Strasseriolides A-D, A Family of Antiplasmodial Macrolides Isolated from the Fungus Strasseria geniculata CF-247251.

A novel family of four potent antimalarial macrolides, strasseriolides A-D (1-4), has been isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. The structures of these compounds, including their absolute configurations, were elucidated by HRMS, NMR spectroscopy, and X-ray single-crystal diffraction. The four compounds gave respective IC50 values of 9.810, 0.013, 0.123, and 0.128 µM against Plasmodium falciparum 3D7 parasites with no significant cytotoxicity against the HepG2 cell line.

Determination of Gymnemic Acid I as a Protein Biosynthesis Inhibitor Using Chemical Proteomics.

The plant Gymnema sylvestre has been used widely in traditional medicine as a remedy for several diseases, and its leaf extract is known to contain a group of bioactive triterpene saponins belonging to the gymnemic acid class. Gymnemic acid I (1) is one of the main components among this group of secondary metabolites and is endowed with an interesting bioactivity profile. Since there is a lack of information about its specific biological targets, the full interactome of 1 was investigated through a quantitative chemical proteomic approach, based on stable-isotope dimethyl labeling. The ribosome complex was found to be the main partner of compound 1, and a full validation of the proteomics results was achieved by orthogonal approaches. Further biochemical and biological investigations revealed an inhibitory effect of 1 on the ribosome machinery.

Cell-Based Fluorescent Screen to Identify Inhibitors of Bacterial Translation Initiation.

A strategy that can be applied to the research of new molecules with antibacterial activity is to look for inhibitors of essential bacterial processes within large collections of chemically heterogeneous compounds. The implementation of this approach requires the development of proper assays aimed at the identification of molecules interfering with specific cell pathways and potentially applicable to the high throughput analysis of large chemical library. Here, I describe a fluorescence-based whole-cell assay in Escherichia coli devised to find inhibitors of the translation initiation pathway. Translation is a complex and essential mechanism. It involves numerous sub-steps performed by factors that are in many cases sufficiently dissimilar in bacterial and eukaryotic cells to be targetable with domain-specific drugs. As a matter of fact, translation has been proven as one of the few bacterial mechanisms pharmacologically tractable with specific antibiotics. The assay described in this chapter is tailored to the identification of molecules affecting the first stage of translation initiation, which is the most dissimilar step in bacteria vs. mammals. The effect of the compounds under analysis is assayed in living cells, thus allowing evaluating their in vivo performance as inhibitors of translation initiation. Compared with other assays for antibacterials, the major advantages of this screen are its simplicity and high mechanism specificity.

Hydrolysis of amphenicol and macrolide antibiotics: Chloramphenicol, florfenicol, spiramycin, and tylosin.

Antibiotics that enter the environment can present human and ecological health risks. An understanding of antibiotic hydrolysis rates is important for predicting their environmental persistence as biologically active contaminants. In this study, hydrolysis rates and Arrhenius constants were determined as a function of pH and temperature for two amphenicol (chloramphenicol and florfenicol) and two macrolide (spiramycin and tylosin) antibiotics. Antibiotic hydrolysis rates in pH 4-9 buffer solutions at 25°C, 50°C, and 60°C were quantified, and degradation products were characterized. All of the antibiotics tested remained stable and exhibited no observable hydrolysis under ambient conditions typical of aquatic ecosystems. Acid- and base-catalyzed hydrolysis occurred at elevated temperatures (50-60°C), and hydrolysis rates increased considerably below pH 5 and above pH 8. Hydrolysis rates also increased approximately 1.5- to 2.9-fold for each 10°C increase in temperature. Based on the degradation product masses found, the functional groups that underwent hydrolysis were alkyl fluoride, amide, and cyclic ester (lactone) moieties; some of the resultant degradation products may remain bioactive, but to a lesser extent than the parent compounds. The results of this research demonstrate that amphenicol and macrolide antibiotics persist in aquatic systems under ambient temperature and pH conditions typical of natural waters. Thus, these antibiotics may present a risk in aquatic ecosystems depending on the concentration present.

Identification of cardiac glycoside molecules as inhibitors of c-Myc IRES-mediated translation.

Translation initiation is a fine-tuned process that plays a critical role in tumorigenesis. The use of small molecules that modulate mRNA translation provides tool compounds to explore the mechanism of translational initiation and to further validate protein synthesis as a potential pharmaceutical target for cancer therapeutics. This report describes the development and use of a click beetle, dual luciferase cell-based assay multiplexed with a measure of compound toxicity using resazurin to evaluate the differential effect of natural products on cap-dependent or internal ribosome entry site (IRES)-mediated translation initiation and cell viability. This screen identified a series of cardiac glycosides as inhibitors of IRES-mediated translation using, in particular, the oncogene mRNA c-Myc IRES. Treatment of c-Myc-dependent cancer cells with these compounds showed a decrease in c-Myc protein associated with a significant modulation of cell viability. These findings suggest that inhibition of IRES-mediated translation initiation may be a strategy to inhibit c-Myc-driven tumorigenesis.

High-content screening for compounds that affect mtDNA-encoded protein levels in eukaryotic cells.

Compounds that interfere with the synthesis of either mitochondrial DNA or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial adenosine triphosphate (ATP) production. Toxicity caused by these compounds is seldom identified in 24- to 72-h cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. To address this problem, the authors developed a 96-well format, high-content screening (HCS) assay that measures, in eukaryotic cells, the level of Complex IV-subunit 1, an mtDNA-encoded protein synthesized on mitochondrial ribosomes, and the level of Complex V-alpha subunit, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The effect of several antibiotics and antiretrovirals on these 2 proteins was assessed, in transformed human liver epithelial cells, 6 days after compound treatment. The results confirmed effects of drugs known to reduce mtDNA-encoded protein levels and also revealed novel information showing that several fluoroquinolones and a macrolide, josamycin, impaired expression of mtDNA-encoded proteins. The HCS assay was robust with an average Z' factor of 0.62. The assay enables large-scale screening of compounds to identify those that potentially affect mtDNA-encoded protein levels and can be implemented within a screening paradigm to minimize compound attrition.

A chemical screen for suppressors of the avrRpm1-RPM1-dependent hypersensitive cell death response in Arabidopsis thaliana.

Arabidopsis thaliana RPM1 encodes an intracellular immune sensor that conditions disease resistance to Pseudomonas syringae expressing the type III effector protein AvrRpm1. Conditional expression of this type III effector in a transgenic line carrying avrRpm1 under the control of a steroid-inducible promoter results in RPM1-dependent cell death that resembles the cell death response of the incompatible RPM1-avrRpm1 plant-bacterium interaction. This line was previously used in a genetic screen, which revealed two genes that likely function in the folding of pre-activation RPM1. We established a chemical screen for small molecules that suppress steroid-inducible and RPM1-avrRpm1-dependent cell death in Arabidopsis seedlings. Screening of a library comprising 6,800 compounds of natural origin identified two trichothecene-type mycotoxins, 4,15-diacetoxyscirpenol (DAS) and neosolaniol (NEO), which are synthesized by Fusarium and other fungal species. However, protein blot analysis revealed that DAS and NEO inhibit AvrRpm1 synthesis rather than suppress RPM1-mediated responses. This inhibition of translational activity likely explains the survival of the seedlings under screening conditions. Likewise, flg22-induced defense responses are also impaired at the translational, but not the transcriptional, level by DAS or NEO. Unexpectedly, both compounds not only prevented AvrRpm1 synthesis, but rather caused an apparent hyper-accumulation of RPM1 and HSP70. The hyper-accumulation phenotype is likely unrelated to the ribotoxic function of DAS and NEO and could be due to an inhibitory activity on the proteolytic machinery of Arabidopsis or elicitor-like activities of type A trichothecenes.

Three methods to assay inhibitors of ribosomal subunit assembly.

The inhibition of bacterial ribosomal subunit formation is a novel target for translational inhibitors. Inhibition of subunit biogenesis has been shown to be equivalent to the inhibition of protein biosynthesis for many antibiotics. This chapter describes three methods for examining the inhibition of subunit formation in growing bacterial cells. The first method permits the determination of the IC50 value for inhibition of assembly and protein synthesis. The second is a pulse and chase labeling procedure to measure the kinetics of subunit formation. The third procedure allows an examination of ribosome reformation after antibiotic removal as a part of the post-antibiotic effect. Together these procedures give a description of the relative inhibitory effects of an antibiotic on translation and subunit formation.

Assays for the identification of inhibitors targeting specific translational steps.

While bacterial protein synthesis is the target of about half of the known antibiotics, the great structural-functional complexity of the translational machinery still offers remarkable opportunities for identifying novel and specific inhibitors of unexploited targets. We designed a knowledge-based in vitro translation assay to identify inhibitors selectively targeting the bacterial or the yeast translational apparatus, preferentially blocking the early steps of protein synthesis. Using a natural-like, "universal" model mRNA and cell-free extracts prepared from Eschericha coli, Saccharomyces cerevisiae, and HeLa cells, we were able to translate, with comparable yields in the three systems, the immunogenic peptide encoded by this "universal" mRNA. The immuno-enzymatic quantification of the translated peptide in the presence of a potential inhibitor can identify a selective bacterial or fungal inhibitor inactive in the human system. When applied to the high-throughput screening (HTS) of a library of approximately 25,000 natural products, this assay led to the identification of two novel and specific inhibitors of bacterial translation.

Intact cell matrix-assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression.

Here we demonstrate for the first time the application of intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione-S-transferase (GST) gene under an arabinose-inducible promoter as a model system. Using ICM-MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose-induced condition. Furthermore, recombinant GST protein was purified by a single-step affinity purification using a glutathione Sepharose 4B affinity column from arabinose-induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose-induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose-mediated regulation of the ara-operon was followed using the ICM-MS technique. All the results obtained from ICM-MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases.

Analysis of non-suspect samples lacking visually identifiable sperm using a Y-STR 10-plex.

Y-STRs are valuable in the investigation of sexual assaults in which autosomal STR genotype interpretation is challenging. To detect male DNA from compromised sexual assault evidence, 45 non-suspect samples were differentially extracted and analyzed with 10 Y-STRs. These samples were positive for the presence of human seminal fluid, but were negative for spermatozoa by microscopic examination. Y-STR data were obtained in approximately 86.2% of the epithelial or sperm fractions. On samples yielding incomplete profiles, results were obtained on an average of 5 loci per sample. The inability to obtain results may be due to insufficient amplifiable male DNA, PCR inhibition, or unfounded accusations of sexual assault. This study indicates that it is possible to obtain a male STR profile even in the absence of visually identifiable spermatozoa. Furthermore, Y-STR loci should become components of CODIS if they are to be used in solving non-suspect sexual assaults.

Chloramphenicol succinate, a competitive substrate and inhibitor of succinate dehydrogenase: possible reason for its toxicity.

From our previous study [Eur. J. Clin. Pharmacol. 56 (2000) 405] we hypothesized that chloramphenicol succinate (CAPS) may be a competitive substrate for succinate dehydrogenase (SDH). It may be oxidized by SDH to release chloramphenicol (CAP), which may inhibit SDH by feed back mechanism. The present ex-vivo/in vitro study was aimed to investigate this possibility by using human tissues (bone marrow and liver samples) and animal tissues (rat liver and kidney). The effect of different SDH activators and specific inhibitors was studied on CAPS metabolism by SDH. The metabolites and reduction products were detected by using HPLC. In marrow samples, CAPS was slowly oxidized to form CAP. The formation of CAP (oxidation product) was enhanced by FAD and low malonate and inhibited by high malonate and 3-NPA. Similar results were obtained with mitochondria from human and rat tissues. These studies suggest that CAPS could be a competitive oxidative substrate and the metabolite CAP could be an inhibitor at the reduction site. Therefore, SDH could be a target molecule responsible for CAPS induced toxicity.

Isolation of a putative ribosome inactivating protein from dried roots of Trichosanthes kirilowii used in Traditional Chinese Medicine.

A putative ribosome inactivating protein (RIP), named T 33, was isolated from dried roots of Trichosanthes kirilowii Maximovicz which are used in Traditional Chinese Medicine. After chromatographic enrichment a protein band with molecular mass of 33 kDa was found by SDS-PAGE. Partial amino acid sequences of this protein consisting of 8 - 33 amino acids reveal homology to highly conserved regions of different RIPs, but also indicate T 33 to be a novel type 2 RIP. Future heterologous expression of this protein will allow investigation of its biological activities in detail.

Uptake and intracellular fate of gelonin, a ribosome-inactivating protein, in rat liver.

Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.

Cellular biosensing system for discovery of protein synthesis inhibitors with an electrochemical phosphate modulator to regulate the acid phosphatase gene expression of Saccharomyces cerevisiae.

A cellular biosensing system for screening protein synthesis inhibitors has been developed by linking an electrochemical phosphate modulator and matrix-immobilized yeast cells with an optical sensing device. To screen the protein synthesis inhibitors, yeast phosphatase gene regulating system has been employed by linking an electrochemical phosphate modulator. Since the yeast phosphatase gene coding gammaAPase is expressed, when the phosphate concentration in solution is lowered below the threshold, the gammaAPase production is triggered by lowering the phosphate concentration with the electrochemical phosphate modulator, and monitored continuously with the photometric device. The electrochemical phosphate modulator was assembled with matrix-immobilized yeast cells. The module could insert to ordinal cuvette to monitor the induced gammaAPase activity in an ordinal photometer. Using the system, induction profile of protein synthesis was easily observed and was affected remarkably by various protein synthesis inhibitors. This seems promising that the system can be applied for first screening process of de novo protein inhibitors. The cellular biosensing system seems promising in screening protein synthesis inhibitors.

Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin.

The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (Cl) = 111-130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5-3.0 microM (200-1200 micrograms/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r2 = 0.99) and HPLC (r2 = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts.

Efectos de paratión sobre la síntesis proteica testicular / Parathion effects in testicular protein synthesis

El paratión como insecticida de uso masivo en la agricultura, puede afectar el potencial reproductivo de la población humana y animal expuesta. Sus propiedades bioquímicas potencialmente alteral los mecanismos de síntesis proteica testicular, afectando la morfofuncionalidad del aparato reproductor. El objetivo del presente trabajo, es cuantificar la síntesis de proteínas testicular, bajo la presencia de paratión, en ratón CF1, in vitro. Se cultivaron túbulos seminífero de ratones CF-1 e incubaron con concentraciones decrecientes (0,4; 0,04 y 0,004 mM) de paratión (PT) por 4 horas. Leucina tritiada (2 µ Ci) fue agregada después de una hora. Posteriormente se evaluó la síntesis proteica como actividad específica (cpm/µgr proteínas). Los resultados mostraron que el paration induce inhibición significativa de la síntesis proteica en la concentración 0,4 mM, mientras que a dosis menores de paratión disminuye el efecto sobre la inhibición de la síntesis de proteínas

Heterogeneity of the ribosome-inactivating protein trichosanthin in Trichosanthes kirilowii tubers.

The ribosome-inactivating protein trichosanthin isolated from the tubers of Trichosanthes kirilowii, the Chinese drug Tianhuafen, has a molecular mass of approximately 26 kDa. We show here that T. kirilowii tubers also contain ribosome-inactivating proteins with a small extent of structural variation from and a larger molecular mass than trichosanthin.

Crystallization and preliminary crystallographic analyses of pokeweed antiviral protein from seeds.

Pokeweed antiviral protein from seeds (PAP-S) is a ribosome inactivating protein which has lowest toxicity and highest inhibition activity as opposed to other pokeweed antiviral proteins and its three potential glycosylation sites (10, 44, 255) were shown to bind to N-acetylglucosamine. Good quality crystals of PAP-S were grown at high protein concentration (100 mg ml-1) and high temperature (306 K). The crystals have space group I222 and cell parameters a = 78.7, b = 85.2 and c = 93.0 A. An X-ray diffraction data set with resolution up to 1.8 A was collected. This high-resolution data will help to locate the sugars bound to the protein and provide accurate structural data for understanding structure-function relationships of PAP-S.

Reproducibility of alkaline antigens of Leishmania major-like and Leishmania (V.) braziliensis evaluated by IgG-ELISA: comparison of antigens added of a protein inhibitor (PMSF) or not

Neste trabalho estudou-se o comportamento de 10 partidas de antigenos alcalinos de L. major-like e L. (V.) braziliensis adicionados ou nao de um inibidor de protease (PMSF) avaliados em tres dias consecutivos por meio de ELISA-IgG empregando soros padrao positivo de pacientes com diagnostico de leishmaniose mucocutanea previamente testados para a presenca de anticorpos anti-leishmania por ELISA. A analise estatistica mostrou que para o antigeno de L. (V.) braziliensis adicionado de PMSF nao houve diferenca significante entre as partidas ou dias de teste. Para o antigeno sem PMSF houve diferenca entre as partidas mas nao entre os dias de teste. Uma ANOVA bi-caudal mostrou diferencas entre os antigenos com e sem PMSF. Os antigenos de L. major-like preparados com e sem adicao de PMSF mostraram diferencas significativas entre as partidas; os tres dias de teste foram significativamente diferentes para o antigeno preparado com PMSF mas somente os dias 1 e 2 o foram com o antigeno sem adicao de inibidor...