Discovery of aminopyridine-based inhibitors of bacterial enoyl-ACP reductase (FabI)

Bacterial enoyl-ACP reductase (FabI) catalyzes the final step in each cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. Our efforts to identify potent, selective FabI inhibitors began with screening of the GlaxoSmithKline proprietary compound collection, which identified several small-molecule inhibitors of Staphylococcus aureus FabI. Through a combination of iterative medicinal chemistry and X-ray crystal structure based design, one of these leads was developed into the novel aminopyridine derivative 9, a low micromolar inhibitor of FabI from S. aureus (IC(50) = 2.4 microM) and Haemophilus influenzae (IC(50) = 4.2 microM). Compound 9 has good in vitro antibacterial activity against several organisms, including S. aureus (MIC = 0.5 microg/mL), and is effective in vivo in a S. aureus groin abscess infection model in rats. Through FabI overexpressor and macromolecular synthesis studies, the mode of action of 9 has been confirmed to be inhibition of fatty acid biosynthesis via inhibition of FabI. Taken together, these results support FabI as a valid antibacterial target and demonstrate the potential of small-molecule FabI inhibitors for the treatment of bacterial infections.     

Chalcomoracin and moracin C, new inhibitors of Staphylococcus aureus enoyl-acyl carrier protein reductase from Morus alba

Bacterial enoyl-acyl carrier protein (ACP) reductase has been confirmed as a novel target for antibacterial drug development. In the screening of inhibitors of Staphylococcus aureus enoyl-ACP reductase (FabI), we found that a methanol extract of leaves of Morus alba L. potently inhibited S. aureus FabI as well as growth of S. aureus. The active principles were identified as chalcomoracin and moracin C by MS and NMR analysis. Chalcomoracin and moracin C inhibited S. aureus FabI with IC(50) of 5.5 and 83.8 μM, respectively. They also prevented the growth of S. aureus with minimum inhibitory concentration (MIC) of 4 and 32 μg/mL, respectively. Consistent with their inhibition against FabI and bacterial growth, they prevented (14)C]acetate incorporation into fatty acid in S. aureus while didn't affect protein synthesis. In this study, we reported that chalcomoracin and moracin C, potent antibacterial compounds from Morus alba, inhibited FabI and fatty acid synthesis.     

Discovery of 4-Pyridone derivatives as specific inhibitors of enoyl-acyl carrier protein reductase (FabI) with antibacterial activity against Staphylococcus aureus

4-Pyridone derivatives were identified as potent inhibitors of FabI, the enoyl-acyl carrier protein reductase in Escherichia coli and Staphylococcus aureus. 1-Substituted derivatives of a hit compound exhibited potent antibacterial activities against S. aureus. Target specificity of 4-pyridone derivatives was confirmed by the strong inhibition of lipid synthesis in macromolecular biosynthesis assay and also by the reduced antimicrobial activity against triclosan-resistant S. aureus isolates possessing a point mutation (Ala95Val) in FabI. Two 4-pyridone compounds exhibited strong antibacterial activities against 30 clinical isolates of methicillin-resistant S. aureus (MRSA) with MIC(90) of 0.5 and 2 mug/ml, respectively. Moreover, they retained activity against S. aureus with a mutation affecting FabI residue 204, which was recently found to be associated with triclosan resistance in clinical isolates of S. aureus. In conclusion, we have identified a novel chemical series, 4-pyridone derivatives, as specific inhibitors of FabI with potent antibacterial activity against S. aureus.     

Identification of inhibitors of bacterial enoyl-acyl carrier protein reductase

The FabI-related enoyl-ACP reductase enzymes of bacteria meet many of the criteria for antibacterial targets. These enzymes are essential for the growth of several pathogenic species, have no significant mammalian homologs, catalyze a rate-limiting step in a vital macromolecular biosynthetic pathway, and are already the targets of antibacterials used in the clinic (isoniazid) and in consumer products (triclosan). The suitability of FabI as an antibiotic target is diminished somewhat by the discovery that many pathogens carry an alternate unrelated enoyl-ACP reductase (FabK) or both reductases. However, a key human pathogen, Staphylococcus aureus and its increasingly common drug-resistant derivative MRSA are sensitive to FabI inhibitors. Screening for inhibitors of this target has resulted in the identification of five chemical classes of potent inhibitors. In addition, analogs of triclosan with increased potency and with pro-drug features have been engineered. At least one of these classes of inhibitors has been optimized and tested in animals for pharmacokinetic properties and efficacy. Further development of one or more of these classes and further screening are expected to generate new FabI inhibitors for application in the clinic against drug-resistant S. aureus.     

Structural and enzymatic analyses reveal the binding mode of a novel series of Francisella tularensis enoyl reductase (FabI) inhibitors

Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.     

Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching

Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with submicromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented.     

Structure‐based drug design and in vitro testing reveal new inhibitors of enoyl‐acyl carrier protein reductases

The need for new antibacterial agents is increasingly becoming of great importance as bacterial resistance to current drugs is quickly spreading. Enoyl‐acyl carrier protein reductases (FabI) are important enzymes for fatty acid biosynthesis in bacteria and other micro‐organisms. In this project, we conducted structure‐based virtual screening against the FabI enzyme, and accordingly, 37 compounds were selected for experimental testing. Interestingly, five compounds were able to demonstrate antimicrobial effect with variable inhibition activity against various strains of bacteria and fungi. Minimum inhibitory concentrations of the active compounds were determined and showed to be in low to medium micromolar range. Subsequently, enzyme inhibition assay was carried out for our five antimicrobial hits to confirm their biological target and determine their IC₅₀ values. Three of these tested compounds exhibited inhibition activity for the FabI enzyme where our best hit MN02 had an IC₅₀ value of 7.8 μM. Furthermore, MN02 is a small bisphenolic compound that is predicted to have all required features to firmly bind with the target enzyme. To sum up, hits discovered in this work can act as a good starting point for the future development of new and potent antimicrobial agents.     

Phenylimidazole derivatives of 4-pyridone as dual inhibitors of bacterial enoyl-acyl carrier protein reductases FabI and FabK

FabI and FabK are bacterial enoyl-acyl carrier protein (ACP) reductases that catalyze the final and rate-limiting step of bacterial fatty acid biosynthesis (FAS) and are potential targets of novel antibacterial agents. We have reported 4-pyridone derivative 3 as a FabI inhibitor and phenylimidazole derivative 5 as a FabK inhibitor. Here, we will report phenylimidazole derivatives of 4-pyridone as FabI and FabK dual inhibitors based on an iterative medicinal chemistry and crystallographic study of FabK from Streptococcus pneumoniae/compound 26. A representative compound 6 showed strong FabI inhibitory (IC50 = 0.38 microM) and FabK inhibitory (IC50 = 0.0045 microM) activities with potent antibacterial activity against S. pneumoniae (MIC = 0.5 microg/mL). Since elevated MIC value was observed against S. pneumoniae mutant possessing one amino acid substitution in FabK, the antibacterial activity of the compound was considered to be due to the inhibition of FabK. Moreover, this compound showed no significant cytotoxicity (IC50 > 69 microM). These results support compound 6 as a novel agent for the treatment of bacterial infections.     

Identification of borinic esters as inhibitors of bacterial cell growth and bacterial methyltransferases, CcrM and MenH

As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. We report herein a class of boron-containing compounds termed borinic esters that have broad spectrum antibacterial activity with minimum inhibitory concentrations (MIC) in the low microgram/mL range. These compounds were identified by screening for inhibitors against Caulobacter crescentus CcrM, an essential DNA methyltransferase from gram negative alpha-proteobacteria. In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. Our data demonstrate the potential for further development of borinic esters as antibacterial agents as well as leads to explore more specific inhibitors against two essential bacterial enzymes.     

Novel approaches to antimicrobial therapy: peptide deformylase

Peptide deformylase (PDF) represents one of the most exciting new targets for the development of novel antimicrobial chemotherapies. PDF is an essential bacterial metalloenzyme that deformylates the N-formylmethionine of newly synthesized bacterial polypeptides. Recent progress in understanding the structure and function of PDF has greatly facilitated the drug discovery process. In this article, the potential of PDF as an antimicrobial target is reviewed, and progress in the development of PDF inhibitors (PDFIs) is highlighted. Several structural classes of compounds have been reported as inhibitors of PDF. However, the real challenge has been in obtaining molecules with potent in vivo antibacterial activity against a range of drug-resistant pathogens. One of the more encouraging compounds reported, BB-83698 (British Biotech plc/Genesoft Inc), has shown in vivo efficacy against Streptococcus pneumoniae in both mouse thigh and lung infection models at doses equivalent to existing therapies. The published data suggest that PDFIs are a promising new class of antimicrobial agent best suited to treat respiratory tract infections (RTIs), but with the potential for activity against a variety of other pathogens. It is anticipated that the first PDFI targeting RTIs will enter the clinic soon.     

CG400462, a new bacterial enoyl-acyl carrier protein reductase (FabI) inhibitor

CG400462, a novel FabI inhibitor, has a potent antibacterial activity against staphylococci. The minimal inhibitory concentration at which 90% of bacterial strains tested were inhibited (MIC(90)) of CG400462 was 0.5 microg/mL against 238 strains of Staphylococcus aureus and 1.0 microg/mL against 51 strains of coagulase-negative staphylococci, irrespective of whether the strains were methicillin-susceptible or -resistant. CG400462 was also effective by subcutaneous administration against systemic infections in mice. In time-kill studies, CG400462 at concentrations of 1x, 2x and 4x MIC had a bacteriostatic activity over 24h. Genetic approaches have confirmed that the mode of action of CG400462 is via inhibition of FabI, which is involved in the biosynthesis of fatty acids in bacteria. Study of the resistance mechanism of S. aureus showed that CG400462-resistant mutants had an alteration in FabI of Met99-->Thr or Tyr147-->His.     

Potent antimicrobial action of triclosan–lysozyme complex against skin pathogens mediated through drug-targeted delivery mechanism

Triclosan (TCS), an antimicrobial agent that inhibits bacterial fatty acid synthesis by blocking the active site of enoyl-ACP reductase (FabI), is a water-insoluble agent that limits its therapeutic candidacy. We have recently shown that the water solubility and antimicrobial activity of TCS were greatly enhanced when complexed to lysozyme (LZ). This study is to examine the therapeutic potential of triclosan–lysozyme (T–LZ) complex against common skin pathogens expressing different levels of FabI, and to delineate the drug-targeting mechanism by lysozyme. The T–LZ exhibited superior antimicrobial activity against two bacterial skin pathogens, Propionibacterium acnes and Corynebacterium minutissimum, while yeast pathogens, Candida albicans and Malassezia furfur lacking FabI enzyme were insensitive to the complex. Unlike free TCS or LZ, the T–LZ complex exhibited a potent antibacterial activity under a wide range of pH condition and salt concentration. Interestingly, P. acnes expressing greater amount of FabI was more susceptible to the T–LZ complex than C. minutissimum that produces lesser amount of the enzyme. A sensitive assay of FabI activity revealed that P. acnes and C. minutissimum treated with the complex exhibited significant inhibition of the intracellular FabI activity than cells treated with free TCS, indicating the efficiency of lysozyme to specifically deliver TCS to its target (FabI) in the cytoplasm of bacterial cells. These results demonstrate, for the first time, that lysozyme is a potential drug carrier that allows specific targeting to the microbial cells of the water-insoluble triclosan and highlights the potency of the complex for the treatment of skin bacterial infections.     

Antimicrobial activity and phytochemical screening of serial extracts from leaves of Aegle marmelos (Linn.)

The in vitro antimicrobial activity of serial petroleum ether, chloroform and methanol extracts from leaves of Aegle mawmelos were investigated against bacterial and fungal species. All the extracts exhibited broad spectrum antimicrobial activity with zones of inhibition ranging from 10 to 22 mm against bacteria: Staphylococcus aureus, beta Streptococcus haemolyticus group A, Proteus mimrabilis, Klebsiella pneumoniae, Pseudomonas aenrginosa, Escherichia coli, Salmonella typhi, fungi: Candida albicans, Candida tropicalis and Aspergillusflavus. The minimal inhibitory concentrations (MIC) and the minimal microbicidal concentrations (MMC) of the extracts ranged from 1.25 to 10 mg/mL and 2.5 to 20 mg/mL respectively. Assessment of antibacterial efficacy of different extract revealed that Staphylococcus aureus, beta Streptococcus haemolyticus group A, Pseudomonas aeruginosa and Escherichia coli showed high susceptibility to petroleum ether extract. Proteus mimrabilis, Klebsiella pneumoniae showed high susceptibility to chloroform extract and Salmonella typhi showed high susceptibility to methanol extract. Petroleum ether extract exhibited the highest antifungal efficacy against all tested fungal species. Phytochemical screening revealed the presence of phenols, sterols in petroleum ether and chloroform extracts, whereas tannins, flavonoids, coumarins, saponins and triterpenoids in methanol extract. The ability of the leaf extracts of Aegle manmelos to inhibit growth of bacteria and fungi is an indication of its broad spectrum antimicrobial activity which could be a potential source for development of novel bioactive antimicrobial agents.     

Antibacterial activity of wild Xylaria sp. strain R005 (Ascomycetes) against multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa

There is a growing need for new and effective antibiotic agents due to the recent emergence of life-threatening, multidrug-resistant bacterial infections such as methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. In the present study, the antimicrobial potential of mushroom was investigated against multidrug-resistant bacterial strains. The mushroom was identified as Xylaria sp. strain R005 based on the morphological characteristics and confirmed by 18S ribosomal RNA sequence comparisons. The crude ethyl acetate extracts of culture filtrate and fruiting bodies of Xylaria sp. showed significant antibacterial activity against multidrug-resistant S. aureus strains (1-10) and P. aeruginosa strains (1-8). The minimum inhibitory concentration of the ethyl acetate extracts of culture filtrate and fruiting bodies ranged from 225 μg/mL to 625 μg/mL, and 120 μg/mL to 625 μg/mL, respectively, against clinical strains of S. aurues and P. aeruginosa. The synergistic action of extracts of Xylaria sp. with vancomycin and ciprofloxacin was observed against S. aureus strain 6 and P. aeruginosa strain 3, respectively. The fractional inhibitory concentration indices (FICIs) of culture filtrate extract with vancomycin and ciprofloxacin were 0.5 and 0.18, respectively. The FICI of fruiting body extract with vancomycin and ciprofloxacin were 0.5 and 0.375, respectively. These results clearly indicate that the metabolites of culture filtrate and fruiting bodies of Xylaria sp. are the potential source for production of new antimicrobial compounds.     

7-(7-氨甲基-5-氮杂螺［2,4］庚烷-5-基)-1-环丙基-6-氟-8-甲氧基-1,4-二氢-4-氧代喹啉-3-羧酸及其类似物的合成与抗菌作用7-(7-氨甲基-5-氮杂螺［2,4］庚烷-5-基)-1-环丙基-6-氟-8-甲氧基-1,4-二氢-4-氧代喹啉-3-羧酸及其类似物的合成与抗菌作用

目的寻找新的广谱、高效、低毒喹诺酮类抗菌药物。方法设计合成7-(7-氨甲基-5-氮杂螺［2,4］庚烷-5-基)-1-环丙基-6-氟-8-甲氧基-1,4-二氢-4-氧代喹啉-3-羧酸及其类似物,测定其体内外活性。结果共合成了20个新化合物,经¹HNMR,MS和HRMS确证其结构。其中5个目标化合物(22～26)有广谱活性,尤其对革兰氏阳性菌具有很强的活性。其中化合物24对所试的13株革兰氏阳性菌的MIC值均0.03 mg·L^-1,其活性优于对照药克林沙星和加替沙星,对所试的6株革兰氏阴性菌,其活性相当于或低于对照药。结论化合物(22～26)值得进一步评价。     

Bacterial ribosomal subunit assembly is an antibiotic target

A substantial number of antimicrobial agents target some activity of the bacterial ribosome for inhibition. Mechanistic studies and recent structural investigations of the ribosome have identified the binding sites and presumed mechanism of inhibitory activity for some compounds. A second target for many of these antibiotics has recently been examined. Formation of both 30S and 50S ribosomal subunits in bacterial cells is impaired by translational inhibitors. For many antimicrobial agents, inhibition of this target is equivalent to inhibition of translation in preventing cell growth. This review will describe features of this new target including the types of compounds which affect particle assembly and differences in the process in different microorganisms. The characteristics of this new target will be identified and aspects of a model to explain this new inhibitory activity will be explored.     

Nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase with potent antibacterial activity against gram-positive pathogens

Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.     

New drugs for Gram-positive uropathogens

Complicated urinary tract infections (UTIs) are frequent nosocomial infections. The bacterial spectrum encompasses Gram-negative but also Gram-positive pathogens in up to 30-40%. The existing treatment for Gram-positive pathogens is not always optimal. Antimicrobials for the treatment of Gram-positive uropathogens comprise older agents, such as aminopenicillins with or without beta-lactamase inhibitors and vancomycin, as well as newer fluoroquinolones, such as levofloxacin or gatifloxacin. However, resistant bacteria such as vancomycin-resistant enterococci (VRE) or methicillin-resistant Staphylococcus aureus (MRSA) (except vancomycin-resistant) are generally also not susceptible to the fluoroquinolones. Therefore new agents need to be assessed in the treatment of UTI. Daptomycin and linezolid are new antimicrobial agents with good efficacy against Gram-positive uropathogens as shown by their minimal inhibitory concentrations. In a phase II study the urinary bactericidal activity of linezolid versus ciprofloxacin in volunteers showed comparable activity of both drugs against fluoroquinolone susceptible Gram-positive uropathogens, whereas linezolid was also as active against fluoroquinolone resistant ones. The pharmacokinetics and the mode of action of these two antibiotics are discussed together with some clinical data in the context of therapeutic use in patients with complicated UTIs.     

Synthesis and antimicrobial activity of 4H-4-oxoquinolizine derivatives: consequences of structural modification at the C-8 position.

The antibacterial 4H-4-oxoquinolizines were introduced recently to overcome bacterial resistance to fluoroquinolones. They exhibit potent antibacterial activity against Gram-positive, Gram-negative, and anaerobic organisms and are highly active against some quinolone-resistant bacteria including quinolone-resistant MRSA. Preliminary studies indicated that oxoquinolizines possess distinct activity and toxicity profiles as compared with their parent quinolones. In order to develop a potent antibacterial agent with the desired spectrum of activity, good tolerability, and balanced pharmacokinetic profile, we synthesized and evaluated a series of oxoquinolizines with various substituents at the C-8 position. Most compounds tested in this study demonstrated better activity against Gram-positive bacteria than ciprofloxacin and exhibited good susceptibility against ciprofloxacin- and methicillin-resistant S. aureus. While maintaining potent in vitro activity, several compounds showed improved in vivo efficacy over ABT-719 as indicated by the mouse protection test. As an example, the oral ED(50) values for the cis-3-amino-4-methylpiperidine analogue 3ss against S. aureus NCTC 10649M, S. pneumoniae ATCC 6303, and E. coli JUHL were 0. 8, 2.0, and 1.4 mg/kg, compared to 3.0, 10.0, and 8.3 mg/kg for ABT-719. The current study revealed that the steric and electronic environment, conformation, and absolute stereochemistry of the C-8 group are very important to the antibacterial profiles. Structural modifications of the C-8 group provide a useful means to improve the antibacterial activities, physicochemical properties, and pharmacokinetic profiles. Manipulation of the C-8 group also allows us to generate analogues with the desired spectrum of activity, such as analogues that are selective against respiratory pathogens.