Efficacy of therapeutically administered gepotidacin in a rabbit model of inhalational anthrax.

Bacillus anthracis is a Gram-positive Centers for Disease Control and Prevention category "A" biothreat pathogen. Without early treatment, inhalation of anthrax spores with progression to inhalational anthrax disease is associated with high fatality rates. Gepotidacin is a novel first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and is being evaluated for use against biothreat and conventional pathogens. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode and has in vitro activity against a collection of B. anthracis isolates including antibacterial-resistant strains, with the MIC90 ranging from 0.5 to 1 µg/mL. In vivo activity of gepotidacin was also evaluated in the New Zealand White rabbit model of inhalational anthrax. The primary endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. The trigger for treatment was the presence of anthrax protective antigen in serum. New Zealand White rabbits were dosed intravenously for 5 days with saline or gepotidacin at 114 mg/kg/d to simulate a dosing regimen of 1,000 mg intravenous (i.v.) three times a day (TID) in humans. Gepotidacin provided a survival benefit compared to saline control, with 91% survival (P-value: 0.0001). All control animals succumbed to anthrax and were found to be blood- and organ culture-positive for B. anthracis. The novel mode of action, in vitro microbiology, preclinical safety, and animal model efficacy data, which were generated in line with Food and Drug Administration Animal Rule, support gepotidacin as a potential treatment for anthrax in an emergency biothreat situation.

Efficacy of Intravenously Administered Gepotidacin in Cynomolgus Macaques following a Francisella tularensis Inhalational Challenge.

Francisella tularensis (F. tularensis) is a Centers for Disease Control (CDC) category "A" Gram-negative biothreat pathogen. Inhalation of F. tularensis can cause pneumonia and respiratory failure and is associated with high mortality rates without early treatment. Gepotidacin is a novel, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode, has activity against multidrug-resistant target pathogens, and has demonstrated in vitro activity against diverse collections of F. tularensis isolates (MIC90 of 0.5 to 1 µg/mL). Gepotidacin was evaluated in the cynomolgus macaque model of inhalational tularemia, using the SCHU S4 strain, with treatment initiated after exposure and sustained fever. Macaques were dosed via intravenous (i.v.) infusion with saline or gepotidacin at 72 mg/kg/day to support a human i.v. infusion dosing regimen of 1,000 mg three times daily. The primary study endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. Gepotidacin treatment resulted in 100% survival compared to 12.5% in the saline-treated control group (P < 0.0001) at Day 43 postinhalational challenge. All gepotidacin-treated animals were blood and organ culture negative for F. tularensis at the end of the study. In contrast, none of the saline control animals were blood and organ culture negative. Gepotoidacin's novel mechanism of action and the efficacy data reported here (aligned with the Food and Drug Administration Animal Rule) support gepotidacin as a potential treatment for pneumonic tularemia in an emergency biothreat situation.

Gepotidacin is efficacious in a nonhuman primate model of pneumonic plague.

Gepotidacin is a first-in-class triazaacenaphthylene antibacterial agent that selectively inhibits bacterial DNA gyrase and topoisomerase IV through a unique binding mode and has the potential to treat a number of bacterial diseases. Development of this new agent to treat pneumonic plague caused by Yersinia pestis depends on the U.S. Food and Drug Administration Animal Rule testing pathway, as testing in humans is not feasible. Here, preclinical studies were conducted in the African green monkey (AGM) inhalational model of pneumonic plague to test the efficacy of gepotidacin. AGMs infected with Y. pestis were dosed intravenously with gepotidacin (48, 36, or 28 milligrams/kilogram per day) for 10 days to provide a plasma concentration that would support a rationale for a 1000 mg twice or thrice daily intravenous dose in humans or saline as a control. The primary end point was AGM survival with predefined euthanasia criteria. Secondary end points included survival duration and bacterial clearance. Gepotidacin showed activity in vitro against diverse Y. pestis isolates including antibiotic-resistant strains. All control animals in the inhalational plague studies succumbed to plague and were blood culture and organ culture positive for Y. pestis. Gepotidacin provided a 75 to 100% survival benefit with all dose regimens. All surviving animals were blood culture and organ culture negative for Y. pestis. Our randomized, controlled efficacy trials in the AGM pneumonic plague nonhuman primate model together with the in vitro Y. pestis susceptibility data support the use of gepotidacin as a treatment for pneumonic plague caused by Y. pestis.

Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production.

Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor GSK1322322 in Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae.

The continuous emergence of multidrug-resistant pathogenic bacteria is compromising the successful treatment of serious microbial infections. GSK1322322, a novel peptide deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated safety and efficacy in human proof-of-concept clinical studies. In vitro studies were performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major pathogens that cause respiratory tract and skin infections. Resistance to GSK1322322 occurred at high frequency through loss-of-function mutations in the formyl-methionyl transferase (FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4 strains) and via missense mutations in Streptococcus pneumoniae (6/21 strains), but the mutations were associated with severe in vitro and/or in vivo fitness costs. The overall FoR to GSK1322322 was very low in Haemophilus influenzae, with only one PDF mutant being identified in one of four strains. No target-based mutants were identified from S. pyogenes, and only one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S. pneumoniae, PDF mutants were isolated from only six of 21 strains tested; an additional 10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants characterized from those three organisms (35/37 mutants) carried mutations in residues at or in close proximity to one of three highly conserved motifs that are part of the active site of the PDF protein, with 30 of the 35 mutations occurring at position V71 (using the S. pneumoniae numbering system).

Bacterial resistance to leucyl-tRNA synthetase inhibitor GSK2251052 develops during treatment of complicated urinary tract infections.

GSK2251052, a novel leucyl-tRNA synthetase (LeuRS) inhibitor, was in development for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. In a phase II study (study LRS114688) evaluating the efficacy of GSK2251052 in complicated urinary tract infections, resistance developed very rapidly in 3 of 14 subjects enrolled, with ≥32-fold increases in the GSK2251052 MIC of the infecting pathogen being detected. A fourth subject did not exhibit the development of resistance in the baseline pathogen but posttherapy did present with a different pathogen resistant to GSK2251052. Whole-genome DNA sequencing of Escherichia coli isolates collected longitudinally from two study LRS114688 subjects confirmed that GSK2251052 resistance was due to specific mutations, selected on the first day of therapy, in the LeuRS editing domain. Phylogenetic analysis strongly suggested that resistant Escherichia coli isolates resulted from clonal expansion of baseline susceptible strains. This resistance development likely resulted from the confluence of multiple factors, of which only some can be assessed preclinically. Our study shows the challenges of developing antibiotics and the importance of clinical studies to evaluate their effect on disease pathogenesis. (These studies have been registered at ClinicalTrials.gov under registration no. NCT01381549 for the study of complicated urinary tract infections and registration no. NCT01381562 for the study of complicated intra-abdominal infections.).

Potent sub-MIC effect of GSK1322322 and other peptide deformylase inhibitors on in vitro growth of Staphylococcus aureus.

Peptide deformylase (PDF), a clinically unexploited antibacterial target, plays an essential role in protein maturation. PDF inhibitors, therefore, represent a new antibiotic class with a unique mode of action that provides an alternative therapy for the treatment of infections caused by drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). GSK1322322 is a novel PDF inhibitor that is in phase II clinical development for the treatment of lower respiratory tract and skin infections. We have discovered that PDF inhibitors can prevent S. aureus in vitro growth for up to 6 h at concentrations 8- to 32-fold below their MICs. This phenomenon seems specific to PDF inhibitors, as none of the antimicrobial agents with alternative mechanisms of action tested show such a potent and widespread effect. It also appears limited to S. aureus, as PDF inhibitors do not show such an inhibition of growth at sub-MIC levels in Streptococcus pneumoniae or Haemophilus influenzae. Analysis of the effect of GSK1322322 on the early growth of 100 randomly selected S. aureus strains showed that concentrations equal to or below 1/8× MIC inhibited growth of 91% of the strains tested for 6 h, while the corresponding amount of moxifloxacin or linezolid only affected the growth of 1% and 6% of strains, respectively. Furthermore, the sub-MIC effect demonstrated by GSK1322322 appears more substantial on those strains at the higher end of the MIC spectrum. These effects may impact the clinical efficacy of GSK1322322 in serious infections caused by multidrug-resistant S. aureus.

Comparative analysis of the antibacterial activity of a novel peptide deformylase inhibitor, GSK1322322.

GSK1322322 is a novel peptide deformylase (PDF) inhibitor being developed for the intravenous and oral treatment of acute bacterial skin and skin structure infections and hospitalized patients with community-acquired pneumonia. The activity of GSK1322322 was tested against a global collection of clinical isolates of Haemophilus influenzae (n = 2,370), Moraxella catarrhalis (n = 115), Streptococcus pneumoniae (n = 947), Streptococcus pyogenes (n = 617), and Staphylococcus aureus (n = 940), including strains resistant to one or more marketed antibiotics. GSK1322322 had an MIC(90) of 1 µg/ml against M. catarrhalis and 4 µg/ml against H. influenzae, with 88.8% of ß-lactamase-positive strains showing growth inhibition at that concentration. All S. pneumoniae strains were inhibited by ≤ 4 µg/ml of GSK1322322, with an MIC(90) of 2 µg/ml. Pre-existing resistance mechanisms did not affect its potency, as evidenced by the MIC(90) of 1 µg/ml for penicillin, levofloxacin, and macrolide-resistant S. pneumoniae. GSK1322322 was very potent against S. pyogenes strains, with an MIC(90) of 0.5 µg/ml, irrespective of their macrolide resistance phenotype. This PDF inhibitor was also active against S. aureus strains regardless of their susceptibility to methicillin, macrolides, or levofloxacin, with an MIC(90) of 4 µg/ml in all cases. Time-kill studies showed that GSK1322322 had bactericidal activity against S. pneumoniae, H. influenzae, S. pyogenes, and S. aureus, demonstrating a ≥ 3-log(10) decrease in the number of CFU/ml at 4× MIC within 24 h in 29 of the 33 strains tested. Given the antibacterial potency demonstrated against this panel of organisms, GSK1322322 represents a valuable alternative therapy for the treatment of infectious diseases caused by drug-resistant pathogens.

Characterization of Streptococcus pneumoniae TrmD, a tRNA methyltransferase essential for growth.

Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, tRNA(Leu)(CAG). Other heterologous nonsubstrate tRNA species, like, tRNA (Thr)(GGT), tRNA(Phe), and tRNA (Ala)(TGC), bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).

Lipopeptide substrates for SpsB, the Staphylococcus aureus type I signal peptidase: design, conformation and conversion to alpha-ketoamide inhibitors.

Pre-protein sequence data was used to design substrates for SpsB, the bacterial signal peptidase I enzyme from Staphylococcus aureus. Key elements were an alkyl membrane anchor, proline at P5 and lysine at P2. The proline at P5 induced a helical turn in the lipopeptide, as deduced from NMR studies, from P6 to P2 in membrane mimetic solvents. The substrate Decanoyl-LTPTAKAASKIDD-OH was cleaved by SpsB, as expected, between the P1 and P1' alanines with a k(cat)/K(m) of 2.3x10(6) M(-1)s(-1) at pH 8.5. Insertion of proline at P1' converted substrates to competitive inhibitors, whilst the incorporation of an alpha-ketoamide at the cleavage site transformed substrates to time dependent inhibitors of SpsB.

Characterization of a novel fucose-regulated promoter (PfcsK) suitable for gene essentiality and antibacterial mode-of-action studies in Streptococcus pneumoniae.

The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P(fcsK), fucose-dependent growth of the S. pneumoniae (P(fcsK)::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function.

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.