Engineered peptide PLG0206 overcomes limitations of a challenging antimicrobial drug class.

The absence of novel antibiotics for drug-resistant and biofilm-associated infections is a global public health crisis. Antimicrobial peptides explored to address this need have encountered significant development challenges associated with size, toxicity, safety profile, and pharmacokinetics. We designed PLG0206, an engineered antimicrobial peptide, to address these limitations. PLG0206 has broad-spectrum activity against >1,200 multidrug-resistant (MDR) ESKAPEE clinical isolates, is rapidly bactericidal, and displays potent anti-biofilm activity against diverse MDR pathogens. PLG0206 displays activity in diverse animal infection models following both systemic (urinary tract infection) and local (prosthetic joint infection) administration. These findings support continuing clinical development of PLG0206 and validate use of rational design for peptide therapeutics to overcome limitations associated with difficult-to-drug pharmaceutical targets.

Synthetic group A streptogramin antibiotics that overcome Vat resistance.

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.

In vitro activity of omadacycline and levofloxacin against Escherichia coli, Klebsiella pneumoniae and Staphylococcus saprophyticus in human urine supplemented with calcium and magnesium.

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.

In vitro activity of the novel antibacterial agent ibezapolstat (ACX-362E) against Clostridioides difficile.

BACKGROUND: Ibezapolstat (ACX-362E) is the first DNA polymerase IIIC inhibitor undergoing clinical development for the oral treatment of Clostridioides difficile infection (CDI). METHODS: In 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. RESULTS: The 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. CONCLUSIONS: The 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.

Comparison of the in vitro antibacterial activity of Ramizol, fidaxomicin, vancomycin, and metronidazole against 100 clinical isolates of Clostridium difficile by broth microdilution.

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.

Aminomethyl Spectinomycins as Therapeutics for Drug-Resistant Gonorrhea and Chlamydia Coinfections.

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.

In Vitro Activities of Omadacycline and Comparators against Anaerobic Bacteria.

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 MIC90 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.

Evaluation of robenidine analog NCL195 as a novel broad-spectrum antibacterial agent.

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.

In vitro potency and fungicidal activity of CD101, a novel echinocandin, against recent clinical isolates of Candida spp.

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.

Evaluation of the bactericidal activity of Telavancin against Staphylococcus aureus using revised testing guidelines.

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.

Comparative in vitro activity of oritavancin and other agents against methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

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.

Preclinical development of Ramizol, an antibiotic belonging to a new class, for the treatment of Clostridium difficile colitis.

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 <0.1% of Ramizol is absorbed systemically. We show this non-systemic nature of the antibiotic translates to over 70% survival of hamsters in a Clostridium difficile colitis model. Lastly, initial in vitro data indicate that resistance to Ramizol occurs at a low frequency. In conclusion, we establish the potential of Ramizol as an effective new treatment for C. difficile associated disease.

Tricyclic GyrB/ParE (TriBE) inhibitors: a new class of broad-spectrum dual-targeting antibacterial agents.

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.

MBX-500, a hybrid antibiotic with in vitro and in vivo efficacy against toxigenic Clostridium difficile.

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.

Comparative in vitro activity profiles of novel bis-indole antibacterials against gram-positive and gram-negative clinical isolates.

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 microg/ml. One bis-indole, MBX 1162, exhibited potent activity against all gram-negative strains, with MIC(90) values of 0.12 to 4 microg/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.

In vitro activity of TR-700, the active ingredient of the antibacterial prodrug TR-701, a novel oxazolidinone antibacterial agent.

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.

Accuracy of six antimicrobial susceptibility methods for testing linezolid against staphylococci and enterococci.

A challenge panel of enterococci (n = 50) and staphylococci (n = 50), including 17 and 15 isolates that were nonsusceptible to linezolid, respectively, were tested with the Clinical and Laboratory Standards Institute broth microdilution and disk diffusion reference methods. In addition, all 100 isolates were tested in parallel by Etest (AB Biodisk, Solna, Sweden), MicroScan WalkAway (Dade, West Sacramento, CA), BD Phoenix (BD Diagnostic Systems, Sparks, MD), VITEK (bioMérieux, Durham, NC), and VITEK 2 (bioMérieux) by using the manufacturers' protocols. Compared to the results of the broth microdilution method for detecting linezolid-nonsusceptible staphylococci and enterococci, MicroScan results showed the highest category agreement (96.0%). The overall categorical agreement levels for VITEK 2, Etest, Phoenix, disk diffusion, and VITEK were 93.0%, 90.0%, 89.6%, 88.0%, and 85.9%, respectively. The essential agreement levels (results within +/-1 doubling dilution of the MIC determined by the reference method) for MicroScan, Phoenix, VITEK 2, Etest, and VITEK were 99.0%, 95.8%, 92.0%, 92.0%, and 85.9%, respectively. The very major error rates for staphylococci were the highest for VITEK (35.7%), Etest (40.0%), and disk diffusion (53.3%), although the total number of resistant isolates tested was small. The very major error rate for enterococci with VITEK was 20.0%. Three systems (MicroScan, VITEK, and VITEK 2) provided no interpretations of nonsusceptible results for staphylococci. These data, from a challenge panel of isolates, illustrate that the recent emergence of linezolid-nonsusceptible staphylococci and enterococci is providing a challenge for many susceptibility testing systems.

The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria.

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.

Characterization of a high-throughput screening assay for inhibitors of elongation factor p and ribosomal peptidyl transferase activity.

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 microM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent K(m)s of 19 microM and 0.5 microM, 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.