ABSTRACT The novel ribosome inhibitors (NRIs) are a broad-spectrum naphthyridine class that selectively inhibits bacterial protein synthesis (P. J. Dandliker et al., Antimicrob. Agents Chemother. 47:3831-3839, 2003). Footprinting experiments, using a range of NRIs and chemical modification agents on Escherichia coli ribosomes, revealed no evidence for direct protection of rRNA. In the presence of tRNA, however, we found that NRIs enhanced the known ribosomal footprinting pattern of tRNA in a dose-dependent manner. The most prominent increase in protection, at A1492/3 and A1413 in helix-44 of 16S RNA, strictly required the presence of tRNA and poly(U), and the effect was correlated with the potency of the inhibitor. Radioligand binding studies with inhibitor [ 3 H]A-424902 showed that the compound binds to tRNA, either in its charged or uncharged form. The dissociation constant for [ 3 H]A-424902 binding to Phe-tRNA Phe was determined to be 1.8 μM, near its translation inhibition potency of 1.6 μM in a cell-free S. pneumoniae extract assay. The compound did not change the binding of radiolabeled tRNA to the 30S ribosomal subunit. Taken together, these results imply that the NRIs exert their effects on protein synthesis by structurally perturbing the tRNA/30S complex at the decoding site.

A novel class of MurF inhibitors was discovered and structure-activity relationship studies have led to several potent compounds with IC(50)=22 approximately 70 nM. Unfortunately, none of these potent MurF inhibitors exhibited significant antibacterial activity even in the presence of bacterial cell permeabilizers.

Structure-activity relationships for a recently discovered novel ribosome inhibitor (NRI) class of antibacterials were investigated. Preliminary efforts to optimize protein synthesis inhibitory activity of the series through modification of positions 3 and 4 of the naphthyridone lead template resulted in the identification of several biochemically potent analogues. A lack of corresponding whole cell antibacterial activity is thought to be a consequence of poor cellular penetration as evidenced by the enhancement of activity observed for a lead analogue tested in the presence of a cell permeabilizing agent.

ABSTRACT ABT-492 demonstrated potent antibacterial activity against most quinolone-susceptible pathogens. The rank order of potency was ABT-492 > trovafloxacin > levofloxacin > ciprofloxacin against quinolone-susceptible staphylococci, streptococci, and enterococci. ABT-492 had activity comparable to those of trovafloxacin, levofloxacin, and ciprofloxacin against seven species of quinolone-susceptible members of the family Enterobacteriaceae, although it was less active than the comparators against Citrobacter freundii and Serratia marcescens. The activity of ABT-492 was greater than those of the comparators against fastidious gram-negative species, including Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae, and Legionella spp. and against Pseudomonas aeruginosa and Helicobacter pylori. ABT-492 was as active as trovafloxacin against Chlamydia trachomatis, indicating good intracellular penetration and antibacterial activity. In particular, ABT-492 was more active than trovafloxacin and levofloxacin against multidrug-resistant Streptococcus pneumoniae, including strains resistant to penicillin and macrolides, and H. influenzae, including β-lactam-resistant strains. It retained greater in vitro activity than the comparators against S. pneumoniae and H. influenzae strains resistant to other quinolones due to amino acid alterations in the quinolone resistance-determining regions of the target topoisomerases. ABT-492 was a potent inhibitor of bacterial topoisomerases, and unlike the comparators, DNA gyrase and topoisomerase IV from either Staphylococcus aureus or Escherichia coli were almost equally sensitive to ABT-492. The profile of ABT-492 suggested that it may be a useful agent for the treatment of community-acquired respiratory tract infections, as well as infections of the urinary tract, bloodstream, and skin and skin structure and nosocomial lung infections.

ABSTRACT We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae , Haemophilus influenzae , Staphylococcus aureus , and Moraxella catarrhalis , and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens.

Several naturally occurring coumarins previously found to be potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons (PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide-N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.

Several naturally occurring coumarins to which humans are routinely exposed have been previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872-879 and Cai et al. (1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were investigated for their inhibitory effects on the metabolism and metabolic activation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols). However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and syn-diol-epoxide-DNA adducts using immobilized boronate chromatography. The current study demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current data also suggest that certain naturally occurring coumarins may possess anticarcinogenic activity toward polycyclic aromatic hydrocarbons.

Several naturally occurring coumarins contained in the human diet have been found to be effective inhibitors and inactivators of murine hepatic ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase in vitro [Cai, Y., Bennett, D., Nair, R.V., Ceska, O., Ashwood-Smith, M., and DiGiovanni, J. (1993) Chem. Res. Toxicol. 6, 872-879]. In the present study, these same coumarins decreased the content of cytochrome P450 (P450) in either 3-methylcholanthrene (MC)- or phenobarbital-induced murine hepatic microsomes but did not have a major effect on heme content. Detailed in vitro studies with [14C]coriandrin, which selectively inhibits and inactivates P450 1A1-mediated EROD activity, demonstrated that it covalently bound, in a preferential manner, to hepatic microsomal protein from MC-pretreated mice. A linear relationship was observed between covalent binding and loss of EROD activity. The inclusion of electrophile trapping agents in the incubations significantly inhibited the covalent binding of [14C]coriandrin to microsomal protein. In addition, the covalent binding of [14C]coriandrin was decreased 46% by 7,8-benzoflavone (7,8-BF), 58% by a monoclonal antibody with specificity toward MC-induced form(s) of P450, and 60% by ethoxyresorufin, implicating the bioactivation of coriandrin by P450 1A1. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [14C]coriandrin-bound microsomal protein from MC-pretreated mice showed that [14C]coriandrin bound covalently to a protein with an approximate molecular mass of 49 kDa. Again, addition of 7,8-BF or polyclonal antibody against P450 1A1 reduced the covalent binding of [14C]coriandrin to this specific protein band. Interestingly, coriandrin was also found to be a potent inhibitor and inactivator of purified human P450 1A1. These results demonstrate that certain coumarins to which humans are exposed in the diet are bioactivated by P450 1A1 to reactive intermediates that subsequently form covalent adducts with the apoprotein, effectively destroying enzyme activity. Thus, certain naturally occurring coumarins may have a significant effect on human health.

The present study was designed to evaluate the effects of a series of natural coumarins on ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities in vitro using hepatic tissues from SENCAR mice. Fifteen different coumarins were examined for potential modulating activities. Several naturally occurring coumarins, found in the human diet, were effective inhibitors of hepatic EROD activity in vitro, including coriandrin, bergamottin, isoimperatorin, and ostruthin. Notably, coriandrin and bergamottin were approximately as potent as 7,8-benzoflavone, a relatively selective inhibitor of cytochrome P450 1A1. Several naturally occurring coumarins were also potent inhibitors of hepatic PROD activity, including imperatorin, bergamottin, isopimpinellin, and angelicin. Kinetic studies of the type of inhibition revealed that these compounds inhibited hepatic EROD and PROD activity by a variety of modes rather than by a uniform one. Furthermore, experiments using a two-stage incubation assay revealed that coriandrin, imperatorin, ostruthin, and several other natural coumarins inactivated hepatic EROD activity (i.e., predominantly cytochrome P450 1A1-mediated) and that isopimpinellin inactivated hepatic PROD activity (i.e., predominantly cytochrome P450 2B1-mediated). Finally, the results indicate that some coumarins had selective inhibitory effects for EROD vs PROD and preliminary analyses suggested a possible structural basis for the observed differences. The current data suggest that certain naturally occurring coumarins, to which humans are exposed in the diet, are potent modulators of cytochrome P450. Furthermore, these compounds may be capable of influencing the metabolic activation of other xenobiotics, including chemical carcinogens.