Discovery of piperazic acid peptide deformylase inhibitors with in vivo activity for respiratory tract and skin infections.

The discovery of a novel series of peptide deformylase inhibitors incorporating a piperazic acid amino acid found in nature is described. These compounds demonstrated potent in vitro enzymatic potency and antimicrobial activity. Crystal structure analysis revealed the piperazic acid optimized a key contact with the PDF protein that accounted for the increased enzymatic potency of these compounds. We describe lead optimization of the P3' region of the series that resulted in a compound with good potency against three target organisms. One molecule showed in vivo efficacy in a rat respiratory infection model but ultimately did not meet candidate progression criteria.

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

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.

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).

In vitro and intracellular activities of peptide deformylase inhibitor GSK1322322 against Legionella pneumophila isolates.

GSK1322322, a novel peptide deformylase inhibitor currently in development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia, showed poor in vitro activity against a panel of 50 Legionella pneumophila strains, with MICs ranging from 1 to 16 µg/ml and an MIC90 of 16 µg/ml, but very potent intracellular activity, with the minimum extracellular concentrations capable of inhibiting intracellular proliferation (MIECs) ranging from 0.12 to 2 µg/ml and 98% of the strains being inhibited by concentrations of ≤ 1 µg/ml.

Microbiome changes in healthy volunteers treated with GSK1322322, a novel antibiotic targeting bacterial peptide deformylase.

GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study Register under study identifier PDF 113376.).

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.

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

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.

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.

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

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.

Design, synthesis and antibacterial activity of 3-methylenepyrrolidine formyl hydroxyamino derivatives as novel peptide deformylase inhibitors.

The synthesis and antibacterial activity of 3-methylenepyrrolidine formyl hydroxyamino derivatives are reported. The antibacterial activities of these derivatives were evaluated to discover SAR at P(1') and P(3') positions, and most of these derivatives exhibit better in vitro antibacterial activity than existing drugs against drug-resistant clinical isolates including MRSA, PRSP, and Haemophilus influenzae.

Phylogenomic and biochemical characterization of three Legionella pneumophila polypeptide deformylases.

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.

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

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*

Structural variation and inhibitor binding in polypeptide deformylase from four different bacterial species.

Polypeptide deformylase (PDF) catalyzes the deformylation of polypeptide chains in bacteria. It is essential for bacterial cell viability and is a potential antibacterial drug target. Here, we report the crystal structures of polypeptide deformylase from four different species of bacteria: Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. Comparison of these four structures reveals significant overall differences between the two Gram-negative species (E. coli and H. influenzae) and the two Gram-positive species (S. pneumoniae and S. aureus). Despite these differences and low overall sequence identity, the S1' pocket of PDF is well conserved among the four enzymes studied. We also describe the binding of nonpeptidic inhibitor molecules SB-485345, SB-543668, and SB-505684 to both S. pneumoniae and E. coli PDF. Comparison of these structures shows similar binding interactions with both Gram-negative and Gram-positive species. Understanding the similarities and subtle differences in active site structure between species will help to design broad-spectrum polypeptide deformylase inhibitor molecules.

A Biomimetic Approach to the Discorhabdin Alkaloids: Total Syntheses of Discorhabdins C and E and Dethiadiscorhabdin D.

The characteristic spirodienone structure of the discorhabdin alkaloids is readily formed by reaction of the tyramine-substituted indoloquinonimines 26, 35, and 36 with cupric chloride, triethylamine, and oxygen. This cyclization provides a possibly biomimetic route to discorhabdins C and E (41 and 42). The unbrominated spirodienone 40 reacts with hydrogen over Pd/C to give enone 46. Bromination at the alpha position gives a mixture of bromoenones that undergo smooth conversion to dethiadiscorhabdin D (4) upon treatment with basic alumina.