Comparative Analysis of the Antibacterial Activity of a Novel Peptide

Transcription

1 AAC Accepts, published online ahead of print on 11 March 2013 Antimicrob. Agents Chemother. doi: /aac Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 Comparative Analysis of the Antibacterial Activity of a Novel Peptide Deformylase Inhibitor, GSK Karen O Dwyer 1, Meredith Hackel 2, Sarah Hightower 1, Daryl Hoban 2, Samuel Bouchillon 2, Donghui Qin 1, Kelly Aubart 1, Magdalena Zalacain 1* and Deborah Butler 1 # 1 Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area, GlaxoSmithKline, Collegeville, PA, USA; 2 International Health Management Associates, Inc., Schaumburg, IL, USA Running Title: Antibacterial activity of GSK # corresponding author, address, Deborah.L.Butler@gsk.com *Present address: Zala Drug Discovery Consulting LLC, West Chester, PA 19380, USA 1

2 ABSTRACT GSK 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 community-acquired pneumonia. The activity of GSK was tested against a global collection of clinical isolates of Haemophilus influenzae (2370), Moraxella catarrhalis (115), Streptococcus pneumoniae (947), Streptococcus pyogenes (617), and Staphylococcus aureus (940), including strains resistant to one or more marketed antibiotics. GSK had an MIC 90 of 1 μg/ml against M. catarrhalis and 4 μg/ml against H. influenzae, with 88.8% of β-lactamase positive strains showing growth inhibition at that concentration. All S. pneumoniae strains were inhibited by <4 μg/ml of GSK , 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. GSK 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 MIC 90 values of 4 μg/ml in all cases. Time-kill studies showed that GSK had bactericidal activity against S. pneumoniae, H. influenzae, S. pyogenes, and S. aureus demonstrating a >3 log 10 decrease in CFU/mL at 4X MIC within 24 hours in 29 of the 33 strains tested. Given the antibacterial potency demonstrated against this panel of organisms, GSK represents a valuable alternative therapy for the treatment of infectious diseases caused by drug-resistant pathogens. 2

3 INTRODUCTION The spread of multidrug resistance among major causative agents of hospitalized community-acquired pneumonia (CAP) and skin and skin structure infections (SSSI), such as Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus pyogenes, has become a serious public health concern. S. pneumoniae resistance to β-lactams, macrolides, tetracyclines, and trimethoprim/sulfamethoxazole (TMP-SMX) was reported as a worldwide problem in the early 2000s (1) and in % of the ~ 40,000 cases of invasive infections reported in the USA were caused by a strain resistant to at least one drug and 11% by a multidrug-resistant S. pneumoniae (MDRSP) strain (2). In H. influenzae, resistance has developed not only to β-lactams (1) but also to tetracyclines, chloramphenicol, and TMP-SMX (3), and there are reservations about the clinical efficacy of macrolides (4). The level of macrolide-resistant S. pyogenes, the least common but deadliest of these pathogens, is variable with an overall rate of 6-7% in the USA (5, 6) and 3 to 31% in Europe (7). In the case of S. aureus, the number of invasive infections caused by methicillin-resistant S. aureus (MRSA) is high in a hospital setting (8) and is increasing in the community (9). The appearance of MRSA strains resistant to cephalosporins, tetracyclines, sulphur drugs, and quinolones has reduced the list of treatment options for this serious pathogen. Hospitalizations associated with drug-resistant infections have considerable implications for the healthcare system compared to susceptible infections, including increased risk of patient mortality and higher hospital costs and length of stay. In fact, based on a 2009 study, the medical costs attributable only to MRSA infections in the US are estimated to exceed $900 million a year (10). New treatment options, particularly those with oral and intravenous formulations appropriate for both adult and pediatric populations are critically needed. 3

4 Peptide deformylase (PDF), a metalloprotease that removes the N-formyl group present in all newly synthesized bacterial polypeptides (11-13), plays an essential role in protein maturation and is a highly conserved, broad spectrum, target (14-17). PDF inhibitors represent, therefore, a new type of antibacterial agent with a novel mode of action and provide an alternative for the treatment of hospitalized CAP and SSSIs caused by pathogens resistant to current therapies. The design of PDF inhibitors for potential clinical use has been the subject of research in a number of laboratories over the past decade, partly inspired by the discovery that actinonin, a naturally occurring antibacterial agent, is an inhibitor of PDF (18, 19). A large number of chemically diverse PDF inhibitors have been discovered through these efforts and compounds with good antibacterial activity and in vivo efficacy have been reported (20). BB (21) and LBM415 (22) also progressed to Phase I clinical trials although they were not further developed. GSK (Figure 1), a novel PDF inhibitor from the hydrazide class, has shown good safety and pharmacokinetic properties in a Phase I clinical trial and promising proof-of-concept results in a Phase IIa study ( GSK is currently being developed for the oral and intravenous treatment of acute bacterial SSSI and hospitalized CAP. In this report we summarize the spectrum of activity of GSK and selected comparator agents against a worldwide collection of H. influenzae, M. catarrhalis, S. pneumoniae, S. aureus and S. pyogenes strains. In addition, we analyze the cell-killing activity of GSK with respect to that of other commonly used antibiotics. 4

5 MATERIALS AND METHODS Bacterial Strains. Organisms (4989 strains) used in this study included 2370 H. influenzae (517 β-lactamase positive), 115 M. catarrhalis, 947 S. pneumoniae (230 penicillin-intermediate and 165 penicillin-resistant; 329 macrolide-resistant and 45 levofloxacin-resistant), 940 S. aureus (414 methicillin-resistant, 482 macrolide-resistant, and 308 levofloxacin-resistant) and 617 S. pyogenes (62 macrolide-resistant). All study organisms were clinical strains isolated in the years 2006 through 2008 and frozen at -70 C, with the exception of the H. influenzae strains which were collected from 2001 through H. influenzae, S. pneumoniae, and M. catarrhalis were from communityassociated respiratory tract infections (RTIs) from a multi-national population, one isolate per patient. S. aureus isolates were 66% community-associated and 34% hospital-associated, 238 strains from RTIs and 702 strains from SSSIs. S. pyogenes isolates were all community-associated, 65% from RTIs and 35% from SSSIs. Fifty four percent of the study isolates were obtained from sites in North America with an additional 32% from 23 European countries. The remaining 14% were divided between 24 other countries. Antimicrobial Agents and Susceptibility Testing. GSK , obtained from GlaxoSmithKline Pharmaceuticals (Collegeville, PA, USA) was dissolved in DMSO and then further diluted 1:10 in sterile water to make a working stock solution. Antibacterial agents used for comparative analysis included amoxicillin, azithromycin, ceftriaxone, clindamycin, levofloxacin, linezolid, penicillin, tigecycline, and vancomycin and were provided by their manufacturers in the USA or by Fluka (Fluka, Buchs, Switzerland) or Sigma (Sigma Pharmaceuticals, LLC. Monticello, IA). Antibiotics were solubilized in the appropriate solvents as recommended by their respective manufacturers. Each resulting solution was further diluted into the appropriate broth medium for the sequential dilutions used in the broth microdilution panels. 5

6 Minimum inhibitory concentration (MIC) endpoints were determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines (23). Cation-adjusted Mueller Hinton (CAMH) broth was used for S. aureus and M. catarrhalis, with 3% lysed horse blood added for S. pneumoniae and S. pyogenes. Haemophilus Test Medium (HTM) broth was used for H. influenzae. Testing was performed using panels freshly prepared on the same day of testing. Beta-lactamase testing was performed on all H. influenzae and S. aureus using the cefinase disk methodology (Nitrocef Disks, Hardy Diagnostics). Methicillin phenotypes were determined for all S. aureus isolates using cefoxitin disks (30 μg). Colonies were taken directly from a second-pass culture plate and prepared to a suspension equivalent to the 0.5 McFarland standard using normal saline. Inoculation of MIC plates took place within 15 minutes after adjustment of the inoculum suspension turbidity. Trays were incubated at 35 C for 20 to 24 hours except for S. aureus and M. catarrhalis which were incubated for 16 to 20 hours before reading the MIC endpoints. Quality control (QC) testing was performed each day of testing as specified by CLSI using the following isolates: S. pneumoniae ATCC 49619, S. aureus ATCC 29213, and H. influenzae ATCC and MIC50, MIC90 and MIC ranges were determined for all antimicrobial agents tested. Resistant phenotypes were defined by susceptibility to the corresponding antimicrobial agent according to CLSI interpretive criteria (24). Methicillin phenotypes were defined based upon the susceptibility results of the cefoxitin disk test. Macrolide phenotypes were based on azithromycin susceptibility. Time-kill Studies. Time-kill studies were conducted to assess the inhibitory activity of GSK compared to linezolid, moxifloxacin, or azithromycin, depending on the organism. Seven strains of S. pneumoniae, eight strains of H. influenzae, and nine strains of S. aureus and S. pyogenes were used in this study. All isolates were obtained from the GSK Anti-Infectives culture 6

7 collection. S. pneumoniae and S. aureus strains were cultured on Trypticase Soy Agar (TSA) with 5% sheep blood or in CAMH broth. S. pneumoniae strains were grown in Todd Hewitt with Yeast Extract broth for inoculum preparation. H. influenzae strains were cultured on Chocolate Agar II plates or in HTM broth. S. pyogenes strains were grown on TSA with 5% sheep blood or MH with 3% lysed horse blood broth. Compounds were diluted into 20 ml broth media in a 50 ml Erlenmeyer flask to give final concentrations of 4X and 10X MIC for each organism/compound combination. For each isolate, a control flask containing no compound was also prepared. Cells from a second-pass plate were used to inoculate broth and grown to mid-logarithmic phase. The appropriate amount of inoculum was added to the pre-warmed 20 ml broth/compound solutions and the drug-free controls to achieve a final concentration of ~ 5x10 5 CFU/mL. In order to minimize the occurrence of resistance to GSK in S. aureus by inactivation of formyl-methionyl transferase (FMT) (16) that could potentially affect interpretation of results, the inoculum in this case was reduced to ~ 5x10 4 CFU/mL and recovered organisms were assessed for FMT mutant characteristics (T. Lewandowski, J. Huang, F. Fan, S, Rogers, D. gentry, R. Holland, P. DeMarsh, K. Aubart, and M. Zalacain, submitted for publication). Flasks were placed on a shaker at 35 C and viable counts were performed at 0, 2, 4, 6, 8, 10, 12, and 24 hours by plating three 20 µl aliquots of 10-fold serial dilutions of 100 µl from each flask onto agar plates. The plates were incubated overnight at 35 C and counts were done at the dilution that provided distinguishable colonies. An average of the three aliquots was used to estimate the number of colony forming units (CFU) in the original sample. In order to minimize the possibility of an inhibitory effect of the compound during growth on the agar plate, one 20 µl aliquot was taken from the flask, diluted 5 fold into appropriate broth and spread onto a plate, starting at time 4 hour (8 7

8 hours for S. aureus). For S. aureus only, at 10 hours onward, this was done three times for each sample to lower the limit of detection from 50 CFU/mL to 16 CFU/mL. The level of bacterial killing by the different compounds was determined by the log 10 decrease in CFU/mL achieved after a specific incubation time with respect to the original inoculum. Strains were grouped according to their Δlog 10 values. Group (-1) contained strains that showed >1 and <2 log 10 decrease in CFU/mL (>90% but <99% killing). Group (-2) contained strains that showed >2 and <3 log 10 decrease in CFU/mL (>99% but <99.9% killing). Group (-3) contained strains that showed >3 log 10 decrease in CFU/mL (>99.9% killing). A compound was considered bactericidal at a specific time and multiple of the MIC if there was a >3 log 10 decrease in CFU/mL with respect to the original inoculum (25). A reduction of <3 log 10 CFU/mL was considered a bacteriostatic effect. In those studies where counts were reduced to the limit of detection and represented a >2.5 log 10 decrease in CFU/mL, the effect was considered to be bactericidal. Downloaded from on November 12, 2018 by guest 8

9 RESULTS Overall antimicrobial activity of GSK The novel PDF inhibitor GSK showed good antibacterial activity against common RTI and SSSI pathogens (Table 1). GSK had an MIC 90 of 1 μg/ml against M. catarrhalis and of 4 μg/ml against H. influenzae with 88.8% of β-lactamase positive strains showing growth inhibition at that concentration. Growth of 88% of the S. pneumoniae strains tested was inhibited by 1 μg/ml of GSK and 98.7% of them could not grow at 2 μg/ml. In fact, 1 μg/ml prevented growth of 97%, 93% and 91% of penicillin, levofloxacin, and macrolide-resistant S. pneumoniae, respectively. Over 80% of the S. aureus strains analyzed were inhibited by GSK at 2 μg/ml and over 95% by 4 μg/ml, regardless of their resistance phenotype to other marketed antibiotics. This molecule also showed potent activity against S. pyogenes with growth of 48.8% of the strains inhibited by 0.25 μg/ml and of nearly 100% of the strains by 1 μg/ml. GSK showed similar potency against isolates of S. aureus or S. pyogenes irrespective of their RTI or SSSI origin. Antibacterial activity of GSK against Gram negative pathogens. The activity of GSK was tested against 2370 H. influenzae of which 20% were resistant to ampicillin (Table 2). Beta-lactamase negative strains had GSK MIC 50 and MIC 90 values of 2 and 4 μg/ml respectively, whereas for β-lactamase positive strains those values were 2 and 8 μg/ml respectively (Table 2). Over 99% of the strains were susceptible to azithromycin and levofloxacin with MIC 90 values of 2 and 0.03 μg/ml, respectively, against this panel of organisms. The GSK MIC 90 for M. catarrhalis (115 isolates) was 1 μg/ml compared to 0.06 μg/ml for azithromycin and levofloxacin and 2 μg/ml for clindamycin (Table 2). 9

10 Antibacterial activity of GSK against Gram positive pathogens. The activity of GSK and of a number of marketed antibiotics was tested against S. pneumoniae (947 strains), S. pyogenes (617 strains) and S. aureus (940 strains) (Table 3). All GSK MICs were <4 μg/ml for S. pneumoniae with MIC 50 and MIC 90 values of 1 and 2 μg/ml, respectively. Pre-existing resistance mechanisms did not affect the potency of this compound, as evidenced by an MIC 90 for penicillin-, levofloxacin-, and macrolide-resistant S. pneumoniae of 1 μg/ml. The activity of comparator antibiotics varied and while >95% of the strains were still susceptible to ceftriaxone and levofloxacin, 17% of the strains were resistant to penicillin, 19% to clindamycin, and nearly 35% to azithromycin. This PDF inhibitor was also consistently active against all S. aureus strains regardless of their susceptibility to methicillin, macrolide, or levofloxacin with MIC 50 and MIC 90 values of 2 and 4 μg/ml, respectively, in all cases. Linezolid and vancomycin, with MIC 90 of 4 and 1 μg/ml respectively, were the only two comparator agents fully effective against this panel of organisms. Resistance to all other antibiotics tested was high and translated into MIC 90 >4 μg/ml for clindamycin, >8 μg/ml for azithromycin and levofloxacin, >16 μg/ml for ampicillin, and >64 μg/ml for ceftriaxone. GSK was very potent against S. pyogenes strains with both MIC 50 and MIC 90 values of 0.5 μg/ml. No difference in activity was observed between macrolide-susceptible and -resistant strains or with the source of isolation. Comparator antibiotics were effective against these S. pyogenes strains with susceptibilities ranging from 90% for azithromycin to nearly 100% for levofloxacin. 10

11 Time-kill analysis of GSK A summary of the cell-killing activity of GSK and comparative agents in different H. influenzae, S. pneumoniae, S. aureus, and S. pyogenes strains is presented in Table 4. GSK showed bactericidal activity by 24 hours at both 4X and 10X MIC against all eight H. influenzae strains tested. Azithromycin also showed bactericidal activity in all strains at 10X MIC and in six of eight strains at 4X MIC. Regrowth was observed with this antibiotic after 24 hours in seven of eight strains treated with 4X MIC and in two of eight strains treated with 10X MIC but it was not due to development of resistance. Moxifloxacin was bactericidal at both concentrations in all strains by 24 hours. In fact, this antibiotic reduced growth by more than 3 log 10 by 8 hours in five of eight strains at both 4X and 10X MIC. Bactericidal activity was observed with GSK at 4X MIC within 24 hours for six of the seven S. pneumoniae strains tested as well as a significant cell-killing activity against the last strain (2.66 log 10 decrease in cell numbers with respect to baseline). At 10X MIC, GSK was bactericidal for all strains tested. Linezolid was bactericidal for all strains at both concentrations within 24 hours and moxifloxacin showed a very rapid cell-killing effect with bactericidal activity in all cases within 8 hours at 4X MIC and within 4 hours at 10X MIC. Time-kill analysis of GSK against nine strains of S. aureus showed that this PDF inhibitor was bactericidal within 24 hours for seven of the nine organisms at 4X and 10X MIC. In general, cell-killing activity was slow with counts decreasing over time and a full bactericidal effect seen at 12 or 24 hours. In contrast, linezolid was clearly bacteriostatic against all the strains in this study with eight of nine strains showing <2 log 10 decreases in cell counts at 24 hours at both 4X and 10X MIC, and eight or six of the nine strains displaying <1 log 10 reduction in bacterial counts at 12 hours at 4X or 10X MIC, respectively. Moxifloxacin at 4X MIC was rapidly bactericidal, with cell counts reducing to the limit of detection within 4 hours against the four strains tested. 11

12 GSK was bactericidal at 4X MIC by 24 hours versus eight of the nine S. pyogenes strains tested. Linezolid showed bactericidal activity against five of nine strains at 4X MIC and six of nine strains at 10X MIC within 24 hours and moxifloxacin was bactericidal within 24 hours against all strains tested and within 8 hours against seven of nine and eight of nine at 4X and 10X MIC, respectively. Downloaded from on November 12, 2018 by guest 12

13 DISCUSSION With the appearance of resistance to most marketed antibiotics in all bacterial pathogens causing serious human diseases, the need for new therapies has become a pressing reality. The development of new agents against previously unexploited targets provides the additional advantage of avoiding potential cross-resistance with antibiotics already in use. One such target is peptide deformylase. PDF is ubiquitous in bacteria where it plays an essential role in protein maturation, as removal of the N-formyl group is an indispensable step prior to the cleavage of the methionine residue by methionine aminopeptidase in the majority of newly synthesized proteins (26, 27). Deformylation is not necessary in eukaryotic cytoplasmic protein synthesis and, although a mitochondrial PDF has been identified (28), it is much less active than its bacterial counterpart even when N-formylated peptides corresponding to the N-terminal sequence of human mitochondrial proteins are used as substrate (29). Thus, inhibitors of PDF can be broad spectrum, selective antibacterial agents not affected by pre-existing resistance mechanisms. The fact that PDF is amenable to X-ray crystallography (30) has facilitated the combination of classical screening methods with a more rational structure-based design approach in the discovery of novel PDF inhibitors. As a result, a considerable number of chemically diverse series of potent PDF inhibitors has been reported over the years, containing both peptidic and non-peptidic scaffolds, with variable antibacterial activity and in vivo efficacy in animal models of infection (20). Moreover, two pseudopeptidic PDF inhibitors, BB (21) and LBM415 (22) showed pharmacokinetic and toxicology properties that supported their progression to Phase I clinical trials. GSK , a non-peptidic PDF inhibitor from the hydrazide family and structurally distinct from the previous molecules, has successfully demonstrated safety and efficacy in human proof-ofconcept clinical studies ( GSK is being progressed as an oral and intravenous agent for the treatment of hospitalized CAP and acute bacterial SSSI and is 13

14 currently in Phase IIb clinical trials. The study reported here, assessing the in vitro activity of GSK against a large international collection of clinical isolates, demonstrates that the compound has potent antibacterial activity against key respiratory and skin pathogens including those resistant to penicillin, methicillin, azithromycin, and levofloxacin. GSK shows an MIC 90 of 2 μg/ml for S. pneumoniae, 4 μg/ml for H. influenzae and S. aureus, 1 μg/ml for M. catarrhalis, and 0.5 μg/ml for S. pyogenes and demonstrates excellent activity against resistant organisms. In fact, the GSK MIC 90 for 165 penicillin-resistant, 329 macrolide-resistant and 45 levofloxacin-resistant S. pneumoniae strain sets is 1 μg/ml, including some strains which are resistant to more than one comparator antibiotic. For example, 7.9%, 11.5%, 58.2% and 84.2% of the penicillin-resistant S. pneumoniae strains are also resistant to ceftriaxone, levofloxacin, clindamycin, or azithromycin, respectively. Equally, a percentage of the 329 macrolide-resistant strains are non-susceptible to ceftriaxone (4.3%), levofloxacin (8.5%) or penicillin (42.2%) and 7.9% of them are resistant to azithromycin, penicillin, and ceftriaxone or levofloxacin. GSK s MIC 90 is 4 μg/ml for 414 methicillin-resistant, 308 levofloxacin-resistant and 482 macrolide-resistant S. aureus. Most MRSA strains are also resistant to azithromycin (88.2%), levofloxacin (65.7%), or ceftriaxone (28.5%). In fact, 52.7% of the MRSA isolates are non-susceptible to two of these antibiotics and 38.9% are resistant to all. Nearly 100% of the strains are susceptible to linezolid, which has an MIC 90 of 4 μg/ml, and vancomycin is effective in all cases with an MIC 90 of 1 μg/ml. Curiously, perhaps as a reflection of the antibiotic therapy used to treat both infections, resistance to ceftriaxone, clindamycin and levofloxacin seems to be more prevalent within the set of RTI strains than the SSSI, while the level of ampicillin and azithromycin resistance is similar amongst both the RTI and SSSI isolates. The activity of GSK against S. pyogenes, including the 62 macrolide-resistant strains 14

15 tested, is similar to that of levofloxacin with MIC 90 s of 0.5 μg/ml and 1 μg/ml, respectively. The resistance phenotype of the S. pyogenes strains tested is not affected by the source of the isolates. Growth of 94.2% of β-lactamase negative and 88.8% of β-lactamase producing H. influenzae strains is suppressed with 4 μg/ml of GSK Of all organisms tested, the weakest activity and the widest range of MICs is observed against H. influenzae probably due to the presence of efflux pumps that have been shown to affect susceptibility to PDF inhibitors (22). Although the efficacy of this compound against H. influenzae strains with MIC of 4-8 μg/ml has been demonstrated in RTI animal models (T. Lewandoswki, unpublished data), pharmacokinetic/pharmacodynamic studies will be necessary to provide additional information on the potential effectiveness of GSK in the clinic. Time-kill studies show that GSK is slowly bactericidal against S. pneumoniae, H. influenzae, S. pyogenes, and S. aureus demonstrating a >3 log 10 decrease in CFU/mL at 4X MIC within 24 hours in 29 of the 33 strains tested. The activity is comparable to that of linezolid in S. pneumoniae where moxifloxacin shows a very fast killing effect. In H. influenzae the cell-killing activity is similar to that of azithromycin and only slightly slower than moxifloxacin. GSK shows more cell-killing activity than linezolid against S. pyogenes and S. aureus, as linezolid is bacteriostatic against four of the nine S. pyogenes strains and all of the S. aureus strains tested. Moxifloxacin, on the other hand, shows a much faster killing effect than GSK against both organisms. These results are similar to those observed with PDF inhibitor LBM415 in H. influenzae (31) and S. pneumoniae (32). However, GSK seems to demonstrate more of a bactericidal effect against S. aureus than previously reported for LBM415 (22, 33) and is clearly differentiated from linezolid. In summary, GSK has the spectrum of activity and in vitro potency necessary for therapeutic use in respiratory tract and skin and soft tissue infections. Furthermore, its unique mode 15

16 of action provides a clear advantage in the treatment of multi-drug resistant pathogens and ensures lack of cross-resistance with any other marketed antibiotics. This PDF inhibitor has completed early clinical trials and has shown pharmacokinetic and safety parameters warranting progression to Phase IIb studies ( If clinically efficacious, GSK will provide a much-needed alternative therapy for the treatment of hospitalized patients with multi-drug resistant infections caused by the bacterial pathogens described in this report. Downloaded from on November 12, 2018 by guest 16

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22 FIGURE LEGENDS Fig. 1. Chemical structure of GSK

23 TABLE 1. Summary of antimicrobial activity at individual MIC values of GSK Organism Name (N) Cumulative percentage of strains inhibited by GSK at MIC (μg/ml) H. influenzae (2370) β-lactamase positive (517) M. catarrhalis (115) S. pneumoniae (947) Penicillin-resistant (165) Levofloxacin-resistant (45) Macrolide-resistant (329) S. aureus (940) MRSA (414) Levofloxacin-resistant (308) Macrolide-resistant (482) RTI (238) SSSI (702) S. pyogenes (617) Macrolide-resistant (62) RTI (218) SSSI (399) MIC 50 values highlighted in light grey MIC 90 values highlighted in dark grey 23

24 TABLE 2. Antibacterial activity of GSK and selected comparators against a global collection of H. influenzae and M. catarrhalis strains MIC (μg/ml) % Category a Organism (N) Drug 50% 90% Range Susceptible Resistant H. influenzae (2370) GSK b Ampicillin > Azithromycin > Levofloxacin β-lactamase negative (1853) GSK Ampicillin > Azithromycin > Levofloxacin β-lactamase positive (517) GSK Ampicillin > c Azithromycin > Levofloxacin M. catarrhalis (115) GSK Azithromycin Clindamycin Levofloxacin a Susceptibility criteria of CLSI. For interpretation of M. catarrhalis results, breakpoints for H. influenzae have been applied b No criteria have been established c Based on β-lactamase test 24

25 TABLE 3. Antimicrobial activity of GSK and selected comparators against Gram positive organisms MIC (μg/ml) % Category a Organism (N) Drug 50% 90% Range Susceptible Resistance S. pneumoniae (947) GSK b Azithromycin 0.12 > > Ceftriaxone > Clindamycin 0.06 > > Levofloxacin > Penicillin > Penicillin-susceptible (552) GSK Azithromycin > Ceftriaxone Clindamycin > Levofloxacin > Penicillin Penicillin-intermediate (230) GSK Azithromycin 4 > > Ceftriaxone Clindamycin 0.06 > > Levofloxacin > Penicillin Penicillin-resistant (165) GSK Azithromycin >8 > > Ceftriaxone > Clindamycin >4 > > Levofloxacin > Penicillin > Levofloxacin-susceptible (900) GSK Azithromycin 0.12 > > Ceftriaxone > Clindamycin 0.06 > > Levofloxacin Penicillin > Levofloxacin-resistant (45) GSK Azithromycin 8 > > Ceftriaxone Clindamycin 0.06 > > Levofloxacin >8 >8 8-> Penicillin Macrolide-susceptible (609) GSK

26 Azithromycin Ceftriaxone Clindamycin Levofloxacin > Penicillin > Macrolide-resistant (329) GSK Azithromycin >8 >8 2-> Ceftriaxone > Clindamycin >4 > > Levofloxacin > Penicillin > MDR c (153) GSK S. aureus (940) GSK Ampicillin 16 > > Azithromycin >8 >8 0.5-> Ceftriaxone 4 > > Clindamycin 0.12 > > Levofloxacin 0.25 > > Linezolid > Tigecycline Vancomycin MSSA (526) GSK Ampicillin 4 > > Azithromycin 1 >8 0.5-> Ceftriaxone > Clindamycin > Levofloxacin > Linezolid > Tigecycline Vancomycin MRSA (414) GSK Ampicillin >16 > >16 0 d 100 d Azithromycin >8 >8 0.5-> Ceftriaxone 16 > >64 0 d 100 d Clindamycin 0.12 > > Levofloxacin 8 > > Linezolid Tigecycline Vancomycin Levofloxacin-susceptible (628) GSK Ampicillin 8 > > Azithromycin 2 >8 0.5-> Ceftriaxone >

27 Clindamycin > Levofloxacin Linezolid > Tigecycline Vancomycin Levofloxacin-resistant (308) GSK Ampicillin 16 > > Azithromycin >8 >8 0.5-> Ceftriaxone 16 > > Clindamycin 0.25 > > Levofloxacin >8 >8 4-> Linezolid Tigecycline Vancomycin Macrolide-susceptible (456) GSK Ampicillin 4 > > Azithromycin Ceftriaxone > Clindamycin Levofloxacin > Linezolid > Tigecycline Vancomycin Macrolide-resistant (482) GSK Ampicillin >16 > > Azithromycin >8 >8 8-> Ceftriaxone 16 > > Clindamycin 0.12 > > Levofloxacin 8 > > Linezolid Tigecycline Vancomycin RTI (238) GSK Ampicillin 16 > > Azithromycin 2 >8 0.5-> Ceftriaxone 4 > > Clindamycin 0.12 > > Levofloxacin 0.5 > > Linezolid > Tigecycline Vancomycin SSSI (702) GSK Ampicillin 16 > >

28 Azithromycin >8 >8 0.5-> Ceftriaxone > Clindamycin 0.12 > > Levofloxacin 0.25 > > Linezolid Tigecycline Vancomycin S. pyogenes (617) GSK Azithromycin 0.12 > > Clindamycin > Levofloxacin > Macrolide-susceptible (555) GSK Azithromycin Clindamycin Levofloxacin > Macrolide-resistant (62) GSK Azithromycin >8 >8 >8-> Clindamycin 0.06 > > Levofloxacin RTI (218) GSK Azithromycin 0.12 > > Clindamycin > Levofloxacin SSSI (399) GSK Azithromycin > Ceftriaxone Clindamycin > Levofloxacin > Linezolid Penicillin a Susceptibility criteria of CLSI b No criteria has been established c Strains resistant to two or more antibiotic classes d Based upon oxacillin susceptibility results (27) 28

29 TABLE 4. Cell-killing activity of GSK and comparator antibiotics at different multiples of MIC against H. influenzae, S. pneumoniae, S. aureus and S. pyogenes strains* N o of strains with the indicated level of killing a at different time Drug and multiple of MIC H. influenzae (8) 4 hr 8 hr 12 hr 24 hr GSK (MICs, μg/ml) 4XMIC XMIC Azithromycin (MICs, μg/ml) 4XMIC b 6 b 6 b 10XMIC Moxifloxacin (MICs, μg/ml) 4XMIC XMIC S. pneumoniae (7) 4 hr 8 hr 12 hr 24 hr GSK (MICs, μg/ml) 4XMIC XMIC Linezolid (MICs, μg/ml) 4XMIC XMIC Moxifloxacin (MICs, μg/ml) 4XMIC XMIC S. aureus (9) 4 hr 8 hr 12 hr 24 hr GSK (MICs, μg/ml) 4XMIC c 3 c 1 c 8 c 7 c 7 c 10XMIC Linezolid (MICs, 2-4 μg/ml) 4XMIC XMIC Moxifloxacin d (MICs, μg/ml) 4XMIC S. pyogenes (9) 4 hr 8 hr 12 hr 24 hr GSK (MICs, μg/ml) 4XMIC XMIC Linezolid (MICs, 1-4 μg/ml) 4XMIC XMIC Moxifloxacin (MICs, μg/ml) 4XMIC XMIC

30 Grey shade denotes a bactericidal effect of GSK against a number of strains at the time specified *Mean starting inocula s value used with the different bacterial species in this study were: H. influenzae, 1.2x10 6 ; S. pneumoniae, 9x10 5 ; S. aureus, 4.4x10 4 ; S. pyogenes, 1.4x10 5 a (-1), >90% but <99% killing; (-2), >99% but <99.9% killing; (-3), >99.9% killing b Regrowth, not due to resistance, observed in 2 strains c Regrowth of S. aureus FMT mutant observed in one strain d Only four strains were used in this study Downloaded from on November 12, 2018 by guest 30

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