolysis and epoxide ring opening/macrocyclization

Thiostrepton (TSR), an archetypal bimacrocyclic thiopeptide antibiotic that arises from complex posttranslational modifications of a genetically encoded precursor peptide, possesses a quinaldic acid (QA) moiety within the side-ring system of a thiopeptide-characteristic framework. Focusing on selective engineering of the QA moiety, i.e., by fluorination or methylation, we have recently designed and biosynthesized biologically more active TSR analogs. Using these analogs as chemical probes, we uncovered an unusual indirect mechanism of TSR-type thiopeptides, which are able to act against intracellular pathogens through host autophagy induction in addition to direct targeting of bacterial ribosome. Herein, we report the accumulation of 6'-fluoro-7', 8'-epoxy-TSR, a key intermediate in the preparation of the analog 6'-fluoro-TSR. This unexpected finding led to unveiling of the TSR maturation process, which involves an unusual dual activity of TsrI, an α/β-hydrolase fold protein, for cascade C-N bond cleavage and formation during side-ring system construction. These two functions of TsrI rely on the same catalytic triad, Ser72-His200-Asp191, which first mediates endopeptidyl hydrolysis that occurs selectively between the residues Met-1 and Ile1 for removal of the leader peptide and then triggers epoxide ring opening for closure of the QA-containing side-ring system in a regio- and stereo-specific manner. The former reaction likely requires the formation of an acyl-Ser72 enzyme intermediate; in contrast, the latter is independent of Ser72. Consequently, C-6' fluorination of QA lowers the reactivity of the epoxide intermediate and, thereby, allows the dissection of the TsrI-associated enzymatic process that proceeds rapidly and typically is difficult to be realized during TSR biosynthesis.

New aristolochic acid and other chemical constituents of Aristolochia maurorum growing wild in Jordan

Investigation of the chemical constituents of Aristolochia maurorum growing wild in Jordan resulted in the isolation and characterisation of one new compound in addition to 19 known compounds. The new compound was identified as aristolochic acid II alanine amide (14). The other known compounds were the following: palmitic acid (1), β-sitosterol (2), E-ethyl-p-coumarate (3), Z-ethyl-p-coumarate (4), aristolochic acid IV methyl ester (5), aristolactam I (6), loliolide (7), (+)-dehydrovomifoliol (8), glycerol-1-palmitate (9), aristolochic acid I (10), E-p-coumaric acid (11), E-N-coumaroyltyramine (12), β-sitosteryl glucoside (13), aristolochic acid IV (15), aristolochic acid III (16), esculetin (17), uracil (18), shepherdine (19) and adenosine (20). The isolated compounds were characterised by different spectroscopic methods including NMR (1D and 2D), UV, IR and HRESIMS.

Precursor-Directed Mutational Biosynthesis Facilitates the Functional Assignment of Two Cytochromes P450 in Thiostrepton Biosynthesis.

Side-ring-modified thiostrepton (TSR) derivatives that vary in their quinaldic acid (QA) substitution possess more potent biological activities and better pharmaceutical properties than the parent compound. In this work, we sought to introduce fluorine onto C-7' or C-8' of the TSR QA moiety via precursor-directed mutational biosynthesis to obtain new TSR variants. Unexpectedly, instead of the target product, the exogenous chemical feeding of 7-F-QA into the ΔtsrT mutant strain resulted in a unique TSR analog with an incomplete side-ring structure and an unoxidized QA moiety (1). Accordingly, two cytochrome P450 genes, tsrP and tsrR, were in-frame deleted to elucidate the candidate responsible for the monooxidation of the QA moiety in TSR. The unfluorinated analog of compound 1 that was thus isolated from ΔtsrP (2) and the abolishment of TSR production in ΔtsrR revealed not only the biosynthetic logic of the TSR side-ring but also the essential checkpoint in TSR maturation before macro-ring closure.

Molecular engineering of thiostrepton via single “base”-based mutagenesis to generate side ring-derived variants

Six thiostrepton (TSR) analogs, siomycin (SIO) and SIO-Dha2Ser were produced in the engineered TSR-producing strain by using a single “base”-based mutagenesis strategy.

Synthesis and Biochemical Evaluation of 3-Phenoxy-1,4-diarylazetidin-2-ones as Tubulin-Targeting Antitumor Agents.

Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated, leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl "A" ring and 4-phenyl "B" ring for potent antiproliferative activity and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC50 values of 38 and 19 nM, respectively, in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular structure in MCF-7 cells as visualized by confocal microscopy, and caused G2/M arrest and apoptosis. Compound 90b possessed a mean GI50 value of 22 nM in the NCI60 cell line screen, displayed minimal cytotoxicity, and was shown to interact at the colchicine-binding site on β-tubulin. Phosphate and amino acid prodrugs of both 78b and 90b were synthesized, of which the alanine amide 102b retained potency and is a promising candidate for further clinical development.

Thiopeptide Antibiotics Exhibit a Dual Mode of Action against Intracellular Pathogens by Affecting Both Host and Microbe

Thiostrepton (TSR) is an archetypal thiopeptide antibiotic possessing a quinaldic acid (QA) moiety in the side ring system. According to the mechanism of TSR previously known to target bacterial ribosome, we recently designed and biosynthesized several TSR derivatives that varied in QA substitution. Utilizing these thiopeptide antibiotics to treat the intracellular pathogen Mycobacterium marinum, we herein report a novel mode of action of TSRs, which induce ER stress-mediated autophagy to enhance host cell defense. This intracellular response, which is sensitive to the modification of the QA group, serves as an indirect but unignorable mechanism for eliminating intracellular pathogens. TSRs are thus the only type of antibiotics, to our knowledge, with thedual action on both the parasitic bacteria and the infected host cells. The newly observed mechanism of TSRs may inspire the future change in the treatment of intracellular pathogens, by taking host response into account.

Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma.

Dysregulation of signaling pathways and energy metabolism in cancer cells enhances production of mitochondrial hydrogen peroxide that supports tumorigenesis through multiple mechanisms. To counteract the adverse effects of mitochondrial peroxide many solid tumor types up-regulate the mitochondrial thioredoxin reductase 2 - thioredoxin 2 (TRX2) - peroxiredoxin 3 (PRX3) antioxidant network. Using malignant mesothelioma cells as a model, we show that thiostrepton (TS) irreversibly disables PRX3 via covalent crosslinking of peroxidatic and resolving cysteine residues in homodimers, and that targeting the oxidoreductase TRX2 with the triphenylmethane gentian violet (GV) potentiates adduction by increasing levels of disulfide-bonded PRX3 dimers. Due to the fact that activity of the PRX3 catalytic cycle dictates the rate of adduction by TS, immortalized and primary human mesothelial cells are significantly less sensitive to both compounds. Moreover, stable knockdown of PRX3 reduces mesothelioma cell proliferation and sensitivity to TS. Expression of catalase in shPRX3 mesothelioma cells restores defects in cell proliferation but not sensitivity to TS. In a SCID mouse xenograft model of human mesothelioma, administration of TS and GV together reduced tumor burden more effectively than either agent alone. Because increased production of mitochondrial hydrogen peroxide is a common phenotype of malignant cells, and TS and GV are well tolerated in mammals, we propose that targeting PRX3 is a feasible redox-dependent strategy for managing mesothelioma and other intractable human malignancies.

PH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements

The pK a values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the (1)H, (13)C, and (15)N nuclei in the seven common ionizable amino acids (X=Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein pK a measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain (15)N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.

Over-expression of FoxM1 is associated with adverse prognosis and FLT3-ITD in acute myeloid leukemia

Forkhead box M1 (FoxM1) drives cell cycle progression and the prevention of growth arrest and is over-expressed in many human malignancies. However, the characteristics of FoxM1 in acute myeloid leukemia (AML) are not clearly understood. We investigated the expression level of FoxM1 and analyzed the correlation of FoxM1 expression with AML patient characteristics and prognoses. Changes in FoxM1 expression were detected after MV4–11 cells, which have an internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 gene (FLT3-ITD), and control THP1 cells (encoding wild-type FLT3) were treated with the FLT3 receptor tyrosine kinase inhibitor AC220 (quizartinib) or FLT3 ligand (FL). Finally, we determined the apoptosis rates after the addition of the FoxM1 inhibitor thiostrepton (TST) to AML cells with or without FLT3-ITD. The expression of FoxM1 in AML patients was correlated with the presence of FLT3-ITD, genetic groups, and possibly overall survival. Inhibition of FLT3-ITD by AC220 down-regulated FoxM1 expression in MV4–11 cells, and stimulation of FLT3 by FL up-regulated FoxM1 expression in MV4–11 and THP1 cells. TST induced the apoptosis of MV4–11 and THP1 cells in a dose-dependent manner. Thus, FoxM1 is a potential prognostic marker and a promising therapeutic target in AML.

Role of FOXM1 expression inbibition regulated by thiostrepton in the proliferation and apoptosis of lung cancer cells

Objective To investigate the effect of thiostrepton (TST) on cell proliferation,apoptosis and the chemosensitivity of non-small cell cancer (NSCLC) cell lines A549 to AG1478 (EGFR-TKI).Methods NSCLC cell A549 was treated with TST,AG1478 or the combination of the two drugs.CCK-8 cell viability assay was performed to determine the relative growth suppression rate of A549 cell.The expression level of forkhead transcription factors M1 (FOXM1) was studied by western blot.Caspase-3 colorimetric assay was employed to evaluate the effect of TST on Caspase-3 activity.A siRNA targeting FOXM1 was designed and transfected into A549 cells.RT-PCR and western blot were used to examine the expressions of FOXM1 and proliferation and apoptosis related molecules.Results TST increased the chemosensitivity of A59 cells to AG1478,a kind of molecular-targeted agent to advanced lung cancer,which made the IC50 value reduce from (4.35 ±0.45) μmol/L to (0.73 ± 0.05) μmol/L (t =11.02,P ＜ 0.05).Besides,TST inhibited the expressions of FOXM1 and its effectors including c-Myc,Cyclin B1 and Bcl-2,while up-regulated P21,Cleaved Caspase-3 and Cleaved PARP.Meanwhile,TST improved the activity of Caspase-3,which showed time-and dose-dependent effects.FOXM1 siRNA changes the expression of the downstream effectors such as c-Myc,Cyclin B1,Bcl-2,P21 and Cleaved PARP,which was similar to the effect of TST.Also,the fluorescent expression of Cleaved Caspase-3 significantly increased in A549 cells.Conclusion TST can significantly inhibit proliferation and induce apoptosis by downregulating the expression of FOXM1,and then increase chemosensitivity of A549 cell to AG1478. Key words: Lung neoplasms; RNA interference ; Forkhead transcription factors M1 ; Thiostrepton; Drug sensitivity

Peroxiredoxin 3 Is a Redox-Dependent Target of Thiostrepton in Malignant Mesothelioma Cells

Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC), indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2 network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-dependent perturbations in mitochondrial function as a therapeutic approach in mesothelioma.

Insights into Quinaldic Acid Moiety Formation in Thiostrepton Biosynthesis Facilitating Fluorinated Thiopeptide Generation

Thiostrepton (TSR), often referred as to a parent compound in the thiopeptide family, is a bimacrocyclic member that features a quinaldic acid (QA) moiety-containing side ring appended to the characteristic core system. QA biosynthesis requires an unusual ring-expanding conversion, showing a methyl transfer onto and a rearrangement of the indole part of L-tryptophan to give a quinoline ketone. Herein, we report that the process involves the activities of the radical methyltransferase TsrT, aminotransferase TsrA, dehydrogenase TsrE, and cyclase TsrD. TsrU, a stereospecific oxidoreductase, catalyzes the further conversion of the ketone into an enantiomerically pure S-alcohol. Elucidation of this chemistry, which is common in the biosynthesis of a number of thiopeptides sharing a QA side ring system, facilitates analog generation, as shown by the achievement of region-specific fluorination of thiostrepton with the improved antibacterial activity.

Correlations between conformational isomerism and chromatographic diastereoselectivity of bis amino amide s-triazine derivatives

NMR spectroscopy was used to probe the conformational behavior of diastereomeric s-triazine derivatives containing two chiral amino amide substituents, in order to shed light onto the mechanism of chromatographic diastereoselectivity. Utilizing the amino hydrogen signals in the proton NMR spectrum, the population of the conformations caused by rotation about the bond between the amino nitrogen and aromatic carbon atoms could be observed. The population distribution between the three possible conformations was similar but not identical between the two diastereomers, with similar trends being observed for both bis alanine amide and bis valine amide derivatives. Based on a simple model in which it is assumed that adsorption to the hydrophobic RP-LC stationary phase occurs only for the conformations having both amino amide R-groups on the same side of the triazine ring plane, the different conformation populations between the two diastereomers obtained by NMR was consistent with the observed RP-LC elution order (L-L diastereomer followed by L-D). The predicted diastereoselectivity values from NMR data were compared to RP-LC diastereoselectivity values obtained using both C18 and polymeric columns, with both acetonitrile/water and DMSO/water mobile phases. Values obtained with the polymeric column were in better agreement with calculated values than those obtained with the C18 column, suggesting that the simple adsorption model used to calculate the diastereoselectivity is more relevant towards a simple hydrophobic polymeric surface rather than a more complex C18 stationary phase. This study indicates that proton NMR is a useful tool for studying the diastereoselective mechanism of these derivatives, due to the relatively slow C-N bond rotation caused by the significant sp(2) character of the amino nitrogen atoms.