The Molecular Pharmacology of Pateamine A

<p>Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found.</p>

The Molecular Pharmacology of Pateamine A

<p>Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found.</p>

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence

Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents which increase intra-oocyte cAMP or prevent its degradation have been predominantly used, however agents such as kinase and protein synthesis inhibitors have also been trialled. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

Is the Antibacterial Activity of Multi-Walled Carbon Nanotubes (MWCNTs) Related to Antibiotic Resistance? An Assessment in Clinical Isolates

Antimicrobial resistance has spread globally, compromising the treatment of common infections. This feature is particularly harmful for nosocomial pathogens that can survive on hospital surfaces. Research studies have been conducted to evaluate new materials that are able to counteract the microbial growth and the colonization of the hospital environment. In this context, nanotechnologies have showed encouraging applications. We investigated the antibacterial activity of multi-walled carbon nanotubes (MWCNTs), both pristine (p) and functionalized (f), at concentrations of 50 and 100 μg mL−1, against bacterial strains isolated from hospital-acquired infections, and this activity was correlated with the antibiotic susceptibility of the strains. The inhibiting effect of MWCNTs occurred for both types and doses tested. Moreover, f-MWCNTs exerted a greater inhibiting effect, with growth decreases greater than 10% at 24 h and 20% at 48 h compared to p-MWCNTs. Moreover, a lower inhibitory effect of MWCNTs, which was more lasting in Gram-positives resistant to cell wall antibiotics, or temporary in Gram-negatives resistant to nucleic acid and protein synthesis inhibitors, was observed, highlighting the strong relation between antibiotic resistance and MWCNT effect. In conclusion, an antimicrobial activity was observed especially for f-MWCNTs that could therefore be loaded with bioactive antimicrobial molecules. However, this potential application of CNTs presupposes the absence of toxicity and therefore total safety for patients.

Oxazolidinone Antibiotics: Chemical, Biological and Analytical Aspects

This review covers the main aspects concerning the chemistry, the biological activity and the analytical determination of oxazolidinones, the only new class of synthetic antibiotics advanced in clinical use over the past 50 years. They are characterized by a chemical structure including the oxazolidone ring with the S configuration of substituent at C5, the acylaminomethyl group linked to C5 and the N-aryl substituent. The synthesis of oxazolidinones has gained increasing interest due to their unique mechanism of action that assures high antibiotic efficiency and low susceptibility to resistance mechanisms. Here, the main features of oxazolidinone antibiotics licensed or under development, such as Linezolid, Sutezolid, Eperezolid, Radezolid, Contezolid, Posizolid, Tedizolid, Delpazolid and TBI-223, are discussed. As they are protein synthesis inhibitors active against a wide spectrum of multidrug-resistant Gram-positive bacteria, their biological activity is carefully analyzed, together with the drug delivery systems recently developed to overcome the poor oxazolidinone water solubility. Finally, the most employed analytical techniques for oxazolidinone determination in different matrices, such as biological fluids, tissues, drugs and natural waters, are reviewed. Most are based on HPLC (High Performance Liquid Chromatography) coupled with UV-Vis or mass spectrometer detectors, but, to a lesser extent are also based on spectrofluorimetry or voltammetry.

Abstract OR-4: New Antibiotic Binding Site on the 30S Ribosomal Subunit

Background: Antibiotic resistance becomes one of the main problems of modern medicine; therefore, the development of new antibacterial compounds is absolutely necessary. The ribosome is the target for a lot of different antibiotics; there are several main binding sites on the ribosome – decoding center, peptidyl-transferase center, and ribosome exit tunnel. Modification or mutation of nucleotides in these sites could make cells resistant to structurally different antibiotics. Methods: pDualrep2 reporter system was used for detection of the protein synthesis inhibitors in cultural broths of new soil bacteria. By means of a cell-free translation system, the inhibitory activity and mechanism of action of Auraplanin were estimated. CryoEM data collection was performed on a Titan Krios operated at 300 kV, equipped with a Falcon II direct electron detector. Results: In this work, we have found a new inhibitor of protein synthesis, which binds in a completely new binding site. This compound is produced by Actinoplanes sp. VKM Ac-2862 and by Cryo-EM study of its complex with E.coli ribosome, it was shown, that it binds close to 560 loop of 30S ribosomal subunit. The new compound is a derivative of tetramic acid and we called it Auraplanin, because of bright orange color of the producer strain. Structural data are in good agreement with genetic results – resistant mutations were located close determined binding site. Substitutions C564G, G558U, and G566A significantly increase minimal inhibitory concentration, all these mutations were not detected previously. We also observed resistant mutation in ribosomal protein S4, this mutation was previously identified as error-prone. Interestingly, ribosomal ambiguity mutations, G299A and G347U, also increased resistance to Auraplanin. Conclusion: On the basis of the genetic, structural and biochemical studies we hypothesized that Auraplanin acts prevent the transfer from an open to a closed conformation of 30S subunit, in contrast to streptomycin, which promotes the formation of a closed state.

Aminoglycosides antagonize bacteriophage proliferation, attenuating phage suppression of bacterial growth, biofilm formation, and antibiotic resistance

The common co-occurrence of antibiotics and phages in both natural and engineered environments underscore the need to understand their interactions and implications for bacterial control and antibiotic resistance propagation. Here, aminoglycoside antibiotics that inhibit protein synthesis (e.g., kanamycin and neomycin) impeded replication of coliphage T3 and Bacillus phage BSP, reducing their infection efficiency and mitigating their hindrance of bacterial growth, biofilm formation and tolerance to antibiotics. For example, treatment with phage T3 reduced subsequent biofilm formation by E. coli liquid cultures to 53 ± 5% of the no-phage control, but a smaller reduction of biofilm formation (89 ± 10%) was observed for combined exposure to phage T3 and kanamycin. Despite sharing a similar mode of action with aminoglycosides (i.e., inhibiting protein synthesis) and antagonizing phage replication –albeit to a lesser degree, tetracyclines did not inhibit phages for bacterial control. Phage T3 combined with tetracycline showed higher suppression of biofilm formation than when combined with aminoglycosides (25 ± 6% of no-phage control). Addition of phage T3 to E. coli suspensions with tetracycline also suppressed the development of tolerance to tetracycline. However, this suppression of antibiotic tolerance development disappeared when tetracycline was replaced with 3 mg/L kanamycin, corroborating greater antagonism with aminoglycosides. Overall, this study highlights this overlooked antagonistic effect on phage proliferation, which may attenuate phage suppression of bacterial growth, biofilm formation, antibiotic tolerance, and maintenance of antibiotic resistance genes. Importance Co-existence of residual antibiotics and phages is common in many environments, which underscores the need to understand their interactive effects on bacteria and the implications for antibiotic resistance propagation. Here, aminoglycosides acting as bacterial protein synthesis inhibitors impeded replication of various phages. This alleviated the suppressive effects of phages against bacterial growth and biofilm formation, and diminished bacterial fitness cost that suppress emergence of tolerance to antibiotics. We show that changes in bacteria caused by environmentally relevant concentrations of sublethal antibiotics can affect phage-host dynamics that are commonly overlooked in vitro, but can result in unexpected environmental consequences.

Aerobacterial Vaginosis among Women Attending an Infertility Clinic at a Tertiary Care Hospital in Chennai, India and Susceptibility Pattern of Isolates

Genital infections and subsequent vaginosis diagnosed through high vaginal swab in women is caused due to fungi or bacteria. The presented study focused on determining the types, numbers and antibacterial susceptibility pattern of aerobic bacteria causing vaginosis in 147 female patients attending infertility centre in Chennai, Tamil Nadu, India. Candida spp. caused 17% of infections with 15.7% of vaginosis caused by E.coli, Klebsiella spp., Acinetobacter spp., Citrobacter spp. and Gram positive cocci – Methicillin Sensitive Staphylococcus aureus (MSSA), Coagulase Negative Staphylococci(CONS), Staphylococcus aureus and Enterococcus spp. causing 12.9% of vaginitis in the study population. A total of 20 different antibiotics – cell wall inhibitors, protein synthesis inhibitors and nucleic acid synthesis inhibitors; were tested to determine the response of bacterial isolates by Kirby-Bauer disc diffusion method. The study result determined that the most effective drug for treating Gram positive bacterial vaginitis as per CLSI guidelines based on susceptibility pattern as: Linezolid(100%), Gentamycin(91.6%), Amikacin(87.5%),Erythromycin(79.2%), Co-Trimoxazole(72.2%), Ciprofloxacin(65.6%) and least Chloramphenicol(44.3%). High level gentamycin(83.3%) was found to be effective in treating Enterococci. The descending order of susceptibility of Gram negative aerobacteria causing vaginitis as per CLSI guidelines are: Amikacin(87.5%), Gentamycin(82.5%), Cefoperazone sulbactam (76.3%), Ciprofloxacin(68.5%), Ceftazidime(62.5%) and least Amoxyclav(25%).All Gram negative bacteria tested were susceptible to– Imipenem and Meropenem as well as Chloramphenicol. Ceftriaxone (87.5%) and Nitrofurantoin(72.3%) among other antibiotics was effective against Gram negative bacteria while all Enterobacteriaceae members were found to be resistant to tetracycline.

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P &lt; 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P &lt; 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P &lt; 0.05) and its activity at 4 and 1 hpa, respectively (P &lt; 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P &lt; 0.05); however, its kinase activity decreased at 6 hpf (P &lt; 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P &lt; 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.