Publications by authors named "Magdalena Zalacain"

28 Publications

  • Page 1 of 1

Novel Specific Metallo-ß-lactamase Inhibitor, ANT2681, Restores Meropenem Activity to Clinically Effective Levels Against NDM-Positive Enterobacterales.

Antimicrob Agents Chemother 2021 Apr 5. Epub 2021 Apr 5.

Antabio SAS, Labège, France.

The global dissemination of metallo-ß-lactamase (MBL)-producing carbapenem resistant Enterobacterales (CRE) is a serious public health concern. Specifically, NDM (New Delhi MBL) has been a major cause of carbapenem therapy failures in recent years, particularly as effective treatments for serine-ß-lactamase (SBL)-producing Enterobacterales are now commercially available. Since the NDM gene is carried on promiscuous plasmids encoding multiple additional resistance determinants, a large proportion of NDM-CREs are also resistant to many commonly used antibiotics, resulting in limited and sub-optimal treatment options. ANT2681 is a specific, competitive inhibitor of MBLs with potent activity against NDM enzymes, progressing to clinical development in combination with meropenem (MEM). Susceptibility studies have been performed with MEM-ANT2681 against 1,687 MBL-positive Enterobacterales, including 1,108 NDM-CRE. Addition of ANT2681 at 8 μg/ml reduced MEM MIC/MIC from >32/>32 μg/ml to 0.25/8 μg/ml. Moreover, the combination of 8 μg/ml of both MEM and ANT2681 inhibited 74.9% of the VIM-positive and 85.7% of the IMP-positive Enterobacterales tested. The antibacterial activity of MEM-ANT2681 against NDM-CRE compared very favourably to that of cefiderocol (FDC) and cefepime (FEP)-taniborbactam, which displayed MIC values of 8 μg/ml and 32 μg/ml, respectively, whereas aztreonam-avibactam (ATM-AVI) had an MIC of 0.5 μg/ml. Particularly striking was the activity of MEM-ANT2681 against NDM-positive (MIC 1 μg/ml), in contrast to ATM-AVI (MIC 4 μg/ml), FDC (MIC >32 μg/ml) and FEP-taniborbactam (MIC >32 μg/ml) which were less effective due to the high incidence of resistant PBP3-insertion mutants. MEM-ANT2681 offers a potential new therapeutic option to treat serious infections caused by NDM-CRE.
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http://dx.doi.org/10.1128/AAC.00203-21DOI Listing
April 2021

Elastase Contributes to the Establishment of Chronic Lung Colonization and Modulates the Immune Response in a Murine Model.

Front Microbiol 2020 12;11:620819. Epub 2021 Jan 12.

Antabio SAS, Labège, France.

Chronic infection by in cystic fibrosis (CF) patients is a major contributor to progressive lung damage and is poorly treated by available antibiotic therapy. An alternative approach to the development of additional antibiotic treatments is to identify complementary therapies which target bacterial virulence factors necessary for the establishment and/or maintenance of the chronic infection. The elastase (LasB) has been suggested as an attractive anti-virulence target due to its extracellular location, its harmful degradative effects on host tissues and the immune system, and the potential to inhibit its activity using small molecule inhibitors. However, while the relevance of LasB in acute infection has been demonstrated, it is still unclear whether this elastase might also play a role in the early phase of chronic lung colonization. By analyzing clinical clonal isolates from a CF patient, we found that the isolate RP45, collected in the early phase of persistence, produces large amounts of active LasB, while its clonal variant RP73, collected after years of colonization, does not produce it. When a mouse model of persistent pneumonia was used, deletion of the gene in RP45 resulted in a significant reduction in mean bacterial numbers and incidence of chronic lung colonization at Day 7 post-challenge compared to those mice infected with wild-type (wt) RP45. Furthermore, deletion of in strain RP45 also resulted in an increase in immunomodulators associated with innate and adaptive immune responses in infected animals. In contrast, deletion of the gene in RP73 did not affect the establishment of chronic infection. Overall, these results indicate that LasB contributes to the adaptation of to a persistent lifestyle. In addition, these findings support pharmacological inhibition of LasB as a potentially useful therapeutic intervention for -infected CF patients prior to the establishment of a chronic infection.
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http://dx.doi.org/10.3389/fmicb.2020.620819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836092PMC
January 2021

Discovery of , a Novel Broad-Spectrum Serine β-Lactamase Inhibitor of the Diazabicyclooctane Class, Which Strongly Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacterales and .

J Med Chem 2020 12 11;63(24):15802-15820. Epub 2020 Dec 11.

Antabio SAS, 436 rue Pierre et Marie Curie, 31670 Labège, France.

The diazabicyclooctanes (DBOs) are a class of serine β-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of β-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of β-lactam antibiotics against carbapenem-resistant (CRAB), a WHO "critical priority pathogen" producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, , which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01535DOI Listing
December 2020

Reply to Asempa et al., "The Ongoing Challenge with NDM-Harboring in Murine Infection Models".

Antimicrob Agents Chemother 2021 01 20;65(2). Epub 2021 Jan 20.

Antimicrobial Pharmacodynamics and Therapeutics, University of Liverpool, Liverpool Health Partners, Liverpool, United Kingdom

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http://dx.doi.org/10.1128/AAC.02249-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848994PMC
January 2021

ANT2681: SAR Studies Leading to the Identification of a Metallo-β-lactamase Inhibitor with Potential for Clinical Use in Combination with Meropenem for the Treatment of Infections Caused by NDM-Producing .

ACS Infect Dis 2020 09 20;6(9):2419-2430. Epub 2020 Aug 20.

Antabio SAS, 436 rue Pierre et Marie Curied, 31670 Labège, France.

The clinical effectiveness of the important β-lactam class of antibiotics is under threat by the emergence of resistance, mostly due to the production of acquired serine- (SBL) and metallo-β-lactamase (MBL) enzymes. To address this resistance issue, multiple β-lactam/β-lactamase inhibitor combinations have been successfully introduced into the clinic over the past several decades. However, all of those combinations contain SBL inhibitors and, as yet, there are no MBL inhibitors in clinical use. Consequently, there exists an unaddressed yet growing healthcare problem due to the rise in recent years of highly resistant strains which produce New Delhi metallo (NDM)-type metallo-carbapenemases. Previously, we reported the characterization of an advanced MBL inhibitor lead compound, ANT431. Herein, we discuss the completion of a lead optimization campaign culminating in the discovery of the preclinical candidate ANT2681, a potent NDM inhibitor with strong potential for clinical development.
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http://dx.doi.org/10.1021/acsinfecdis.0c00207DOI Listing
September 2020

Pharmacodynamics of the Novel Metallo-β-Lactamase Inhibitor ANT2681 in Combination with Meropenem for the Treatment of Infections Caused by NDM-Producing .

Antimicrob Agents Chemother 2020 10 20;64(11). Epub 2020 Oct 20.

Antimicrobial Pharmacodynamics and Therapeutics, University of Liverpool, Liverpool Health Partners, Liverpool, United Kingdom

that produce metallo-β-lactamases (MBLs) are an emerging threat to public health. The metallo-β-lactamase inhibitor (MBLi) ANT2681 inhibits the enzymatic activity of MBLs through interaction with the dinuclear zinc ion cluster present in the active site that is common to these enzymes. ANT2681 is being codeveloped, with meropenem as the partner β-lactam, as a novel combination therapy for infections caused by MBL-producing bacteria. The pharmacokinetics/pharmacodynamics of meropenem-ANT2681 were studied in a murine neutropenic thigh model of NDM-producing Dose-ranging studies were performed with both meropenem and ANT2681. Dose fractionation experiments were performed to identify the relevant pharmacodynamic index of ANT2681 when coadministered with meropenem. A background of meropenem at 50 mg/kg of body weight every 4 h (q4h) subcutaneously (s.c.) had minimal antibacterial effect. On this background, half-maximal effect was observed with an ANT2681 dose of 89 mg/kg q4h intravenously (i.v.). The dose fractionation study showed that area under the concentration-time curve (AUC) was the relevant pharmacodynamic index for the inhibitor. The magnitude of the meropenem-ANT2681 exposure required to achieve stasis was explored using 5 NDM-producing strains. A 3-dimensional surface fitted to the pharmacodynamic data from the 5 strains suggested that stasis was achieved with an T > potentiated meropenem MIC of 40% and ANT2681 AUC of 700 mg · h/liter. These data and analyses provide the underpinning evidence for the combined use of meropenem and ANT2681 for clinical infections.
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http://dx.doi.org/10.1128/AAC.01076-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577127PMC
October 2020

SAR Studies Leading to the Identification of a Novel Series of Metallo-β-lactamase Inhibitors for the Treatment of Carbapenem-Resistant Enterobacteriaceae Infections That Display Efficacy in an Animal Infection Model.

ACS Infect Dis 2019 01 30;5(1):131-140. Epub 2018 Nov 30.

Antabio SAS , 436 rue Pierre et Marie Curie , 31670 Labège , France.

The clinical effectiveness of carbapenem antibiotics such as meropenem is becoming increasingly compromised by the spread of both metallo-β-lactamase (MBL) and serine-β-lactamase (SBL) enzymes on mobile genetic elements, stimulating research to find new β-lactamase inhibitors to be used in conjunction with carbapenems and other β-lactam antibiotics. Herein, we describe our initial exploration of a novel chemical series of metallo-β-lactamase inhibitors, from concept to efficacy, in a survival model using an advanced tool compound (ANT431) in conjunction with meropenem.
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http://dx.doi.org/10.1021/acsinfecdis.8b00246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332448PMC
January 2019

Discovery of a Novel Metallo-β-Lactamase Inhibitor That Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacteriaceae.

Antimicrob Agents Chemother 2018 05 26;62(5). Epub 2018 Apr 26.

Antabio SAS, Labège, France.

Infections caused by carbapenem-resistant (CRE) are increasingly prevalent and have become a major worldwide threat to human health. Carbapenem resistance is driven primarily by the acquisition of β-lactamase enzymes, which are able to degrade carbapenem antibiotics (hence termed carbapenemases) and result in high levels of resistance and treatment failure. Clinically relevant carbapenemases include both serine β-lactamases (SBLs; e.g., KPC-2 and OXA-48) and metallo-β-lactamases (MBLs), such as NDM-1. MBL-producing strains are endemic within the community in many Asian countries, have successfully spread worldwide, and account for many significant CRE outbreaks. Recently approved combinations of β-lactam antibiotics with β-lactamase inhibitors are active only against SBL-producing pathogens. Therefore, new drugs that specifically target MBLs and which restore carbapenem efficacy against MBL-producing CRE pathogens are urgently needed. Here we report the discovery of a novel MBL inhibitor, ANT431, that can potentiate the activity of meropenem (MEM) against a broad range of MBL-producing CRE and restore its efficacy against an NDM-1-producing strain in a murine thigh infection model. This is a strong starting point for a chemistry lead optimization program that could deliver a first-in-class MBL inhibitor-carbapenem combination. This would complement the existing weaponry against CRE and address an important and growing unmet medical need.
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http://dx.doi.org/10.1128/AAC.00074-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923143PMC
May 2018

Reducing Antibacterial Development Risk for GSK1322322 by Exploring Potential Human Dose Regimens in Nonclinical Efficacy Studies Using Immunocompetent Rats.

Antimicrob Agents Chemother 2017 11 24;61(11). Epub 2017 Oct 24.

GlaxoSmithKline, Collegeville, Pennsylvania, USA.

Directly testing proposed clinical dosing regimens in nonclinical studies can reduce the risk during the development of novel antibacterial agents. Optimal dosing regimens can be identified in animal models by testing recreated human pharmacokinetic profiles. An example of this approach using continuous intravenous infusions of GSK1322322 in immunocompetent rats to evaluate recreated human exposures from phase I trials in pneumonia models with and and an abscess model with is presented. GSK1322322 was administered via continuous intravenous infusion to recreate 1,000- or 1,500-mg oral doses every 12 h in humans. Significant reductions ( ≤ 0.05 for all comparisons) in bacterial numbers compared with those for the baseline controls were observed for and (mean log reductions, 1.6 to ≥2.7 and 1.8 to 3.3 CFU/lungs, respectively) with the recreated 1,000-mg oral dose. This profile was also efficacious against (mean log reduction, 1.9 to 2.4 CFU/abscess). There was a nonsignificant trend for improved efficacy against with the 1,500-mg oral dose (mean log reduction, 2.4 to 3.1 CFU/abscess). These results demonstrate that the human oral 1,000- or 1,500-mg exposure profiles of GSK1322322 recreated in rats were effective against representative community-associated pathogens and supported selection of the 1,500-mg oral dose given every 12 h for a phase II clinical skin infection study. Furthermore, this work exemplifies how the testing of recreated human pharmacokinetic profiles can be incorporated into the development process and serve as an aid for selecting optimal dosing regimens prior to conducting large-scale clinical studies.
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http://dx.doi.org/10.1128/AAC.00959-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655044PMC
November 2017

Pathogen Prevalence and Antimicrobial Susceptibility Among Enterobacteriaceae Causing Hospital-associated Intra-abdominal Infections in Adults in the United States (2012-2013).

Clin Ther 2016 Jun 24;38(6):1510-1521. Epub 2016 May 24.

International Health Management Associates, Schaumburg, Illinois.

Purpose: Selection and prompt initiation of the appropriate empiric antimicrobial therapy are critical to decrease morbidity and mortality and shorten the length of hospitalization among patients with hospital-associated intra-abdominal infections (HA-IAIs). Therapeutic choices for the treatment of patients with HA-IAI require careful consideration. This study was conducted to evaluate the antimicrobial susceptibility of common pathogens collected from adult patients with HA-IAI in the United States.

Methods: Gram-negative bacilli (N = 1285) were collected during 2012-2013 from SMART (Study for Monitoring Antimicrobial Resistance Trends). Isolates were tested at a central laboratory by using Clinical and Laboratory Standards Institute methods and interpretation of susceptibility to 12 antimicrobial agents.

Findings: Most of the isolates (80.8%) were Enterobacteriaceae, and Escherichia coli was the most common species. Susceptibility to frequently used antimicrobial agents for treating IAI showed that ertapenem, imipenem, and amikacin were more active than other agents against Enterobacteriaceae, including multidrug-resistant isolates. More than 92% of E coli, including extended-spectrum β-lactamase (ESBL) producers, and Klebsiella pneumoniae isolates were susceptible to ertapenem, imipenem, and amikacin. Cefepime was the most active (>90% susceptibility) cephalosporin against all species except K pneumoniae (86.6%) but with much reduced activity against isolates with ESBLs. Piperacillin/tazobactam had reduced activity against Enterobacter species (70.4%-76.4% susceptible) and ESBL-producing K pneumoniae (22.5% susceptible). Fluoroquinolones exhibited poor activity against E coli (overall susceptibility <70%).

Implications: Proper empiric antimicrobial treatment, including combining appropriate agents, of HA-IAI requires detailed understanding of the epidemiology of common pathogens and antimicrobial resistance patterns. In light of rising rates of antimicrobial resistance, ongoing surveillance is critical for clinical decision-making.
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http://dx.doi.org/10.1016/j.clinthera.2016.04.035DOI Listing
June 2016

Pharmacokinetics/Pharmacodynamics of Peptide Deformylase Inhibitor GSK1322322 against Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in Rodent Models of Infection.

Antimicrob Agents Chemother 2016 01 19;60(1):180-9. Epub 2015 Oct 19.

Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

GSK1322322 is a novel inhibitor of peptide deformylase (PDF) with good in vitro activity against bacteria associated with community-acquired pneumonia and skin infections. We have characterized the in vivo pharmacodynamics (PD) of GSK1322322 in immunocompetent animal models of infection with Streptococcus pneumoniae and Haemophilus influenzae (mouse lung model) and with Staphylococcus aureus (rat abscess model) and determined the pharmacokinetic (PK)/PD index that best correlates with efficacy and its magnitude. Oral PK studies with both models showed slightly higher-than-dose-proportional exposure, with 3-fold increases in area under the concentration-time curve (AUC) with doubling doses. GSK1322322 exhibited dose-dependent in vivo efficacy against multiple isolates of S. pneumoniae, H. influenzae, and S. aureus. Dose fractionation studies with two S. pneumoniae and S. aureus isolates showed that therapeutic outcome correlated best with the free AUC/MIC (fAUC/MIC) index in S. pneumoniae (R(2), 0.83), whereas fAUC/MIC and free maximum drug concentration (fCmax)/MIC were the best efficacy predictors for S. aureus (R(2), 0.9 and 0.91, respectively). Median daily fAUC/MIC values required for stasis and for a 1-log10 reduction in bacterial burden were 8.1 and 14.4 for 11 S. pneumoniae isolates (R(2), 0.62) and 7.2 and 13.0 for five H. influenzae isolates (R(2), 0.93). The data showed that for eight S. aureus isolates, fAUC correlated better with efficacy than fAUC/MIC (R(2), 0.91 and 0.76, respectively), as efficacious AUCs were similar for all isolates, independent of their GSK1322322 MIC (range, 0.5 to 4 μg/ml). Median fAUCs of 2.1 and 6.3 μg · h/ml were associated with stasis and 1-log10 reductions, respectively, for S. aureus.
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http://dx.doi.org/10.1128/AAC.01842-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704172PMC
January 2016

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

Antimicrob Agents Chemother 2015 Aug 26;59(8):4644-52. Epub 2015 May 26.

Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

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).
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http://dx.doi.org/10.1128/AAC.00484-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505285PMC
August 2015

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

Antimicrob Agents Chemother 2014 28;58(1):290-6. Epub 2013 Oct 28.

Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

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.
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http://dx.doi.org/10.1128/AAC.01292-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910740PMC
September 2014

Staphylococcus aureus formyl-methionyl transferase mutants demonstrate reduced virulence factor production and pathogenicity.

Antimicrob Agents Chemother 2013 Jul 9;57(7):2929-36. Epub 2013 Apr 9.

Antibacterial Discovery Performance Unit, Infectious Diseases Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection.
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http://dx.doi.org/10.1128/AAC.00162-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697361PMC
July 2013

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

Antimicrob Agents Chemother 2013 May 11;57(5):2333-42. Epub 2013 Mar 11.

Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

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 β-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.
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http://dx.doi.org/10.1128/AAC.02566-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632952PMC
May 2013

Understanding the origins of time-dependent inhibition by polypeptide deformylase inhibitors.

Biochemistry 2011 Aug;50(31):6642-54

Department of Biological Reagents, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.

The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.
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http://dx.doi.org/10.1021/bi200655gDOI Listing
August 2011

A rapid microtiter plate assay for measuring the effect of compounds on Staphylococcus aureus membrane potential.

J Microbiol Methods 2010 Nov 27;83(2):254-6. Epub 2010 Aug 27.

Antibacterial Discovery Performance Unit, Infectious Diseases Center of Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426, United States.

We developed a homogenous microtiter based assay using the cationic dye 3, 3'-Diethyloxacarbocyanine iodide, DiOC2(3), to measure the effect of compounds on membrane potential in Staphylococcus aureus. In a screen of 372 compounds from a synthetic compound collection with anti-Escherichia coli activity due to unknown modes of action at least 17% demonstrated potent membrane activity, enabling rapid discrimination of nuisance compounds.
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http://dx.doi.org/10.1016/j.mimet.2010.08.012DOI Listing
November 2010

Selection of retapamulin, a novel pleuromutilin for topical use.

Antimicrob Agents Chemother 2006 Nov;50(11):3882-5

Department of Microbiology Research, MMPD CEDD, GlaxoSmithKline Pharmaceuticals, 1250 S. Collegeville Rd, Collegeville, PA 19426-0989, USA.

The in vitro activity of retapamulin was determined and compared to that of topical and community antibiotics. The MIC(90)s of retapamulin against Staphylococcus aureus and Streptococcus pyogenes were 0.12 microg/ml and 0.016 microg/ml, respectively. Retapamulin has a low propensity to select resistance and produces an in vitro postantibiotic effect.
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http://dx.doi.org/10.1128/AAC.00178-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635201PMC
November 2006

Phylogenomic and biochemical characterization of three Legionella pneumophila polypeptide deformylases.

J Bacteriol 2006 Jul;188(14):5249-57

Microbiology Department, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.

Legionella pneumophila is a gram-negative facultative intracellular human pathogen that can cause fatal Legionnaires' disease. Polypeptide deformylase (PDF) is a novel broad-spectrum antibacterial target, and reports of inhibitors of PDF with potent activities against L. pneumophila have been published previously. Here, we report the identification of not one but three putative pdf genes, pdfA, pdfB, and pdfC, in the complete genome sequences of three strains of L. pneumophila. Phylogenetic analysis showed that L. pneumophila PdfA is most closely related to the commonly known gamma-proteobacterial PDFs encoded by the gene def. PdfB and PdfC are more divergent and do not cluster with any specific bacterial or eukaryotic PDF. All three putative pdf genes from L. pneumophila strain Philadelphia 1 have been cloned, and their encoded products have been overexpressed in Escherichia coli and purified. Enzymatic characterization shows that the purified PDFs with Ni2+ substituted are catalytically active and able to remove the N-formyl group from several synthetic polypeptides, although they appear to have different substrate specificities. Surprisingly, while PdfA and PdfB with Zn2+ substituted are much less active than the Ni2+ forms of each enzyme, PdfC with Zn2+ substituted was as active as the Ni2+ form for the fMA substrate and exhibited substrate specificity different from that of Ni2+ PdfC. Furthermore, the catalytic activities of these enzymes are potently inhibited by a known small-molecule PDF inhibitor, BB-3497, which also inhibits the extracellular growth of L. pneumophila. These results indicate that even though L. pneumophila has three PDFs, they can be effectively inhibited by PDF inhibitors which can, therefore, have potent anti-L. pneumophila activity.
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http://dx.doi.org/10.1128/JB.00866-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539947PMC
July 2006

Peptide deformylase inhibitors.

Prog Med Chem 2006 ;44:109-43

Microbial, Musculoskeletal, and Proliferative Diseases CEDD, GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426, USA.

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http://dx.doi.org/10.1016/S0079-6468(05)44403-3DOI Listing
November 2007

Mechanism of time-dependent inhibition of polypeptide deformylase by actinonin.

Biochemistry 2005 Jan;44(1):253-60

Enzymology and Mechanistic Pharmacology, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.

Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for the removal of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition of bacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterized using steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observed under steady-state conditions. Progress curve analysis is consistent with a two-step binding mechanism, in which tightening of the initial encounter complex (EI) results in a final complex (EI*) with an extremely slow, but observable, off-rate (t(1/2) for inhibitor dissociation >or=0.77 days). Stopped-flow measurement of PDF fluorescence confirms formation of EI and provides a direct measurement of the association rate. Rapid dilution studies establish that the potency of actinonin is enhanced by more than 2000-fold upon tightening of EI to form EI*, from K(i) = 530 nM (EI) to Ki*
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http://dx.doi.org/10.1021/bi048632bDOI Listing
January 2005

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

J Bacteriol 2004 Apr;186(8):2346-54

Microbial, Musculoskeletal and Proliferative Diseases CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.

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).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC412112PMC
http://dx.doi.org/10.1128/jb.186.8.2346-2354.2004DOI Listing
April 2004

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

J Mol Microbiol Biotechnol 2003 ;6(2):109-26

Microbial, Musculoskeletal and Proliferative Diseases CEDD, Collegeville, PA 17426, USA.

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.
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http://dx.doi.org/10.1159/000076741DOI Listing
June 2004

Insights into catalysis by a knotted TrmD tRNA methyltransferase.

J Mol Biol 2003 Nov;333(5):931-49

GlaxoSmithKline, 709 Swedeland Road, UE0447, King of Prussia, PA 19406, USA.

The crystal structure of Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD) complexed with S-adenosyl homocysteine (AdoHcy) has been determined at 2.5A resolution. TrmD, which methylates G37 of tRNAs containing the sequence G36pG37, is a homo-dimer. Each monomer consists of a C-terminal domain connected by a flexible linker to an N-terminal AdoMet-binding domain. The two bound AdoHcy moieties are buried at the bottom of deep clefts. The dimer structure appears integral to the formation of the catalytic center of the enzyme and this arrangement strongly suggests that the anticodon loop of tRNA fits into one of these clefts for methyl transfer to occur. In addition, adjacent hydrophobic sites in the cleft delineate a defined pocket, which may accommodate the GpG sequence during catalysis. The dimer contains two deep trefoil peptide knots and a peptide loop extending from each knot embraces the AdoHcy adenine ring. Mutational analyses demonstrate that the knot is important for AdoMet binding and catalytic activity, and that the C-terminal domain is not only required for tRNA binding but plays a functional role in catalytic activity.
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http://dx.doi.org/10.1016/j.jmb.2003.09.011DOI Listing
November 2003

Inhibitors of pantothenate kinase: novel antibiotics for staphylococcal infections.

Antimicrob Agents Chemother 2003 Jun;47(6):2051-5

Microbial, Musculoskeletal and Proliferative Diseases and Bioinformatics, GlaxoSmithKline Pharmaceuticals, Collegeville Pennsylvania 19426, USA.

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC155856PMC
http://dx.doi.org/10.1128/aac.47.6.2051-2055.2003DOI Listing
June 2003

The promoter of a cold-shock-like gene has pleiotropic effects on Streptomyces antibiotic biosynthesis.

FEMS Microbiol Lett 2003 Mar;220(2):215-21

Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

We have isolated a Streptomyces hygroscopicus chromosomal DNA fragment able to induce production of the blue-pigmented antibiotic actinorhodin in Streptomyces lividans. The 1.9-kb fragment contains four orfs (orf1-4) of which only orf2 and orf3 were complete. The minimal region involved in activation of actinorhodin production is limited to 165 bp corresponding to the promoter region of orf3. The truncated Orf1 show homologies with threonine synthases, Orf2 is similar to other proteins of unknown function, Orf3 (here named Csp1) is homologous to cold-shock-induced proteins of the Csp family, and Orf4 encodes the N-terminal region of GroEL2. Transcription of csp1 seems to be subjected to temporal control but is not obviously induced by cold shock. Interestingly, the csp1-groEL2 region pleiotropically regulates the production of antibiotics from Streptomyces coelicolor and Streptomyces nodosus.
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http://dx.doi.org/10.1016/S0378-1097(03)00101-0DOI Listing
March 2003

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

J Bacteriol 2003 Mar;185(6):2051-8

Microbial, Musculoskeletal and Proliferative Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426, USA.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150135PMC
http://dx.doi.org/10.1128/jb.185.6.2051-2058.2003DOI Listing
March 2003

Novel antibacterials: a genomics approach to drug discovery.

Curr Drug Targets Infect Disord 2002 Dec;2(4):291-308

Department of Microbiology. Microbial, Musculoskeletal and Proliferative Diseases CEDD, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426-0989, USA.

The appearance of antibiotic resistant pathogens, including vancomycin resistant Staphylococcus aureus, in the clinic has necessitated the development of new antibiotics. The golden age of antibiotic discovery, in which potent selective compounds were readily extracted from natural product extracts is over and novel approaches need to be implemented to cover the therapeutic shortfall. The generation of huge quantities of bacterial sequence data has allowed the identification of all the possible targets for therapeutic intervention and allowed the development of screens to identify inhibitors. Here, we described a number of target classes in which genomics has contributed to its identification. As a result of analyzing sequence data, all of the tRNA synthetases and all of the two-component signal transduction systems were readily isolated; which would not have been easily identified if whole genome sequences were not available. Fatty acid biosynthesis is a known antibacterial target, but genomics showed which genes in that pathway had the appropriate spectrum to be considered as therapeutic targets. Genes of unknown function may seem untractable targets, but if those that are broad spectrum and essential are identified, it becomes valuable to invest time and effort to determine their cellular role. In addition, we discuss the role of genomics in developing technologies that assist in the discovery of new antibiotics including microarray gridding technology. Genomics can also increase the chemical diversity against which the novel targets can be screened.
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http://dx.doi.org/10.2174/1568005023342227DOI Listing
December 2002