Dynamic Control of X Chromosome Conformation and Repression by a Histone H4K20 Demethylase.

Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We have discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C. elegans dosage compensation and demonstrated H4K20me1's pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. The structure and the activity of the dosage compensation complex (DCC) subunit DPY-21 define a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminates H4K20me1 enrichment in somatic cells, elevates X-linked gene expression, reduces X chromosome compaction, and disrupts X chromosome conformation by diminishing the formation of topologically associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression.

Evybactin is a DNA gyrase inhibitor that selectively kills Mycobacterium tuberculosis.

The antimicrobial resistance crisis requires the introduction of novel antibiotics. The use of conventional broad-spectrum compounds selects for resistance in off-target pathogens and harms the microbiome. This is especially true for Mycobacterium tuberculosis, where treatment requires a 6-month course of antibiotics. Here we show that a novel antimicrobial from Photorhabdus noenieputensis, which we named evybactin, is a potent and selective antibiotic acting against M. tuberculosis. Evybactin targets DNA gyrase and binds to a site overlapping with synthetic thiophene poisons. Given the conserved nature of DNA gyrase, the observed selectivity against M. tuberculosis is puzzling. We found that evybactin is smuggled into the cell by a promiscuous transporter of hydrophilic compounds, BacA. Evybactin is the first, but likely not the only, antimicrobial compound found to employ this unusual mechanism of selectivity.

The metabolic landscape of the bacteria Rhodococcus erythropolis used in the biodesulfurization of petroleum products: an emphasis on 2-hydroxybiphenyl.

Microorganisms produce diverse classes of metabolites under various physiological conditions. Many bacterial strains have been reported to carry out the process of desulfurization in a cost-effective manner by converting dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) and then using the 2-HBP as a carbon source for growth and development. Key rate-limiting factors and an increased concentration of 2HBP (400 µM) affect the biodesulfurization activity of bacteria through the produced metabolites. Thus, this study was designed to explore the nature of the metabolites produced by Rhodococcus erythropolis in the presence of DBT and 2HBP supplemented with a culture medium. A total of 330 metabolites were detected, and the key metabolites identified were 11Z-eicosaenoyl-EA, 1-carboxyethylisoleucine, 1(3)-glyceryl-PGF2alpha, taurine, 2-hydroxynicotinic acid, 4,4-dimethyl-14alpha-hydroxymethyl-5alpha-cholest-8-en-3beta-ol, and 10-nitrooleic acid. The supplementation of DBT and DBT-2HBP resulted in the differential regulation of these metabolites, either through downregulation or overexpression. Furthermore, at high concentrations of 2-HBP, 1-carboxyethylisoleucine, taurine, 2-hydroxynicotinic acid, and nicotinic acid were upregulated. This work proposes that the identified metabolites may play a role in bacteria-mediated desulphurization and could be beneficial in developing a cost-effective method of desulphurization for refining petroleum.

Assessment of the dibenzothiophene desulfurization potential of indigenously isolated bacterial consortium IQMJ-5: a different approach to safeguard the environment.

Biodesulfurization is emerging as a valuable technology for the desulfurization of dibenzothiophene (DBT) and its alkylated substitutes, which are otherwise regarded as refractory to other physical and chemical desulfurizing techniques. The inability of the currently identified pure cultures and artificial microbial consortia due to lower desulfurization rate and product inhibition issues has compelled the researcher to look for an alternative solution. Thus, in the present study, an indigenously isolated microbial consortium was employed to tackle the desulfurization issue. Herein, we isolated several kinds of DBT desulfurizing natural microbial consortia from hydrocarbon-contaminated soil samples by conventional enrichment technique. The most effective desulfurizing microbial consortium was sequenced through illumine sequencing technique. Finally, the effect of the products of the desulfurizing pathway (such as 2-hydroxybiphenyl (2-HBP) and sulfate (SO4-2) was evaluated on the growth and desulfurization capability of the isolated consortium. The outcomes of Gibb's assay analysis showed that six isolates followed the "4S" pathway and converted DBT to 2-HBP. Among the isolates, I5 showed maximum growth rate (1.078 g/L dry cell weight) and desulfurization activity (about 77% as indicated by HPLC analysis) and was considered for further in-depth experimentation. The analysis of 16S rRNA by high-throughput sequencing approach of the I5 isolate revealed five types of bacterial phyla including Proteobacteria, Bacteroidetes, Firmicutes, Patescibacteria, and Actinobacteria (in order of abundance). The isolate showed significant tolerance to the inhibitory effect of both 2-HBP and SO4-2 and maintained growth in the presence of even about 1.0 mM initial concentration of both products. This clearly suggests that the isolate can be an efficient candidate for future in-depth desulfurization studies of coal and other fossil fuels.

K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site.

Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK subfamily generate 'leak' currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit. In contrast to other potassium channels, K2P channels use a selectivity filter 'C-type' gate as the principal gating site. Despite recent advances, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate 'leak mode' and provide direct evidence for K2P selectivity filter gating.

Mechanism of ß-arrestin recruitment by the µ-opioid G protein-coupled receptor.

Agonists to the µ-opioid G protein-coupled receptor (µOR) can alleviate pain through activation of G protein signaling, but they can also induce ß-arrestin activation, leading to such side effects as respiratory depression. Biased ligands to µOR that induce G protein signaling without inducing ß-arrestin signaling can alleviate pain while reducing side effects. However, the mechanism for stimulating ß-arrestin signaling is not known, making it difficult to design optimum biased ligands. We use extensive molecular dynamics simulations to determine three-dimensional (3D) structures of activated ß-arrestin2 stabilized by phosphorylated µOR bound to the morphine and D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) nonbiased agonists and to the TRV130 biased agonist. For nonbiased agonists, we find that the ß-arrestin2 couples to the phosphorylated µOR by forming strong polar interactions with intracellular loop 2 (ICL2) and either the ICL3 or cytoplasmic region of transmembrane (TM6). Strikingly, Gi protein makes identical strong bonds with these same ICLs. Thus, the Gi protein and ß-arrestin2 compete for the same binding site even though their recruitment leads to much different outcomes. On the other hand, we find that TRV130 has a greater tendency to bind the extracellular portion of TM2 and TM3, which repositions TM6 in the cytoplasmic region of µOR, hindering ß-arrestin2 from making polar anchors to the ICL3 or to the cytosolic end of TM6. This dramatically reduces the affinity between µOR and ß-arrestin2.

Daily rhythms of REV-ERBα and its role as transcriptional repressor of clock genes in fish hepatic oscillator.

The REV-ERBα nuclear receptor is a key component of the molecular machinery of circadian oscillators in mammals. While the rhythmic expression of this receptor has been described in teleosts, several critical aspects of its regulation remain unknown, such as which synchronizers entrain its rhythm, and whether it can modulate the expression of other clock genes. The objective of this study was to gain deeper understanding of the role of REV-ERBα in the fish circadian system. To this end, we first investigated the cues that entrain the rhythm of rev-erbα expression in the goldfish (Carassius auratus) liver and hypothalamus. A 12-h shift in feeding time induced a parallel shift in the hepatic rhythm of rev-erbα expression, confirming that this gene is food-entrainable in the goldfish liver. In contrast, light seems the main driver of rev-erbα rhythmic expression in the hypothalamus. Next, we examined the effects of REV-ERBα activation on locomotor activity and hepatic expression of clock genes. Subchronic treatment with the REV-ERBα agonist SR9009 slightly decreased locomotor activity anticipating light onset and food arrival, and downregulated hepatic bmal1a, clock1a, cry1a, per1a and pparα expression. This generalized repressing action of REV-ERBα on the expression of hepatic clock genes was confirmed in vitro by using agonists (SR9009 and GSK4112) and antagonist (SR8278) of this receptor. Overall, the present work reveals that REV-ERBα modulates the daily expression of the main genes of the teleostean liver clock, reinforcing its role in the liver temporal homeostasis, which seems highly conserved in both fish and mammals.

Characterization of a dszEABC operon providing fast growth on dibenzothiophene and construction of broad-host-range biodesulfurization catalysts.

A new operon for biodesulfurization (BDS) of dibenzothiophene and derivatives has been isolated from a metagenomic library made from oil-contaminated soil, by selecting growth of E. coli on DBT as the sulfur source. This operon is similar to a dszEABC operon also isolated by metagenomic functional screening but exhibited substantial differences: (i) the new fosmid provides much faster growth on DBT; (ii) associated dszEABC genes can be expressed without the need of heterologous expression from the vector promoter; and (iii) monooxygenases encoded in the fosmid cannot oxidize indole to produce indigo. We show how expression of the new dszEABC operon is regulated by the sulfur source, being induced under sulfur-limiting conditions. Its transcription is activated by DszR, a type IV activator οf σN -dependent promoters. DszR is coded in a dszHR operon, whose transcription is in turn regulated by sulfur and presumably activated by the global regulator of sulfur metabolism CysB. Expression of dszH is essential for production of active DszR, although it is not involved in sulfur sensing or regulation. Two broad-host-range DBT biodesulfurization catalysts have been constructed and shown to provide DBT biodesulfurization capability to three Pseudomonas strains, displaying desirable characteristics for biocatalysts to be used in BDS processes.

Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2.

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

Synthesis and evaluation of RNase L-binding 2-aminothiophenes as anticancer agents.

Aminothiophene is a scaffold that is widely present in drugs and biologically active small molecules as chemical probes. In this study, 43 compounds sharing a 2-aminothiophenone-3-carboxylate (ATPC) scaffold, known to activate the ribonuclease L (RNase L), were synthesized and selected ATPCs showed enhancement of thermal stability of RNase L upon binding. Screening of antiproliferation activities against human cancer cell lines revealed that ATPCs represented by compounds 4l and 50 showed potent single-digit micromolar antiproliferation activity against human cancer cell lines. Compounds 4l and 50 exhibited time- and dose-dependent proliferation inhibition, induced cellular apoptosis measured by cleaved PARP and via flow cytometry, inhibited cell migration, and inhibited cell colony formation. Combining the results reported in this work, ATPCs were evaluated as potential anticancer agents mediated by RNase L-binding and apoptosis induction. The work contributes to the study on the polypharmacological properties of aminothiophene-containing small molecules.

Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.

Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.

Species-dependent hepatic and intestinal metabolism of selective oestrogen receptor degrader LSZ102 by sulphation and glucuronidation.

LSZ102 is an orally bioavailable selective oestrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass.To investigate metabolism and first-pass effects, metabolites were analysed in human plasma samples after oral dosing of LSZ102 to patients, rat plasma samples after oral dosing of [14C]LSZ102, and in vitro incubations of [14C]LSZ102 with human and rat hepatocytes and intestinal S9 fractions. The kinetics of human sulfotransferase (SULT) enzymes potentially involved in metabolism of LSZ102 was characterised.Sulphate metabolites were found to be the major components in human plasma, as well as in human hepatocytes and intestinal S9 fractions. Contrastingly, glucuronidation was predominant in rat plasma, hepatocytes and intestinal S9. LSZ102 was found to be metabolised by several human SULTs expressed in liver and intestine. The combined metabolism data in rat and human provide supporting evidence for an extensive intestinal first-pass metabolism effect via sulphation in human but glucuronidation in rat.As LSZ102 is metabolised by a number of different SULTs, drug-drug interactions resulting from the inhibition of one SULT are unlikely.Despite the observed species difference in metabolism, the major human metabolites of LSZ102, sulphate M5, glucuronide M4, and secondary glucuronide/sulphate metabolite M12, have no or weak pharmacological activity and are not considered a toxicity risk as they are phase II conjugative metabolites.

Monooxygenase- and Dioxygenase-Catalyzed Oxidative Dearomatization of Thiophenes by Sulfoxidation, cis-Dihydroxylation and Epoxidation.

Enzymatic oxidations of thiophenes, including thiophene-containing drugs, are important for biodesulfurization of crude oil and drug metabolism of mono- and poly-cyclic thiophenes. Thiophene oxidative dearomatization pathways involve reactive metabolites, whose detection is important in the pharmaceutical industry, and are catalyzed by monooxygenase (sulfoxidation, epoxidation) and dioxygenase (sulfoxidation, dihydroxylation) enzymes. Sulfoxide and epoxide metabolites of thiophene substrates are often unstable, and, while cis-dihydrodiol metabolites are more stable, significant challenges are presented by both types of metabolite. Prediction of the structure, relative and absolute configuration, and enantiopurity of chiral metabolites obtained from thiophene enzymatic oxidation depends on the substrate, type of oxygenase selected, and molecular docking results. The racemization and dimerization of sulfoxides, cis/trans epimerization of dihydrodiol metabolites, and aromatization of epoxides are all factors associated with the mono- and di-oxygenase-catalyzed metabolism of thiophenes and thiophene-containing drugs and their applications in chemoenzymatic synthesis and medicine.

Benzo[b]naphtho[1,2-d]thiophene Sulfoxides: Biomimetic Synthesis, Photophysical Properties, and Applications.

A practical synthesis of nonsymmetrical thiophene-fused aromatic systems has been developed that was inspired by the biodegradation of benzothiophene. For the first time, the photophysical properties of a series of π-conjugated benzo[b]naphtho[1,2-d]thiophene (BNT) sulfoxides were explored both in solution and in the solid state. The excellent fluorescence characteristics enable various applications of these compounds.

The Luminescent Conjugated Oligothiophene h-FTAA Attenuates the Toxicity of Different Aß Species.

The prevailing opinion is that prefibrillar ß-amyloid (Aß) species, rather than end-stage amyloid fibrils, cause neuronal dysfunction in Alzheimer's disease, although the mechanisms behind Aß neurotoxicity remain to be elucidated. Luminescent conjugated oligothiophenes (LCOs) exhibit spectral properties upon binding to amyloid proteins and have previously been reported to change the toxicity of Aß1-42 and prion protein. In a previous study, we showed that an LCO, pentamer formyl thiophene acetic acid (p-FTAA), changed the toxicity of Aß1-42. Here we investigated whether an LCO, heptamer formyl thiophene acetic acid (h-FTAA), could change the toxicity of Aß1-42 by comparing its behavior with that of p-FTAA. Moreover, we investigated the effects on toxicity when Aß with the Arctic mutation (AßArc) was aggregated with both LCOs. Cell viability assays on SH-SY5Y neuroblastoma cells demonstrated that h-FTAA has a stronger impact on Aß1-42 toxicity than does p-FTAA. Interestingly, h-FTAA, but not p-FTAA, rescued the AßArc-mediated toxicity. Aggregation kinetics and binding assay experiments with Aß1-42 and AßArc when aggregated with both LCOs showed that h-FTAA and p-FTAA either interact with different species or affect the aggregation in different ways. In conclusion, h-FTAA protects against Aß1-42 and AßArc toxicity, thus showing h-FTAA to be a useful tool for improving our understanding of the process of Aß aggregation linked to cytotoxicity.

Modeling the Bioactivation and Subsequent Reactivity of Drugs.

Electrophilically reactive drug metabolites are implicated in many adverse drug reactions. In this mechanism-termed bioactivation-metabolic enzymes convert drugs into reactive metabolites that often conjugate to nucleophilic sites within biological macromolecules like proteins. Toxic metabolite-product adducts induce severe immune responses that can cause sometimes fatal disorders, most commonly in the form of liver injury, blood dyscrasia, or the dermatologic conditions toxic epidermal necrolysis and Stevens-Johnson syndrome. This study models four of the most common metabolic transformations that result in bioactivation: quinone formation, epoxidation, thiophene sulfur-oxidation, and nitroaromatic reduction, by synthesizing models of metabolism and reactivity. First, the metabolism models predict the formation probabilities of all possible metabolites among the pathways studied. Second, the exact structures of these metabolites are enumerated. Third, using these structures, the reactivity model predicts the reactivity of each metabolite. Finally, a feedfoward neural network converts the metabolism and reactivity predictions to a bioactivation prediction for each possible metabolite. These bioactivation predictions represent the joint probability that a metabolite forms and that this metabolite subsequently conjugates to protein or glutathione. Among molecules bioactivated by these pathways, we predicted the correct pathway with an AUC accuracy of 89.98%. Furthermore, the model predicts whether molecules will be bioactivated, distinguishing bioactivated and nonbioactivated molecules with 81.06% AUC. We applied this algorithm to withdrawn drugs. The known bioactivation pathways of alclofenac and benzbromarone were identified by the algorithm, and high probability bioactivation pathways not yet confirmed were identified for safrazine, zimelidine, and astemizole. This bioactivation model-the first of its kind that jointly considers both metabolism and reactivity-enables drug candidates to be quickly evaluated for a toxicity risk that often evades detection during preclinical trials. The XenoSite bioactivation model is available at http://swami.wustl.edu/xenosite/p/bioactivation.

Discovery of 6-substituted thieno[2,3-d]pyrimidine analogs as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis in folate receptor expressing human tumors.

We discovered 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with 3-4 bridge carbons and side-chain thiophene or furan rings for dual targeting one-carbon (C1) metabolism in folate receptor- (FR) expressing cancers. Synthesis involved nine steps starting from the bromo-aryl carboxylate. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRα or FRß, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRα-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds 3-5 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents.

Desulphurisation kinetics of thiophenic compound by sulphur oxidizing Klebsiella oxytoca SOB-1.

AIMS: The major aims of this study are to determine the capability of sulphur oxidizing bacterium (SOB-1) to desulphurize dibenzothiophene (DBT) and crude oil, detection of the reaction kinetics and identify the proposed pathway of DBT desulphurization. METHODS AND RESULTS: The isolate was genetically identified based on 16S rRNA gene sequencing as Klebsiella oxytoca and deposited in the Genebank database under the accession number: MT355440. The HPLC analysis of the remaining DBT concentration revealed that, SOB-1 could desulphurize 90% of DBT (0·25 mmol l-1 ) within 96 h. The maximum production of sulphate ions from the desulphurization of DBT (0·36 mmol l-1 ) and crude oil (0·4 mmol l-1 ) could be quantitatively detected after 48 h of incubation at 30°C. The high values of correlation coefficient (R2 ) obtained at all studied concentrations; suggested that biodesulfurization kinetics of DBT follows the first-order reaction model. The kinetics studies showed that, DBT may have an inhibitory effect on SOB-1 when the initial concentration exceeded 0·75 mmol l-1 . The GC-MS analysis exhibited four main metabolites rather than DBT. The most important ones are 2-hydroxybiphenyl (2-HBP) and methoxybiphenyl n(2-MBP). CONCLUSIONS: Klebsiella oxytoca SOB-1 catalyzes the desulphurization of DBT through 4S pathway and forms four main metabolic products. The release of sulphate ion and formation of 2-HBP indicating the elimination of sulphur group without altering the carbon skeleton of DBT. The bacterial strain could also catalyzes desulphurization of crude oil. The desulphurization kinetics follows the first-order reaction model. SIGNIFICANCE AND IMPACT OF THE STUDY: Klebsiella oxytoca SOB-1 could be used as a promising industrial and environmental biodesulfurizing agent as it is not affecting carbon skeleton of thiophenic compounds and forming less toxic metabolic product (2-MBP).

Pharmacokinetics, mass balance, and metabolism of [14C]vicagrel, a novel irreversible P2Y12 inhibitor in humans.

Vicagrel, a novel irreversible P2Y12 receptor inhibitor, is undergoing phase III trials for the treatment of acute coronary syndromes in China. In this study, we evaluated the pharmacokinetics, mass balance, and metabolism of vicagrel in six healthy male Chinese subjects after a single oral dose of 20 mg [14C]vicagrel (120 µCi). Vicagrel absorption was fast (Tmax = 0.625 h), and the mean t1/2 of vicagrel-related components was ~38.0 h in both plasma and blood. The blood-to-plasma radioactivity AUCinf ratio was 0.55, suggesting preferential distribution of drug-related material in plasma. At 168 h after oral administration, the mean cumulative excreted radioactivity was 96.71% of the dose, including 68.03% in urine and 28.67% in feces. A total of 22 metabolites were identified, and the parent vicagrel was not detected in plasma, urine, or feces. The most important metabolic spot of vicagrel was on the thiophene ring. In plasma pretreated with the derivatization reagent, M9-2, which is a methylated metabolite after thiophene ring opening, was the predominant drug-related component, accounting for 39.43% of the radioactivity in pooled AUC0-8 h plasma. M4, a mono-oxidation metabolite upon ring-opening, was the most abundant metabolite in urine, accounting for 16.25% of the dose, followed by M3-1, accounting for 12.59% of the dose. By comparison, M21 was the major metabolite in feces, accounting for 6.81% of the dose. Overall, renal elimination plays a crucial role in vicagrel disposition, and the thiophene ring is the predominant metabolic site.

Ferredoxin-mediated reduction of 2-nitrothiophene inhibits photosynthesis: mechanism and herbicidal potential.

Searching for compounds that inhibit the growth of photosynthetic organisms highlighted a prominent effect at micromolar concentrations of the nitroheteroaromatic thioether, 2-nitrothiophene, applied in the light. Since similar effects were reminiscent to those obtained also by radicals produced under excessive illumination or by herbicides, and in light of its redox potential, we suspected that 2-nitrothiophene was reduced by ferredoxin, a major reducing compound in the light. In silico examination using docking and tunneling computing algorithms of the putative interaction between 2-nitrothiophene and cyanobacterial ferredoxin has suggested a site of interaction enabling robust electron transfer from the iron-sulfur cluster of ferredoxin to the nitro group of 2-nitrothiophene. ESR and oximetry analyses of cyanobacterial cells (Anabaena PCC7120) treated with 50 µM 2-nitrothiophene under illumination revealed accumulation of oxygen radicals and peroxides. Gas chromatography mass spectrometry analysis of 2-nitrothiophene-treated cells identified cytotoxic nitroso and non-toxic amino derivatives. These products of the degradation pathway of 2-nitrohiophene, which initializes with a single electron transfer that forms a short-live anion radical, are then decomposed to nitrate and thiophene, and may be further reduced to a nitroso hydroxylamine and amino derivatives. This mechanism of toxicity is similar to that of nitroimidazoles (e.g. ornidazole and metronidazole) reduced by ferredoxin in anaerobic bacteria and protozoa, but differs from that of ornidazole in planta.