Peptide Deformylase Inhibitors Research Articles

Synthesis and antimicrobial activities of novel peptide deformylase inhibitors

A new series of N-formylhydroxylamine compounds were designed, optimized with the AutoDock 4.0.1 to investigate the interactions between the target compounds and the amino acid residues of the Escherichia coli PDF center dot Ni enzyme, and then synthesized through multi-step sequence starting from diethyl malonate. The structures of the compounds were characterized on the basis of spectral (FT-IR, 1H NMR and mass) analysis. All the synthesized compounds have been screened for their antimicrobial activities. It was found that the compounds 11c, 11d, 11f and 11g exhibited potent inhibitory activity against S. aureus in vitro.

Peptide deformylase inhibitors with retro-amide scaffold: Synthesis and structure–activity relationships

Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins. Thus inhibition of PDF activity is considered to be one of the most effective antibiotic strategies. Reported herein are the synthesis and structure–activity relationship studies of retro-amide inhibitors based on actinonin, a naturally occurring PDF inhibitor. Analysis of the structure–activity relationships led to the discovery of 7a, which exhibits potent enzyme inhibition and antibacterial activity against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.

Design, synthesis, and antibacterial activity of 2,5-dihydropyrrole formyl hydroxyamino derivatives as novel peptide deformylase inhibitors

The synthesis and antibacterial activity of 2,5-dihydropyrrole formyl hydroxyamino derivatives are reported. The antibacterial activities of these derivatives were evaluated, and some of these derivatives showed better in vitro antibacterial activity than existing drugs, including penicillin, ciprofloxacin, vancomycin, and linezolid.

Up‐regulation and inhibition of human peptide deformylase in cancer cells and tissues

The human isoform of peptide deformylase (PDF) has been recently discovered and found to localize to mitochondria; however, its role in the deformylation of newly‐synthesized mitochondrial polypeptides is uncertain. Our hypothesis is that human mitochondrial PDF (hsPDF) is functional in human cells. The objectives of this study were to quantify hsPDF in normal and cancerous cells and tissues and to determine the effects of hsPDF inhibition on cell proliferation. Quantitative real time PCR was used to measure hsPDF mRNA expression in cancerous and normal cell lines and tissues. Additionally, various cancer and normal cell lines were treated with increasing concentrations of a known PDF inhibitor, actinonin, and cell proliferation was assessed. Increases in hsPDF mRNA levels were seen in most cancer cell lines when compared to the normal. hsPDF was upregulated in most cancer tissues compared to the normal, with the greatest differences observed in breast, colon and lung tissues. The antiproliferative effects of actinonin were greater in prostate, colon, and breast cancer cell lines than in the normal cells. These results suggest that hsPDF is up‐regulated in certain cancers and that it may play an active role in proliferation of human cells. This work is supported by the NDSU Center for Protease Research (NIH 2P20 RR015566) and start‐up funds from NDSU to KR.

Isolation and Identification of FR198248, a Hydroxylated 1,3-Dihydroisobenzofuran, fromAspergillus flavipesas an Inhibitor of Peptide Deformylase

Two highly hydroxylated 1,3-dihydroisobenzofurans, FR198248 (1) and FR202306 (2), were isolated as peptide deformylase (PDF) inhibitors from Aspergillus flavipes. Compounds 1 and 2 inhibited Staphylococus aureus PDF with IC(50) values of 3.6 and 2.5 microM, respectively, and also showed antibacterial activity with an MIC value of 25 microg/ml. In contrast, 6-O-methyl derivative 3 of compound 2 was inactive against both PDF and S. aureus.

Error-prone initiation factor 2 mutations reduce the fitness cost of antibiotic resistance.

Mutations in the fmt gene (encoding formyl methionine transferase) that eliminate formylation of initiator tRNA (Met-tRNAi) confer resistance to the novel antibiotic class of peptide deformylase inhibitors (PDFIs) while concomitantly reducing bacterial fitness. Here we show in Salmonella typhimurium that novel mutations in initiation factor 2 (IF2) located outside the initiator tRNA binding domain can partly restore fitness of fmt mutants without loss of antibiotic resistance. Analysis of initiation of protein synthesis in vitro showed that with non-formylated Met-tRNAi IF2 mutants initiated much faster than wild-type IF2, whereas with formylated fMet-tRNAi the initiation rates were similar. Moreover, the increase in initiation rates with Met-tRNAi conferred by IF2 mutations in vitro correlated well with the increase in growth rate conferred by the same mutations in vivo, suggesting that the mutations in IF2 compensate formylation deficiency by increasing the rate of in vivo initiation with Met-tRNAi. IF2 mutants had also a high propensity for erroneous initiation with elongator tRNAs in vitro, which could account for their reduced fitness in vivo in a formylation-proficient strain. More generally, our results suggest that bacterial protein synthesis is mRNA-limited and that compensatory mutations in IF2 could increase the persistence of PDFI-resistant bacteria in clinical settings.

In Silico Study of the Selective Inhibition of Bacterial Peptide Deformylases by Several Drugs

To counter increasing levels of pathogen resistance new classes of antibiotics are needed without delay. The metalloenzyme peptide deformylase (PDF) correspond to one of the most promising bacterial targets in the search for novel mode of antibiotics action and was firstly selected as a specific bacterial target. Peptide analogs were developed as inhibitors containing a hydroxamate or formyl- hydroxylamine as metal interacting group, and used as inhibitors with in vitro activity against a broad spectrum of organisms and successful antibacterial activity in vivo that is harmonizing with good pharmacokinetic properties and excellent tolerability in diverse species, but a human homologue was recently discovered. A new strategy for selecting highly efficient compounds with low inhibition effect against human PDF was developed. An original class of small, non-peptidic inhibitors of peptide deformylase (PDF) as potent antibiotics such as indol-group and its derivatives with the same mode of action in vivo as previously identified PDF inhibitors but without the apoptotic effects of these inhibitors in human cells, has been discovered. This study has confirmed the selective action of these compounds on bacterial PDFs by docking method using the autodock program. Indeed, a good correlation between IC50 and deltaG values of different complexes PDF-inhibitors was observed. The evaluation of the various molecular properties of these inhibitors lets us conclude that all these compounds are most likely drugable.

Synthesis of Methoxyfumimycin with 1,2-Addition to Ketimines

The synthesis of (+/-)-methoxyfumimycin, a potential new bacterial peptide deformylase (PDF) inhibitor, is reported. To generate the stereogenic fully substituted carbon, the key step is a 1,2-addition of a methyl Grignard reagent to a ketimine. The overall synthetic strategy involves a Dakin oxidation of a vanillin derivative, Friedel-Crafts acylation, Claisen rearrangement, lactonization, and rhodium-catalyzed olefin isomerization.

Solvent-assisted slow conversion of a dithiazole derivative produces a competitive inhibitor of peptide deformylase

Due to its potential as an antibiotic target, E. coli peptide deformylase (PDF Ec) serves as a model enzyme system for inhibitor design. While investigating the structural–functional and inhibitory features of this enzyme, we unexpectedly discovered that 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) served as a slow-binding inhibitor of PDF Ec when the above compound was dissolved only in dimethylformamide (DMF), but not in any other solvent, and allowed to age. The time dependent inhibitory potency of the DMF-dissolved AMT was correlated with the broadening of the inhibitor's 295 nm spectral band toward the visible region, concomitant with the increase in the mass of the parent compound by about 2-fold. These data led to the suggestion that DMF facilitated the slow dimerization of AMT (via the formation of a disulfide bond), and that the dimeric form of AMT served as an inhibitor for PDF Ec. The latter is not caused by the simple oxidation of sulfhydryl groups by oxidizing agents such as H 2O 2. Newly synthesized dimeric/dithiolated form of AMT (“bis-AMT”) exhibited similar spectral and inhibitory features as given by the parent compound when incubated with DMF. The computer graphic modeling data revealed that bis-AMT could be reliably accommodated within the active site pocket of PDF Ec, and the above enzyme–ligand interaction involves coordination with the enzyme resident Ni 2+ cofactor. The mechanism of the DMF-assisted activation of AMT (generating bis-AMT), the overall microscopic pathway for the slow-binding inhibition of PDF Ec by bis-AMT, and the potential of bis-AMT to serve as a new class of antibiotic agent are presented.

Fmt Bypass in Pseudomonas aeruginosa Causes Induction of MexXY Efflux Pump Expression

The intrinsic resistance of P. aeruginosa PAO1 to the peptide deformylase inhibitor (PDF-I) LBM415 was mediated by the MexAB-OprM and MexXY-OprM efflux pumps, the latter of which was strongly induced by LBM415. Single-step exposure of PAO1 deleted for mexAB-oprM (therefore lacking both MexAB-OprM and MexXY-OprM functions) to PDF-Is selected for nfxB mutants, which express the MexCD-OprJ efflux pump, indicating that these compounds are also substrates for this pump. Selection of resistant mutants by use of levels of LBM415 greater than that accommodated by efflux yielded two additional groups of mutations, in the methionyl-tRNA(fmet) formyltransferase (fmt) and folD genes. Both mechanisms are known to impose an in vitro growth deficit (also observed here), presumably due to impairment of protein synthesis. We surmised that this inherent impairment of protein synthesis would upregulate expression of mexXY in a fashion similar to upregulation by LBM415 or by ribosome inhibitory compounds. Transcriptional profiling and/or mexX::lux promoter fusion analysis revealed that fmt and folD mutants were strongly upregulated for mexXY and another gene known to be required for upregulation of the pump, PA5471. Complementation of the fmt mutation in trans reversed this constitutive expression. This supports the notion that MexXY has a natural physiological function responding to impairment of ribosome function or protein synthesis and that fmt mutation (Fmt bypass) and folD mutation generate the intracellular mexXY-inducing signal.

New Antibiotic Molecules: Bypassing the Membrane Barrier of Gram Negative Bacteria Increases the Activity of Peptide Deformylase Inhibitors

BackgroundMulti-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol.Methodology/Principal FindingsTo determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the lastest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step.Conclusions/SignificanceOur results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound.

In Vivo Characterization of the Peptide Deformylase Inhibitor LBM415 in Murine Infection Models

LBM415 is an antibacterial agent belonging to the peptide deformylase inhibitor class of compounds. It has previously been shown to demonstrate good activity in vitro against a range of pathogens. In this study, the in vivo efficacy of LBM415 was evaluated in various mouse infection models. We investigated activity against a systemic infection model caused by intraperitoneal inoculation of Staphylococcus aureus (methicillin [meticillin] susceptible [MSSA] and methicillin resistant [MRSA]) and Streptococcus pneumoniae (penicillin susceptible [PSSP] and multidrug resistant [MDRSP]), a thigh infection model caused by intramuscular injection of MRSA, and a lung infection produced by intranasal inoculation of PSSP. In the systemic MSSA and MRSA infections, LBM415 was equivalent to linezolid and vancomycin. In the systemic PSSP infection, LBM415 was equivalent to linezolid, whereas against systemic MDRSP infection, the LBM415 50% effective dose (ED50) was 4.8 mg/kg (dosed subcutaneously) and 36.6 mg/kg (dosed orally), compared to 13.2 mg/kg for telithromycin and >60 mg/kg for penicillin V and clarithromycin. In the MRSA thigh infection, LBM415 significantly reduced thigh bacterial levels compared to those of untreated mice, with levels similar to those after treatment with linezolid at the same dose levels. In the pneumonia model, the ED50 to reduce the bacterial lung burden by >4 log10 in 50% of treated animals was 23.3 mg/kg for LBM415, whereas moxifloxacin showed an ED50 of 14.3 mg/kg. In summary, LBM415 showed in vivo efficacy in sepsis and specific organ infection models irrespective of resistance to other antibiotics. Results suggest the potential of peptide deformylase inhibitors as a novel class of therapeutic agents against antibiotic-resistant pathogens.

Peptide deformylase – a promising therapeutic target for tuberculosis and antibacterial drug discovery

Background: Tuberculosis (TB) remains the most important infectious disease causing morbidity and death, due to the human pathogen Mycobacterium tuberculosis. The emergence of multi-drug-resistant and extensively-drug-resistant forms of TB have resulted in an increase in the number of TB cases. Thus, there is an urgent need to identify new drugs with novel targets to ensure future therapeutic success. Studies have indicated that peptide deformylase is an interesting potential candidate for discovering antimicrobial agents. Objective: To explore the role of peptide deformylase, a highly conserved metalloprotease and an essential enzyme in bacterial life cycle, as a target for antibacterial as well as antimycobacterial drug development. Methods: This review is based on recent published literature and online resources related to peptide deformylase inhibitors and their anbacterial potential. Results/conclusion: Peptide deformylase is an emerging therapeutic target for the treatment of tuberculosis and peptide deformylase inhibitors can act as potential future antibacterial agents.

In vitro and ex vivo activity of peptide deformylase inhibitors against Mycobacterium tuberculosis H37Rv

Bacterial peptide deformylase (PDF) catalyses removal of the N-terminal formyl group of proteins and is essential for protein maturation, growth and survival of bacteria. Thus, PDF appears to be a good antimycobacterial drug target. In the present study, various well-known PDF inhibitors, such as BB-3497, actinonin, 1,10-phenanthroline, hydroxylamine hydrochloride and galardin, were selected to evaluate their inhibitory activity against Mycobacterium tuberculosis. All compounds were found to be active against M. tuberculosis, with MIC 90 values (lowest drug concentration at which 90% of growth was inhibited on the basis of CFU enumeration) ranging from 0.2 mg/L to 74 mg/L. BB-3497 and 1,10-phenanthroline exhibited potent in vitro antimycobacterial activity, and also showed synergism with isoniazid and rifampicin. All compounds showed a bacteriostatic mode of inhibition. Under ex vivo conditions and short-course chemotherapy, BB-3497 and actinonin were found to be significantly active, with BB-3497 exhibiting comparable efficacy to that of isoniazid. Collectively, promising activities of PDF inhibitors such as BB-3497 and actinonin suggest their potential use against M. tuberculosis.

Cover Picture: Structure–Activity Relationship Analysis of the Peptide Deformylase Inhibitor 5‐Bromo‐1H‐indole‐3‐acetohydroxamic Acid (ChemMedChem 2/2009)

AbstractThe cover picture shows a “reverse” indole derivative in complex with Bacillus stearothermophilus peptide deformylase (PDF). This compound was selected from a structure–activity relationship study as a potent inhibitor of bacterial PDFs and shows antibacterial activity toward Bacillus subtilis as well as other pathogens such as Streptococcus pneumoniae and Staphylococcus aureus. For more details, see the Full Paper by I. Artaud et al. on p. 261 ff.magnified image

Mutations in Three Distinct Loci Cause Resistance to Peptide Deformylase Inhibitors inBacillus subtilis

Bacillus subtilis mutants with resistance against peptide deformylase inhibitors were isolated. All showed a bypass of the pathway through mutations in three genes required for formylation of Met-tRNA(fMet), fmt, folD, and glyA. glyA corresponds to a yet uncharacterized locus inducing resistance. The bypass of formylation caused robust fitness reduction but was not accompanied by alterations of the transcription profile. A subtle adaptation of the enzymes of the intermediary metabolism was observed.

The Use of Structure-Guided Design to Discover New Anti-Microbial Agents: Focus on Antibacterial Resistance

Serious attempts to address antibiotic resistance, a worldwide public health concern, have recently become more intensive. In hospital settings, resistance to antibacterial agents has been recognized by clinicians for several decades. Resistant strains are now isolated on a daily basis from patients with community-acquired infections further elevating the level of concern among public health officials. The pharmaceutical industry has generally focused its attentions on chronic therapeutic indications in recent years (e.g. cardiovascular and metabolic diseases), but will likely be forced to reengage in antibacterial discovery efforts as therapeutic options diminish for the treatment of infections caused by multidrug resistant pathogens. The ability to squeeze additional utility out of known classes of antibacterial agents has become limited and antibacterial discovery scientists will need to focus on new approaches and targets. These new approaches will need to include strategies that explicitly address resistance up front and simultaneously attempt to facilitate the slower development of resistance as new compound classes enter clinical use. One approach that can be a useful component of antibacterial discovery efforts and prospectively address resistance is structure-guided design (SGD). This review will describe several recent examples in which SGD was applied as part of a multidisciplinary effort to address antibacterial resistance. These include dihydrofolate reductase inhibitors, broad-spectrum β-lactamase inhibitors, novel oxazolidinones, aminoglycoside mimetics, peptide deformylase inhibitors, and inhibitors that simultaneously target DNA gyrase and topoisomerase IV. Keywords: Antibiotic resistance, antibiotic drug discovery, resistance mechanisms, structure-guided design, dual targeting

Towards an asymmetric synthesis of the bacterial peptide deformylase (PDF) inhibitor fumimycin

Studies towards the synthesis of the bacterial peptide deformylase (PDF) inhibitor fumimycin are reported. The synthetic approach features an organocatalytic access to the alpha,alpha-disubstituted amino acid unit and results in the synthesis of an advanced intermediate which already contains all functionalities of fumimycin.

Synthesis and In Vitro Antibacterial Activity of (2S)‐N‐(Substitutedphenyl)‐1‐[(2R)‐2‐[(Formylhydroxyamino)Methyl]‐1‐Oxohexyl]‐2‐Pyrrolidinecarboxamides as Potential Peptide Deformylase Inhibitors

Gram-positive organisms have re-emerged as the major hospital pathogens, which make the unmet medical needs for antibacterial therapy even worse. In searching for potent agents against Gram-positive pathogens, novel (2S)-N-(substitutedphenyl)-1-[(2R)-2-[(formylhydroxyamino)methyl]-1-oxohexyl]-2-pyrrolidinecarboxamides, analogues of peptide deformylase inhibitor LBM-415 were designed, synthesized and evaluated for their antibacterial activities in vitro. Many of these compounds exhibited high potency against Gram-positive organisms compared with reference agent: LBM-415.

PP-003 Evaluation on the cellular immunity of M. tuberculosis in HIV/AIDS and tuberculosis co-infected individuals in China

Objectives: The present study explores the potential of known peptide deformylase (PDF) inhibitors as chemotherapeutic agents against experimental tuberculosis. Methods: Several PDF inhibitors like BB-3497, actinonin, 1, 10-phenanthroline, hydroxylamine hydrochloride, and galardin that have shown their potential against other bacteria were selected for their evaluation against Mycobacterium tuberculosis. Results: Based on in vitro studies, the minimum inhibitory concentration (MIC) of PDF inhibitors was found to be in the range of 0.2 74mg/L, and among different PDF inhibitors, BB-3497 and 1, 10-phenanthroline exhibited potent antimycobacterial activity. In addition PDF inhibitors exhibited bacteriostatic mode of inhibition. Ex vivo studies with BB-3497 and actinonin led to reduction of colony forming units (cfu) by almost 2 log10 units at their MIC concentration. Moreover, reduction of cfu by BB3497 was comparable to that of isoniazid. Under short course chemotherapy, BB-3497 was significantly active and comparable to isoniazid. Extended chemotherapy of 6 weeks with BB-3497 and actinonin revealed BB-3497 to be more potent under in vivo condition. Interestingly, BB-3497 and actinonin exhibited synergism with isoniazid and rifampicin under in vitro conditions and during chemotherapy of murine tuberculosis. Conclusions: Peptide deformylase inhibitors like BB-3497 and actinonin showed their efficacy against Mycobacterium tuberculosis under in vitro, ex vivo and in vivo conditions and BB-3497 was found to be the most efficacious inhibitor. Thus PDF from M. tuberculosis appears to be an attractive target for development of new antibiotics against tuberculosis.