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International audience; Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Background Omadacycline, an aminomethylcycline, was approved in 2018 for the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia. In a Phase Ib study, around 34% of the absorbed dose of omadacycline was shown to be excreted in urine—an important property for urinary tract infection (UTI) treatment. Therefore, omadacycline has been studied in two Phase II trials for the treatment of uncomplicated UTIs and acute pyelonephritis. The activity of omadacycline against UTI pathogens in human urine is important to understand in this context. Objectives To study the in vitro activity of omadacycline against UTI pathogens in human urine supplemented with calcium and magnesium. Methods Omadacycline activity was compared with that of levofloxacin against the urinary pathogens Escherichia coli, Klebsiella pneumoniae and Staphylococcus saprophyticus in standard medium, pooled normal human urine and neutral pH-adjusted pooled normal human urine spiked with calcium or magnesium at concentrations consistent with hypercalcaemia and hypermagnesaemia. Results The activities of omadacycline and levofloxacin against these urinary pathogens were lower in urine relative to standard medium; addition of Mg2+ to broth and urine had a further negative impact on omadacycline activity, whereas the addition of Ca2+ had less of an impact. Levofloxacin activity was not substantially reduced in either broth or urine by the addition of divalent cations. Conclusions The activity of omadacycline against UTI organisms was lower in urine relative to standard medium and was negatively impacted by magnesium. Omadacycline displayed slightly reduced activity when excess calcium was present, but, overall, the differences were ≤2-fold. These observations should be considered along with the pharmacokinetics of the agent for clinical context.

BackgroundIbezapolstat (ACX-362E) is the first DNA polymerase IIIC inhibitor undergoing clinical development for the oral treatment of Clostridioides difficile infection (CDI).MethodsIn this study, the in vitro activity of ibezapolstat was evaluated against a panel of 104 isolates of C. difficile, including those with characterized ribotypes (e.g. 027 and 078) and those producing toxin A or B and was shown to have similar activity to those of comparators against these strains.ResultsThe overall MIC50/90 (mg/L) for ibezapolstat against evaluated C. difficile was 2/4, compared with 0.5/4 for metronidazole, 1/4 for vancomycin and 0.5/2 for fidaxomicin. In addition, the bactericidal activity of ibezapolstat was evaluated against actively growing C. difficile by determining the MBC against three C. difficile isolates. Time–kill kinetic assays were additionally performed against the three C. difficile isolates, with metronidazole and vancomycin as comparators.ConclusionsThe killing of C. difficile by ibezapolstat was observed to occur at concentrations similar to its MIC, as demonstrated by MBC:MIC ratios and reflected in time–kill kinetic assays. This activity highlights the therapeutic potential of ibezapolstat for the treatment of CDI.

Natural products serve as chemical blueprints for the majority of classes of antibiotics in our clinical arsenal. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class. Virginiamycin acetyltransferases (Vats) are resistance proteins that provide protection against streptogramins, potent Gram-positive antibiotics that inhibit the bacterial ribosome. Due to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogs that overcome Vat resistance have not been previously accessible. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with unprecedented structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogs to the bacterial ribosome at high resolution (2.4-2.8 Å), revealing new binding interactions that extend into the peptidyl tRNA binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. Two of these analogs have excellent activity against a streptogramin-resistant strain of S. aureus expressing VatA and exhibit decreased acetylation rates in vitro. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Antibiotic-resistant bacteria is a major threat to human health and is predicted to become the leading cause of death from disease by 2050. Despite the recent resurgence of research and development in the area, few antibiotics have reached the market, with most of the recently approved antibiotics corresponding to new uses for old antibiotics, or structurally similar derivatives thereof. We have recently reported an in silico approach that led to the design of an entirely new class of antibiotics for the bacteria-specific mechanosensitive ion channel of large conductance: MscL. Here, we present the preclinical development of one such antibiotic, Ramizol, a first generation antibiotic belonging to that class. We present the lack of interaction between Ramizol and other mammalian channels adding credibility to its MscL selectivity. We determine the pharmacokinetic profile in a rat model and show

The in vitro broth microdilution testing method for telavancin, a lipoglycopeptide active against S. aureus, was revised in 2014 to include polysorbate-80 in the test media. This study evaluates the bactericidal activity of telavancin against S. aureus in media containing polysorbate-80 by in vitro time-kill analysis alongside relevant comparators.

This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This author accepted manuscript is made available following 12 month embargo from date of publication (June 2018) in accordance with the publisher’s archiving policy, Antibiotic drug development remains a major challenge with few candidates in clinical development. Ramizol, a first-in-class styrylbenzene antibiotic, is under development for the treatment of Clostridium difficile associated disease. Here, we investigate the in vitro antibacterial activity of Ramizol in comparison to fidaxomicin, vancomycin and metronidazole against 100 clinical isolates of C. difficile by the broth microdilution method. We show there is no apparent impact of ribotype, toxin-production, or resistance to fidaxomicin, vancomycin or metronidazole on the activity of Ramizol. Moreover, we show Ramizol has a narrower MIC range translating to potentially better control over the therapeutic dose. Together, these results support the further development of Ramizol for the treatment of C. difficile associated disease., The authors acknowledge funding from Boulos & Cooper Pharmaceuticals for this research.

Bacterial sexually transmitted infections are widespread and common, with Neisseria gonorrhoeae (gonorrhea) and Chlamydia trachomatis (chlamydia) being the two most frequent causes. If left untreated, both infections can cause pelvic inflammatory disease, infertility, ectopic pregnancy, and other sequelae. The recommended treatment for gonorrhea is ceftriaxone plus azithromycin (to empirically treat chlamydial coinfections). Antibiotic resistance to all existing therapies has developed in gonorrheal infections. The need for new antibiotics is great, but the pipeline for new drugs is alarmingly small. The aminomethyl spectinomycins, a new class of semisynthetic analogs of the antibiotic spectinomycin, were developed on the basis of a computational analysis of the spectinomycin binding site of the bacterial 30S ribosome and structure-guided synthesis. The compounds display particular potency against common respiratory tract pathogens as well as the sexually transmitted pathogens that cause gonorrhea and chlamydia. Here, we demonstrate the in vitro potencies of several compounds of this class against both bacterial species; the compounds displayed increased potencies against N. gonorrhoeae compared to that of spectinomycin and, significantly, demonstrated activity against C. trachomatis that is not observed with spectinomycin. Efficacies of the compounds were compared to those of spectinomycin and gentamicin in a murine model of infection caused by ceftriaxone/azithromycin-resistant N. gonorrhoeae ; the aminomethyl spectinomycins significantly reduced the colonization load and were as potent as the comparator compounds. In summary, data produced by this study support aminomethyl spectinomycins as a promising replacement for spectinomycin and antibiotics such as ceftriaxone for treating drug-resistant gonorrhea, with the added benefit of treating chlamydial coinfections.

Omadacycline (OMC), a broad-spectrum aminomethylcycline, has shown clinical efficacy in anaerobic acute bacterial skin and skin structure infections (ABSSSI) and in animal models of intra-abdominal anaerobic infections. Here, the in vitro activity of OMC against clinically relevant anaerobes was similar to that of tigecycline, with MIC 90 values of 1 to 8 μg/ml against Bacteroides spp., 0.5 μg/ml against Clostridium difficile , Prevotella spp., and Porphyromonas asaccharolytica , 1 μg/ml against Peptostreptococcus spp., and 16 μg/ml against Clostridium perfringens .

Made available in DSpace on 2018-11-26T17:40:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-05 Australian Research Council (ARC) Neoculi Pty Ltd National Health and Medical Research Council of Australia (NHMRC) University of South Australia Sansom Institute of Health Research Micromyx LLC The spread of multidrug resistance among bacterial pathogens poses a serious threat to public health worldwide. Recent approaches towards combating antimicrobial resistance include repurposing old compounds with known safety and development pathways as new antibacterial classes with novel mechanisms of action. Here we show that an analog of the anticoccidial drug robenidine (4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine; NCL195) displays potent bactericidal activity against Streptococcus pneumoniae and Staphylococcus aureus by disrupting the cell membrane potential. NCL195 was less cytotoxic to mammalian cell lines than the parent compound, showed low metabolic degradation rates by human and mouse liver microsomes, and exhibited high plasma concentration and low plasma clearance rates in mice. NCL195 was bactericidal against Acinetobacter spp and Neisseria meningitidis and also demonstrated potent activity against A. baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. in the presence of sub-inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA) and polymyxin B. These findings demonstrate that NCL195 represents a new chemical lead for further medicinal chemistry and pharmaceutical development to enhance potency, solubility and selectivity against serious bacterial pathogens. Univ Adelaide, Sch Anim & Vet Sci, Australian Ctr Antimicrobial Resistance Ecol, Roseworthy, SA, Australia Univ Newcastle, Sch Environm & Life Sci, Chem, Callaghan, NSW, Australia Univ Adelaide, Sch Anim & Vet Sci, Roseworthy, SA, Australia Neoculi Pty Ltd, Burwood, Vic, Australia Univ South Australia, Sansom Inst Hlth Res, Sch Pharm & Med Sci, Ctr Pharmaceut Innovat & Dev, Adelaide, SA, Australia Univ South Australia, Sansom Inst Hlth Res, Sch Pharm & Med Sci, Adelaide, SA, Australia Monash Univ, Monash Inst Pharmaceut Sci, Ctr Drug Candidate Optimisat, Parkville, Vic, Australia Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Drugs & Med, Sao Paulo, Brazil Fiona Stanley Hosp, Path West Lab Med WA, Dept Microbiol, Murdoch, WA, Australia Murdoch Univ, Sch Vet & Life Sci, Murdoch, WA, Australia Micromyx LLC, Kalamazoo, MI USA Univ Adelaide, Sch Biol Sci, Dept Mol & Cellular Biol, Adelaide, SA, Australia Univ Adelaide, Sch Biol Sci, Dept Mol & Cellular Biol, Res Ctr Infect Dis, Adelaide, SA, Australia Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Drugs & Med, Sao Paulo, Brazil Australian Research Council (ARC): LP110200770 National Health and Medical Research Council of Australia (NHMRC): 565526 National Health and Medical Research Council of Australia (NHMRC): 627142

Candida infections vary in severity and manifestation. Common infections include invasive bloodstream infections among hospitalized/immunocompromised patients and vulvovaginal candidiasis among women. Echinocandins and azoles are commonly utilized to treat Candida infections, although echinocandin use has been restricted to indications amenable to once-daily IV administration. CD101, a novel echinocandin with a long plasma half-life and enhanced stability, is in development for once-weekly IV administration for the treatment of candidemia and invasive candidiasis. In this study, the MIC of CD101 and comparators against 500 recent clinical Candida isolates was determined per Clinical and Laboratory Standards Institute guidelines. For select isolates, the minimum fungicidal concentration (MFC; n=49) and time-kill (n=9) of CD101 and comparators was evaluated. The MIC50/90s (μg/mL; n=100/species) for CD101, anidulafungin, fluconazole, and amphotericin B, respectively, were: C. albicans (0.008/0.03, 0.004/0.008, 0.25/4, 0.25/0.5), C. tropicalis (0.008/0.03, 0.004/0.015, 0.5/2, 0.5/1), C. parapsilosis (1/1, 0.5/2, 0.5/1, 0.5/1), C. glabrata (0.03/0.03, 0.03/0.03, 8/>32, 0.5/0.5), and C. krusei (0.03/0.03, 0.03/0.03, 32/>32, 1/1). CD101 MICs were comparable to anidulafungin and both maintained potency against fluconazole-resistant isolates. Against rare anidulafungin-resistant isolates, the MICs of CD101 and anidulafungin were elevated vs. anidulafungin-susceptible isolates. Similar to anidulafungin, CD101 was fungicidal with an MFC:MIC ratio ≤4 for 95% of evaluable isolates and resulted in 3-log killing by 24-48h for all isolates evaluated by time-kill. The potent fungicidal activity of CD101 highlights the potential clinical utility of CD101 IV for the treatment of invasive candidiasis and candidemia.

Methicillin-resistant Staphylococcus aureus (MRSA) infections constitute a threat to the public health due to their prevalence and associated mortality and morbidity. Several agents have been recently approved to treat MRSA skin infections including lipoglycopeptides (dalbavancin, oritavancin, and telavancin), ceftaroline, and tedizolid. This study compared the MIC, minimum bactericidal concentration (MBC), and time-kill of these agents alongside daptomycin, linezolid, and vancomycin against MRSA (n=15); meropenem, cefazolin, and nafcillin were also included against methicillin-susceptible S. aureus (MSSA [n=12]). MIC and MBC testing was conducted in accordance with Clinical and Laboratory Standards Institute guidelines, and time-kills were evaluated at multiples of the MIC and the free-drug maximum plasma concentration (fCmax) at both standard and high inoculum densities for a subset of MRSA (n=2) and MSSA (n=2). MRSA and MSSA were highly susceptible to all agents, with the lipoglycopeptides having the most potent activity by MIC50/90. All agents excluding tedizolid and linezolid were bactericidal by MBC for MRSA and MSSA, though dalbavancin and telavancin exhibited strain-specific bactericidal activity for MRSA. All agents excluding tedizolid and linezolid were bactericidal by time-kill at their respective fCmax against MRSA and MSSA at standard inoculum density, though oritavancin exhibited the most rapid bactericidal activity. Oritavancin and daptomycin at their respective fCmax maintained similar kill curves at high inoculum density. In contrast, the killing observed with other agents was typically reduced or slowed at high inoculum density. These data demonstrate the rapid bactericidal activity of oritavancin and daptomycin against S. aureus relative to other MRSA agents regardless of bacterial burden.

UNLABELLED The magnitudes of the postantibiotic effect (PAE) and post-β-lactamase-inhibitory effect (PLIE) of ceftazidime-avibactam, ceftaroline-avibactam, and aztreonam-avibactam were determined against isolates of Enterobacteriaceae and Pseudomonas aeruginosa that either harboured genes encoding serine and/or metallo-β-lactamases, or did not harbour bla genes. The bla genes included ones that encoded extended spectrum β-lactamases, AmpC and KPC β-lactamases, and one metallo-β-lactamase, NDM-1. No substantial PAE was observed for any combination against any isolate. One substantial PLIE was found: a value of 1·9 h for ceftazidime-avibactam against Klebsiella pneumoniae (blaKPC-2 ). From comparison with results in the literature, we propose that the existence of a substantial PLIE depends on the bacterial isolate and on the specific β-lactamase inhibitor and β-lactam combination. SIGNIFICANCE AND IMPACT OF THE STUDY A wave of new β-lactamase inhibitors is entering either therapeutic use or clinical trials. The present work characterizes the postantibiotic effect (PAE) and post-β-lactamase-inhibitory effect (PLIE) of the clinically most advanced of these compounds, avibactam. We show that the existence of a measurable PLIE is strain- (and possibly compound-) dependent, and cannot be relied upon as a standard component of the primary pharmacology of a new β-lactamase inhibitor. This variability was not reported in earlier studies of clavulanic acid or sulbactam.

Clostridium difficile infection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, the in vitro and in vivo efficacies of MBX-500 were explored against the Gram-positive anaerobe, C. difficile . MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.

Antimicrobial susceptibilities of 233 Gram-positive and 180 Gram-negative strains to two novel bis-indoles were evaluated. Both compounds were potent inhibitors of Gram-positive bacteria, with MIC 90 values of 0.004 to 0.5 μg/ml. One bis-indole, MBX 1162, exhibited potent activity against all Gram-negative strains, with MIC 90 values of 0.12 to 4 μg/ml, even against high-level-resistant pathogens, and compared favorably to all comparator antibiotics. The bis-indole compounds show promise for the treatment of multidrug-resistant clinical pathogens.

TR-701 is the prodrug of the microbiologically active molecule TR-700, a novel orally and intravenously administered oxazolidinone antibacterial agent. The in vitro activity of TR-700 was evaluated against 1,063 bacterial clinical isolates including staphylococci, enterococci, streptococci, Moraxella catarrhalis , Haemophilus influenzae , and a variety of anaerobic bacterial species. The test strains were recent (2005 to 2008) clinical isolates from diverse U.S. (80%) and non-U.S. (20%) sites. MIC assays were conducted using reference broth microdilution and agar dilution methods with the principal comparators linezolid and vancomycin. TR-700 was four- to eightfold more potent than linezolid against staphylococci and generally fourfold more potent against enterococci and streptococci. TR-700 was less active against M. catarrhalis and H. influenzae but was twofold more active than linezolid. Against anaerobic species, the activity of TR-700 was equivalent to or up to fourfold higher than that of linezolid. These results indicate that TR-700 is a promising new oxazolidinone antibacterial agent with greater in vitro potency than linezolid against clinically important gram-positive bacteria.

TR-701 is the orally active prodrug of TR-700, a novel oxazolidinone that demonstrates four- to eightfold-greater activity than linezolid (LZD) against Staphylococcus and Enterococcus spp. In this study evaluating the in vitro sensitivity of LZD-resistant isolates, TR-700 demonstrated 8- to 16-fold-greater potency than LZD against all strains tested, including methicillin-resistant Staphylococcus aureus (MRSA), strains of MRSA carrying the mobile cfr methyltransferase gene, and vancomycin-resistant enterococci. The MIC 90 for TR-700 against LZD-resistant S. aureus was 2 μg/ml, demonstrating the utility of TR-700 against LZD-resistant strains. A model of TR-700 binding to 23S rRNA suggests that the increased potency of TR-700 is due to additional target site interactions and that TR-700 binding is less reliant on target residues associated with resistance to LZD.

The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.

Elongation Factor P (EF-P) is an essential component of bacterial protein synthesis, enhancing the rate of translation by facilitating the addition of amino acids to the growing peptide chain. Using purified Staphylococcus aureus EF-P and a reconstituted Escherichia coli ribosomal system, an assay monitoring the addition of radiolabeled N-formyl methionine to biotinylated puromycin was developed. Reaction products were captured with streptavidin-coated scintillation proximity assay (SPA) beads and quantified by scintillation counting. Data from the assay were used to create a kinetic model of the reaction scheme. In this model, EF-P binding to the ribosome essentially doubled the rate of the ribosomal peptidyl transferase reaction. As described here, EF-P bound to the ribosomes with an apparent K a of 0.75 µM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent K m s of 19 µM and 0.5 µM, respectively. The assay was shown to be sensitive to a number of antibiotics known to target ribosomal peptide bond synthesis, such as chloramphenicol and puromycin, but not inhibitors that target other stages of protein synthesis, such as fusidic acid or thiostrepton. (Journal of Biomolecular Screening 2006:736-742)

Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.

The key enzymatic activity of the ribosome is catalysis of peptide bond formation. This reaction is a target for many clinically important antibiotics. However, the molecular mechanisms of the peptidyl transfer reaction, the catalytic contribution of the ribosome, and the mechanisms of antibiotic action are still poorly understood. Here we describe a novel, simple, convenient, and sensitive method for monitoring peptidyl transferase activity (SPARK). In this method, the ribosomal peptidyl transferase forms a peptide bond between two ligands, one of which is tritiated whereas the other is biotin-tagged. Transpeptidation results in covalent attachment of the biotin moiety to a tritiated compound. The amount of the reaction product is then directly quantified using the scintillation proximity assay technology: binding of the tritiated radioligand to the commercially available streptavidin-coated beads causes excitation of the bead-embedded scintillant, resulting in detection of radioactivity. The reaction is readily inhibited by known antibiotics, inhibitors of peptide bond formation. The method we developed is amenable to simple automation which makes it useful for screening for new antibiotics. The method is useful for different types of ribosomal research. Using this method, we investigated the effect of mutations at a universally conserved nucleotide of the active site of 23S rRNA, A2602 (Escherichia coli numbering), on the peptidyl transferase activity of the ribosome. The activities of the in vitro reconstituted mutant subunits, though somewhat reduced, were comparable with those of the subunits assembled with the wild-type 23S rRNA, indicating that A2602 mutations do not abolish the ability of the ribosome to catalyze peptide bond formation. Similar results were obtained with double mutants carrying mutations at A2602 and another universally conserved nucleotide in the peptidyl transferase center, A2451. The obtained results agree with our previous conclusion that the ribosome accelerates peptide bond formation primarily through entropic rather than chemical catalysis.

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.

Oxazolidinones represent a novel class of antibiotics that inhibit protein synthesis in sensitive bacteria. The mechanism of action and location of the binding site of these drugs is not clear. A new representative of oxazolidinone antibiotics, linezolid, was found to be active against bacteria and against the halophilic archaeon Halobacterium halobium. The use of H. halobium, which possess only one chromosomal copy of rRNA operon, allowed isolation of a number of linezolid-resistance mutations in rRNA. Four types of linezolid-resistant mutants were isolated by direct plating of H. halobium cells on agar medium containing antibiotic. In addition, three more linezolid-resistant mutants were identified among the previously isolated mutants of H. halobium containing mutations in either 16 S or 23 S rRNA genes. All the isolated mutants were found to contain single-point mutations in 23 S rRNA. Seven mutations affecting six different positions in the central loop of domain V of 23 S rRNA were found to confer resistance to linezolid. Domain V of 23 S rRNA is known to be a component of the ribosomal peptidyl transferase center. Clustering of linezolid-resistance mutations within this region strongly suggests that the binding site of the drug is located in the immediate vicinity of the peptidyl transferase center. However, the antibiotic failed to inhibit peptidyl transferase activity of the H. halobium ribosome, supporting the previous conclusion that linezolid inhibits translation at a step different from the catalysis of the peptide bond formation.

The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging of N -formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N -formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [ 3 H] N -formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits from Escherichia coli . In addition, complex formation with Staphylococcus aureus 70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or N -formylmethionyl-tRNA to E. coli 30S ribosomal subunits, nor did it prevent the formation of the IF2– N -formylmethionyl-tRNA binary complex. The results demonstrate that oxazolidinones inhibit the formation of the initiation complex in bacterial translation systems by preventing formation of the N -formylmethionyl-tRNA–ribosome–mRNA ternary complex.

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.

The soil bacteria Pseudomonas putida can use benzoate or 3-chlorobenzoate as a sole carbon source. Benzoate and 3-chlorobenzoate are converted into catechol and 3-chlorocatechol, respectively, which are in turn converted into tricarboxylic acid cycle intermediates. The catabolic pathways of both compounds proceed through similar intermediates, have similar genetic organization, and have homologous enzymes responsible for different catabolic steps. This has led to suggestions that the plasmid-borne 3-chlorocatechol degradation genes evolved from the chromosomal catechol degradation genes. Both catechol and 3-chlorocatechol pathways are positively regulated by the homologous regulatory proteins CatR and ClcR, respectively. These proteins belong to the LysR family of DNA binding proteins and bind to highly conserved target sequences. We examined the ability of CatR and ClcR to cross-regulate the two pathways. CatR was shown in vitro by DNase I footprinting and gel-shift assays to interact with the clcABD promoter region. Likewise, ClcR was shown to interact in vitro with the catBC promoter region. In in vivo experiments, CatR complemented a ClcR- P. putida strain harboring the clcABD operon for growth on 3-chlorobenzoate. However, ClcR was not capable of complementing a CatR- P. putida strain for growth on benzoate. These observations were confirmed by lacZ-transcriptional fusion expression experiments. Differences in the CatR and ClcR binding sites and their in vitro binding characteristics may explain the ability of CatR and not ClcR to cross-activate. These differences may provide insight about the evolution of regulatory systems in P. putida.

The oxazolidinones are a new class of potent antibiotics that are active against a broad spectrum of Gram-positive bacterial pathogens including those resistant to other antibiotics. These drugs specifically inhibit protein biosynthesis whereas DNA and RNA synthesis are not affected. Although biochemical and genetic studies indicate that oxazolidinones target the ribosomal peptidyltransferase center, other investigations suggest that they interact with different regions of ribosomes. Thus, the exact binding site and mechanism of action have remained elusive. Here, we study, by use of base-specific reagents, the effect of the oxazolidinones on the chemical protection footprinting patterns of the 23S rRNA. We report: (i) reproducible protection of G2607 and G2608 of 23S rRNA by a potent oxazolidinone on a ribosome.tRNA.mRNA complex; (ii) no protections were observed on 70S ribosomes devoid of tRNA and mRNA; (iii) EF-G also weakly protected G2607 and G2608; (iv) mutations at G2608 conferred resistance to the oxazolidinones in Escherichia coli cells; and (v) G2607 and G2608 occur near the exit to the peptide tunnel on the 50S subunit. A mechanism for the pleiotropic action of the oxazolidinones is discussed.

The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium , Staphylococcus aureus , and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.

The oxazolidinones are a novel class of antibiotics that inhibit initiation of protein synthesis in bacteria. In order to investigate their novel mechanism of action, the interactions of several oxazolidinones with bacterial 70S ribosomes, 50S subunits, and 30S subunits have been characterized by 1 H nuclear magnetic resonance (NMR) line-broadening analyses and transferred nuclear Overhauser enhancement (TRNOE) experiments. PNU-177553 and PNU-100592 (eperezolid) and their corresponding enantiomers, PNU-184414 and PNU-107112, were studied. The dissociation constants were determined to be 94 ± 44 μM and 195 ± 40 μM for PNU-177553 and eperezolid, respectively. There was a ∼4-fold decrease in affinity for their corresponding enantiomers. The NMR-derived dissociation constants are consistent with their antibacterial activity. PNU-177553 and eperezolid were found to bind only to the 50S subunit, with similar affinity as to the 70S ribosome, and to have no affinity for the 30S subunit. Specific binding of PNU-177553 was further confirmed in TRNOE experiments in which positive NOEs observed for the small molecule alone were changed to negative NOEs in the presence of bacterial 70S ribosomes. The observed NOEs indicated that PNU-177553 did not adopt a significantly different conformation when bound to the 70S ribosome, compared to the extended conformation that exists when free in solution. Since this is likeliest the case for each of the four compounds included in this study, the A ring C5 side chain may be positioned in the proper orientation for antibacterial activity in PNU-177553 and eperezolid but not in their inactive enantiomers.

In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene ( ypfP ) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419–430, 1998). To define the role of ypfP in this strain of S. aureus , a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP . Chloramphenicol-resistant ( ypfP :: cat ) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either l - or d -alanine. Moreover, viability of the mutant at 37°C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to d -glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP :: cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.

Two strains of Thiothrix nivea were examined to determine their morphological and physiological features. Microculture studies revealed the production of gliding gonidia and possible mechanisms for rosette formation. The strains were able to use only 4 of 41 carbon sources tested, but only if sulfide or thiosulfate was present. Physiologically, Thiothrix may be an obligate mixotroph. The guanine-plus-cytosine content of the deoxyribonucleic acid was 52 mol%. Our strains fit the description of T. nivea, and strain JP2 (= ATCC 31500) was designated as the neotype strain.

The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined by using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing [3-14C]glyphosate revealed that approximately 50 to 59% of the C-3 carbon was oxidized to CO2. Fractionation of stationary-phase cells labeled with [3-14C]glyphosate revealed that from 45 to 47% of the assimilated label is distributed to proteins and that the amino acids methionine and serine are highly labeled. Adenine and guanine received 90% of the C-3 label found in the nucleic acid fraction, and the only pyrimidine base labeled was thymine. These results indicated that C-3 of glyphosate was at some point metabolized to a C-1 compound whose ultimate fate could be both oxidation to CO2 and distribution to amino acids and nucleic acid bases that receive a C-1 group from the C-1-donating coenzyme tetrahydrofolate. Pulse-labeling of PG2982 cells with [3-14C]glyphosate resulted in the isolation of [3-14C]sarcosine as an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of a sarcosine-oxidizing enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. This pathway is supported by the results of [1,2-14C]glyphosate metabolism studies, which show that radioactivity in the proteins of labeled cells is found only in the glycine and serine residues.

The leaky heme mutants G204, G216, and G214 are shown to accumulate exogenous sterols. Unlike hem mutants which have complete blocks in the heme pathway, these strains do not require ergosterol, methionine, or unsaturated fatty acids for growth. The addition of aminolevulinic acid to the growth medium inhibited sterol uptake in G204 96% but had only a slight effect on sterol uptake by strains G214 and G216. Sterol uptake in all three strains was inhibited 83–94% when cells were grown in the presence of hematin. Sterol analysis of these strains grown in the presence and absence of either aminolevulinic acid or hematin revealed that saturation of the cell membrane with ergosterol was not responsible for the dramatic decrease in sterol uptake. These results suggest that sterol uptake by yeast cells is controlled by heme, and explain the non-viability of yeast strains that are heme competent and auxotrophic for sterols.

The glyphosate-degrading Pseudomonas sp. strain PG2982 was found to utilize each of 10 organophosphonate compounds as a sole phosphorus source. Representative compounds tested included alkylphosphonates, 1-amino-substituted alkylphosphonates, amino-terminal phosphonates, and an arylphosphonate. This report demonstrates that PG2982 is capable of utilizing a wider range of structurally different organophosphonate compounds than any organism described to date.