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The echinocandins are a class of semisynthetic natural products that target β-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibited in vitro activity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GS in vitro , and there was a strong correlation between enzyme inhibition and in vitro antifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants of Saccharomyces cerevisiae with reduced susceptibility to the piperazinyl-pyridazinones had substitutions in FKS1 . The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model of Candida glabrata infection.

A novel series of pyridazinone analogs has been developed as potent β-1,3-glucan synthase inhibitors through structure-activity relationship study of the lead 5-[4-(benzylsulfonyl)piperazin-1-yl]-4-morpholino-2-phenyl-pyridazin-3(2H)-one (1). The effect of changes to the core structure is described in detail. Optimization of the sulfonamide moiety led to the identification of important compounds with much improved systematic exposure while retaining good antifungal activity against the fungal strains Candida glabrata and Candida albicans.

A structure-activity relationship study of the lead 5-[4-(benzylsulfonyl)piperazin-1-yl]-4-morpholino-2-phenyl-pyridazin-3(2H)-one 1 has resulted in the identification of 2-(3,5-difluorophenyl)-4-(3-fluorocyclopentyloxy)-5-[4-(isopropylsulfonyl)piperazin-1-yl]-pyridazin-3(2H)-one 11c as a β-1,3-glucan synthase inhibitor. Compound 11c exhibited significant efficacy in an in vivo mouse model of Candida glabrata infection.

Antibiotic resistant hospital acquired infections are on the rise, creating an urgent need for novel bactericidal drugs. Enzymes involved in lipopolysaccharide (LPS) biosynthesis are attractive antibacterial targets since LPS is the major structural component of the outer membrane of Gram-negative bacteria. Lipid A is an essential hydrophobic anchor of LPS and the first committed step in lipid A biosynthesis is catalyzed by a unique zinc dependent metalloamidase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC is an attractive Gram-negative only target that has been chemically validated by potent bactericidal hydroxamate inhibitors that work by coordination of the enzyme's catalytic zinc ion. An exploratory chemistry effort focused on expanding the SAR around hydroxamic acid zinc-binding 'warheads' lead to the identification of novel compounds with enzyme potency and antibacterial activity similar to CHIR-090.

We compared the in vitro activities of posaconazole, voriconazole, itraconazole, and amphotericin B against clinical isolates of Aspergillus spp. and Rhizopus spp., and explored the in vitro interaction between posaconazole and amphotericin B against Rhizopus spp. Clinical strains of 82 Aspergillus spp. (43 Aspergillus fumigatus, 29 A. flavus, 7 A. niger, 2 A. terreus, 1 A. nidulans) and 11 Rhizopus oryzae isolates were tested in accordance with CLSI M38-A microdilution guidelines. In vitro activity of posaconazole against Aspergillus spp. was also investigated with the Etest. The combination of posaconazole and amphotericin B against R. oryzae isolates was investigated by the checkerboard methodology. Voriconazole was the most active drug in vitro against Aspergillus spp., followed by posaconazole, itraconazole, and amphotericin B, in order of decreasing activity. In studies with R. oryzae isolates, posaconazole was found to be the most potent drug followed by itraconazole and amphotericin B. Voriconazole had no meaningful activity against Rhizopus. Posaconazole Etest MICs (microg/ml) with Aspergillus spp. were found to be considerably lower than those obtained with the CLSI microdilution method (4-9 and 3-7 two-fold lower than CLSI MICs at 24 and 48 h, respectively). The interaction between posaconazole and amphotericin B was indifferent for all R. oryzae isolates tested; importantly no antagonism was observed.

We evaluated the in vitro activity of posaconazole against nine Candida species using minimum fungicidal concentration (MFC) measurements and time-kill methods.MFCs of posaconazole were determined for 209 clinical isolates (32 Candida albicans, 30 Candida glabrata, 21 Candida tropicalis, 29 Candida krusei, 28 Candida parapsilosis sensu stricto, 50 Candida inconspicua, 13 Candida kefyr, 3 Candida lusitaniae and 3 Candida guilliermondii) and 7 ATCC Candida strains. The following strains were tested in time-kill studies: 3 strains each of C. glabrata, C. kefyr, C. guilliermondii and C. lusitaniae; 2 C. tropicalis; 4 C. albicans; 4 C. inconspicua; 9 C. krusei; 12 C. parapsilosis; and 7 ATCC strains.Posaconazole was fungicidal in both MFC and time-kill experiments (at 2 mg/L within 48 h in time-kill assays) against each C. krusei, C. inconspicua and C. lusitaniae strain and was fungistatic against each C. albicans, C. glabrata, C. tropicalis and C. guilliermondii strain. For the C. parapsilosis strains, posaconazole MFCs wereor=2 mg/L for 20 of the 28 isolates tested; however, in the time-kill tests, even at drug concentrations of 32-64 x MIC, posaconazole was fungistatic against all C. parapsilosis strains tested.MFC values were good predictors of the fungistatic or fungicidal activity of posaconazole, as determined by time-kill curves, for all tested species except C. parapsilosis. Our results highlight the fungicidal action of posaconazole against a number of clinically relevant Candida species.

Objectives: To determine the activity of newer triazoles against strains of Histoplasma capsulatum resistant to fluconazole. Methods: Susceptibility testing was performed on 17 paired pre- and post-treatment H. capsulatum isolates from patients with AIDS who failed fluconazole. Results: The median MICs of fluconazole, voriconazole, and posaconazole and ravuconazole for the pre-treatment isolates were 1 mg/L, 0.015 mg/L and

Two clinical Candida albicans isolates that exhibited high-level resistance to azoles and modest decreases in susceptibility to amphotericin B were cultured from unrelated patients. Both isolates harbored homozygous nonsense mutations in ERG3 , which encodes an enzyme, sterol Δ 5,6 -desaturase, involved in ergosterol synthesis. Extraction and analysis of the sterols from both isolates confirmed the absence of sterol Δ 5,6 -desaturase activity. Although the loss of sterol Δ 5,6 -desaturase activity is known to confer resistance to azoles, this mechanism of resistance has rarely been seen in clinical isolates, suggesting that such mutants are at a competitive disadvantage. To test this hypothesis, the virulence of the erg3 mutants was assayed by using a mouse systemic infection model. The mutants were significantly less virulent than the wild-type comparator strains. However, the kidney fungal burdens in mice infected with the erg3 mutants were similar to those in mice infected with the wild-type strains. Similar results were obtained by using a laboratory-generated homozygous erg3 deletion mutant (D. Sanglard et al., Antimicrob. Agents Chemother. 47:2404-2412, 2003). Reintroduction of a wild-type ERG3 allele into the homozygous deletion mutant restored virulence, ergosterol synthesis, and susceptibility to azoles, confirming that these phenotypic changes were solely due to the inactivation of Erg3p.

Real-time quantitative PCR was used to measure expression levels of genes encoding efflux pumps, ERG11 and two control genes, ACT1 and PMA1 , in a collection of 14 fluconazole-susceptible Candida albicans isolates. For each gene, average expression levels and variations within the population were determined. These values were then used as reference points to make predictions about the molecular basis of resistance in 38 clinical isolates (the majority of which were resistant to fluconazole) obtained from 18 patients treated with posaconazole for refractory oropharyngeal candidiasis. For each of the 38 isolates, the expression levels of genes encoding efflux pumps, ERG11 and the control genes, were measured as above. Comparison of the two data sets revealed that expression of ACT1 and PMA1 did not vary significantly between the two sets of isolates. In contrast, MDR1, ERG11 , CDR1 , and CDR2 were overexpressed in 3, 4, 14, and 35, respectively, of the isolates from patients treated with azoles. In addition to these changes, the patient isolates all had at least one and often multiple missense mutations in ERG11 . Select ERG11 alleles were expressed in Saccharomyces cerevisiae ; all of the alleles tested conferred reduced susceptibility to fluconazole. Despite both the increases in pump expression and the ERG11 mutations, only one of the patient isolates exhibited a large decrease in posaconazole susceptibility.

OBJECTIVES To characterize the molecular mechanisms responsible for reduced susceptibility to azoles in Candida albicans clinical isolates. MATERIALS AND METHODS Seven sequential C. albicans isolates were cultured from an AIDS patient treated with posaconazole for refractory oropharyngeal candidiasis. Expression levels of the CDR1, CDR2 and MDR1 genes, encoding efflux pumps previously implicated in azole resistance, and ERG11, encoding the azole target site, were monitored using northern blot and real-time PCR. The ERG11 genes from all seven isolates were sequenced. RESULTS The seven closely related isolates exhibited significant decreases in susceptibility to fluconazole (MIC >or= 32 mg/L) and voriconazole (MIC >or= 2 mg/L) and progressive decreases in susceptibility to both posaconazole (isolates 1-4 MIC 0.25 mg/L, isolates 5-7 MIC 2 mg/L) and itraconazole (isolates 1-4 MIC 1 mg/L, isolates 5-7 MIC > 8 mg/L). None of the isolates exhibited any significant changes in the expression levels of ERG11 or the efflux pump genes. All seven isolates had multiple mutations in ERG11; isolates one through four each had five missense mutations; four of the resultant amino acid changes were previously associated with azole resistance. The fifth isolate had an additional novel mutation in one copy of ERG11, resulting in a Pro-230 to Leu substitution. This mutation was present in both ERG11 genes in the last two isolates. Select ERG11 genes were expressed in Saccharomyces cerevisiae, the ERG11 allele with all six mutations conferred the highest level of posaconazole resistance. CONCLUSIONS Multiple mutations in ERG11 are required to confer decreased susceptibility to posaconazole.

Everninomicins are a class of oligosaccharide antibiotics isolated from Micromonospora carbonaceae (25). One such compound, evernimicin ({"type":"entrez-protein","attrs":{"text":"SCH27899","term_id":"1052962409"}}SCH27899), has been evaluated as a therapeutic agent (Ziracin) (6). Evernimicin has been shown to have potent activity against many gram-positive bacteria, including vancomycin-resistant enterococci, methicillin-resistant staphylococci, and penicillin-resistant pneumococci (9). In fact, there were no staphylococcal, enterococcal, or pneumococcal isolates that displayed resistance to evernimicin in either the investigation by Jones and Barrett (9) or a more recent worldwide survey of clinical isolates, including isolates known to be resistant to other antibiotics (R. S. Hare, F. J. Sabatelli, and The Ziracin Susceptibility Testing Group, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., Abstr. E-119, p. 204, 1998). The lack of cross-resistance to evernimicin suggests that the mechanism of action is novel, and prior selection for resistance to antimicrobial agents currently in clinical use will not impact the efficacy of evernimicin. Recently, in Streptococcus pneumoniae, we have shown that mutations in ribosomal protein L16 (encoded by rplP) cause a reduced susceptibility to evernimicin and that evernimicin has an inhibitory effect on in vivo protein synthesis (1). Another study demonstrated evernimicin-mediated inhibition of protein synthesis in cell-free assays derived from Escherichia coli and Staphylococcus aureus (13). Furthermore, radiolabeled evernimicin was found to bind the large ribosomal subunit at a single, unique high-affinity site (13). The rrn operon is unique because unlike most chromosomal genes, which are present in a single copy, rrn operons are typically present as multiple loci in the genome. Therefore, rrn operon mutations that confer resistance to protein synthesis inhibitors are typically recessive or weakly codominant since the majority of the ribosomes must harbor the mutant allele for normal growth to occur in the presence of the inhibitor (19). Consequently, selectable spontaneous mutations in rrn loci in organisms with a high copy number of rrn operons are rare and have not had a clinical impact. Alternative resistance mechanisms, such as alterations to the ribosomal proteins (5), ribosome modification or protection (e.g., rRNA methylation [26]), and drug metabolism or efflux, are more clinically relevant. In contrast, ribosomal DNA (rDNA) mutations resulting in resistance to antimicrobial agents occur more frequently in bacteria that harbor a single rrn operon (e.g., Mycobacterium abscessus, Mycobacterium chelonae [24], Mycobacterium avium [14], and Halobacterium halobium [12]) or two rrn operons (e.g., Helicobacter pylori [23] and Mycobacterium smegmatis [17]). However, even in organisms with two rrn operons, the mode of action of the drug can influence whether a heterogeneous rRNA population confers resistance. Heterogeneous rrn alleles of M. smegmatis conferring erythromycin resistance appear to be a dominant phenotype (18), whereas heterogeneous rrn alleles conferring aminoglycoside resistance appear as a recessive phenotype (17). In a strain of Staphylococcus aureus that harbored an additional rplP allele on a plasmid, mutations to L16 which conferred evernimicin resistance were recessive (unpublished data). In this paper, we report the isolation and characterization of rDNA mutations in S. pneumoniae that confer reduced susceptibility to evernimicin. The locations of the mutations within the 23S rRNA have not been found to confer resistance to other protein synthesis inhibitors. If the positions of the mutations in the rRNA are considered to be potential points of contact with evernimicin or specific modifiers of the ribosomal structure required to interact with evernimicin, then the sites of interaction between evernimicin and the 50S ribosomal subunit appear to be unique. Identification of the unique sites of interaction may help to clarify the detailed mechanism of action of evernimicin.

Evernimicin (SCH 27899) is a new antibiotic with activity against a wide spectrum of gram-positive bacteria and activity against some gram-negative bacteria. Previous metabolic labeling studies indicated that evernimicin specifically inhibited protein synthesis in Staphylococcus aureus . Using a susceptible Escherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli . In cell-free translation assays with extracts from either E. coli or S. aureus , evernimicin had a 50% inhibitory concentration of approximately 125 nM. In contrast, cell-free systems derived from wheat germ and rabbit reticulocytes were inhibited only by very high levels of evernimicin. Evernimicin did not promote transcript misreading. [ 14 C]evernimicin specifically bound to the 50S subunit from E. coli . Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli and S. aureus and 50S subunits from E. coli returned dissociation constants of 84, 86, and 160 nM, respectively. In binding experiments, performed in the presence of excess quantities of a selection of antibiotics known to bind to the 50S subunit, only the structurally similar drug avilamycin blocked binding of [ 14 C]evernimicin to ribosomes.

The Escherichia coli molybdate transporter, encoded by the modABCD operon, is negatively regulated by the modE gene product in response to the intracellular molybdate concentration. Utilizing an in vivo titration assay, we localized the ModE-binding site to the start of modA transcription. This localization was further characterized using in vitro gel-shift assays and DNase I footprinting. ModE bound the wild-type modA promoter with an apparent dissociation constant (Kd) of 45 nM, and addition of molybdate, in physiologically relevant amounts, significantly increased DNA binding. Consistent with these data, modA promoter fragments containing mutations that reduced ModE repression in vivo displayed proportionately higher apparent Kd values in vitro. DNase I footprinting of the modA promoter revealed a single protected region that overlapped the start site of transcription and extended from position -18 to +10, relative to the transcript start site. Gel-shifting assays, employing the promoter regions from the tor, nrf, moa and moe operons, revealed that ModE bound only the moa promoter region, with an apparent Kd of 24nM. Footprint analysis of the moaA promoter revealed a single protected region located immediately upstream of the putative -35 consensus sequence and extending from position -202 to -174, relative to the start of translation. In vivo expression of a moaA-lacZ operon fusion was stimulated twofold by ModE. However, relative to modA, binding of ModE to the moaA promoter appeared to be largely molybdate independent both in vitro and in vivo. These findings demonstrate that ModE acts both as a repressor and activator of the mod and moa operons, respectively, depending on the properties of the binding site.

The structure–activity relationship studies of a novel sulfonylurea series of piperazine pyridazine-based small molecule glucan synthase inhibitors is described. The optimization of PK profiles within the series led to the discovery of several compounds with improved pharmacokinetic profiles which demonstrated in vitro potency against clinically relevant strains. However, the advancement of compounds from this series into a non-lethal systemic fungal infection model failed to show in vivo efficacy.

A detailed structure–activity relationship study of a novel series of pyridazine-based small molecule glucan synthase inhibitors is described. The optimization of the PK profile of this series led to the discovery of compound 11g, which demonstrated in vivo potency ip in a lethal fungal infection model.

Background Vicriviroc (VCV), a small-molecule antagonist of the C–C chemokine receptor 5 (CCR5), blocks HIV's entry into CD4+ cells. Small studies have suggested that resistance to CCR5 antagonists is slow to develop. Objectives To examine resistance to VCV in isolates from treatment experienced patients who experienced virologic failure in two phase 3 trials. Study design Genotypic and phenotypic susceptibility to VCV, and other antiretroviral drugs were evaluated at baseline and at defined intervals during the study. In a post hoc analysis, viral tropism at baseline was evaluated using the Trofile-ES assay. Only subjects with R5-tropic virus were included in the analysis. Viral envelope sequencing was performed on samples from subjects with emergent VCV resistance defined using a relative MPI cutoff. Results 71/486 subjects treated with VCV for 48 weeks met the protocol-defined virologic failure criteria. 7/71 (10%) had DM/X4 virus at the time of virologic failure; VCV resistance was identified in 4/486 treated subjects (1%). No control subject had detectable DM/X4 virus or VCV resistance at virologic failure. Clonal analysis of envelope sequences from VCV-resistant virus identified 2–5 amino acid substitutions at or near the crown of the V3 loop; however, no signature V3 mutations were identified. Changes outside the V3 loop were also observed in resistant clones; no consistent variant pattern was observed. Conclusions In these trials, use of a sensitive tropism assay and potent antiretroviral drug combinations contributed to the infrequent detection of X4-tropic virus and VCV resistance. Substitutions in the V3 loop were associated with VCV resistance, however, no specific pattern of amino acid changes were sufficient to reliably predict VCV susceptibility.

Objectives: In the phase 2 VICTOR-E1 study, treatment-experienced subjects receiving 20 mg or 30 mg of the CCR5 antagonist vicriviroc (VCV), with a boosted protease containing optimized background regimen, experienced significantly greater reductions in HIV-1 viral load compared with control subjects. Among the 79 VCV-treated subjects, 15 experienced virologic failure, and of these 5 had VCV-resistant virus. This study investigated the molecular basis for the changes in susceptibility to VCV in these subjects. Methods: Sequence analysis and phenotypic susceptibility testing was performed on envelope clones from VCV-resistant virus. For select clones, an exchange of mutations in the V3 loop was performed between phenotypically resistant clones and the corresponding susceptible clones. Results: Phenotypic resistance was manifest by reductions in the maximum percent inhibition. Clonal analysis of envelopes from the 5 subjects identified multiple amino acid changes in gp160 that were exclusive to the resistant clones, however, none of the changes were conserved between subjects. Introduction of V3 loop substitutions from the resistant clones into the matched susceptible clones was not sufficient to reproduce the resistant phenotype. Likewise, changing the substitutions in the V3 loops from resistant clones to match susceptible clones only restored susceptibility in 1 clone. Conclusions: There were no clearly conserved patterns of mutations in gpl60 associated with phenotypic resistance to VCV and mutations both within and outside of the V3 loop contributed to the resistance phenotype. These data suggest that genotypic tests for VCV susceptibility may require larger training sets and additional information beyond V3 sequences.

Background. Vicriviroc is a C-C motif chemokine receptor 5 (CCR5) antagonist that is in clinical development for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. This study explored the molecular basis for the development of phenotypically resistant virus. Method. HIV-1 RNA from treatment-naive subjects who experienced virological failure in a phase 2 dose-finding trial was evaluated for coreceptor usage and susceptibility. For viruses that exhibited reduced susceptibility to vicriviroc, envelope clones were phenotypically and genotypically characterized. Results. Twenty-six vicriviroc-treated subjects experienced virological failure; for 24 the virus remained CCR5-tropic, and 2 had dual/X4 virus. Reduced susceptibility to vicriviroc, manifested as decreases in the maximum percent inhibition value (no increase in median inhibitory concentration), was detected in 4 of the 26 subjects who experienced virological failure. Clonal analysis of envelopes in samples from these 4 subjects revealed multiple sequence changes in gp160, principally within the variable domain 1/variable domain 2, variable domain 3, and variable domain 4 loops. However, no consistent pattern of mutations was observed across subjects. Conclusions. In this study, only a small proportion of treatment failures were associated with tropism changes or reduced susceptibility to vicriviroc. Genotypic analysis of cloned env sequences revealed no specific mutational pattern associated with reduced susceptibility to vicriviroc, although numerous changes were observed in the variable domain 3 loop and in other regions of gp160.

Two large studies compared posaconazole and fluconazole or itraconazole for prophylaxis in subjects undergoing allogeneic hematopoietic stem cell transplantation or subjects with acute myelogenous leukemia. To assess the impact of prophylaxis on colonization and the development of resistance in Saccharomyces yeasts, identification and susceptibility testing were performed with yeasts cultured at regular intervals from mouth, throat, and stool samples. Prior to therapy, 34 to 50% of the subjects were colonized with yeasts. For all three drugs, the number of positive Candida albicans cultures decreased during drug therapy. In contrast, the proportion of subjects with positive C. glabrata cultures increased by two- and fourfold in the posaconazole and itraconazole arms, respectively. Likewise, in the fluconazole arm the proportion of subjects with positive C. krusei cultures increased twofold. C. glabrata was the species that most frequently exhibited decreases in susceptibility, and this trend did not differ significantly between the prophylactic regimens. For the subset of subjects from whom colonizing C. glabrata isolates were recovered at the baseline and the end of treatment, approximately 40% of the isolates exhibited more than fourfold increases in MICs during therapy. Molecular typing of the C. albicans and C. glabrata isolates confirmed that the majority of the baseline and end-of-treatment isolates were closely related, suggesting that they were persistent colonizers and not newly acquired. Overall breakthrough infections by Candida species were very rare (∼1%), and C. glabrata was the colonizing species that was the most frequently associated with breakthrough infections.

Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.

Sensititre YeastOne colorimetric antifungal panels were compared with the CLSI (formerly NCCLS) M38-A reference method for testing the susceptibility of filamentous fungi to posaconazole; agreement (±2 log 2 dilutions) between the two methods was 97%. These data confirm the utility of YeastOne panels for measuring the susceptibility of filamentous fungi to posaconazole.

Posaconazole and itraconazole were more potent inhibitors of ergosterol synthesis, in both intact cells and cell extracts from Absidia corymbifera and Rhizopus oryzae , than voriconazole and fluconazole. Similarly, expression of CYP51 from R. oryzae in Saccharomyces cerevisiae significantly increased resistance to fluconazole and voriconazole but not to posaconazole and itraconazole.

The in vitro activity of the novel triazole antifungal agent posaconazole (Noxafil; SCH 56592) was assessed in 45 laboratories against approximately 19,000 clinically important strains of yeasts and molds. The activity of posaconazole was compared with those of itraconazole, fluconazole, voriconazole, and amphotericin B against subsets of the isolates. Strains were tested utilizing Clinical and Laboratory Standards Institute broth microdilution methods using RPMI 1640 medium (except for amphotericin B, which was frequently tested in antibiotic medium 3). MICs were determined at the recommended endpoints and time intervals. Against all fungi in the database (22,850 MICs), the MIC 50 and MIC 90 values for posaconazole were 0.063 μg/ml and 1 μg/ml, respectively. MIC 90 values against all yeasts (18,351 MICs) and molds (4,499 MICs) were both 1 μg/ml. In comparative studies against subsets of the isolates, posaconazole was more active than, or within 1 dilution of, the comparator drugs itraconazole, fluconazole, voriconazole, and amphotericin B against approximately 7,000 isolates of Candida and Cryptococcus spp. Against all molds (1,702 MICs, including 1,423 MICs for Aspergillus isolates), posaconazole was more active than or equal to the comparator drugs in almost every category. Posaconazole was active against isolates of Candida and Aspergillus spp. that exhibit resistance to fluconazole, voriconazole, and amphotericin B and was much more active than the other triazoles against zygomycetes. Posaconazole exhibited potent antifungal activity against a wide variety of clinically important fungal pathogens and was frequently more active than other azoles and amphotericin B.

Objectives: The in vitro antifungal activity of posaconazole was compared with that of fluconazole and amphotericin B. Materials and methods: A microdilution method (M27-A2) was used with 331 clinical yeast isolates. Results: The geometric mean MICs of posaconazole, fluconazole and amphotericin B were 0.16, 0.91 and 0.15 mg/L, respectively. Posaconazole was markedly more active than fluconazole and was active against 9/11 fluconazole-resistant Candida albicans, and five Candida glabrata had an MIC of posaconazole of 16 mg/L. Conclusions: These data indicate that posaconazole is a potentially effective antifungal agent for the treatment of mycoses caused by yeasts.

Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata . In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata , Aspergillus fumigatus , and Aspergillus flavus . Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14α-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14α-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14α-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.

The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole. Mutations in and around the CYP51 active site have resulted in azole resistance. In this work, homology models of the CYP51 enzymes from Aspergillus fumigatus and Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis . Using these models, binding modes for voriconazole (VOR), fluconazole (FLZ), itraconazole (ITZ), and posaconazole (POS) were predicted from docking calculations. Previous work had demonstrated that mutations in the vicinity of the heme cofactor had a greater impact on the binding of FLZ and VOR than on the binding of POS and ITZ. Our modeling data suggest that the long side chains of POS and ITZ occupy a specific channel within CYP51 and that this additional interaction, which is not available to VOR and FLZ, serves to stabilize the binding of these azoles to the mutated CYP51 proteins. The model also predicts that mutations that were previously shown to specifically impact POS susceptibility in A. fumigatus and C. albicans act by interfering with the binding of the long side chain.

The last decade has seen a dramatic increase in serious fungal infections. Those primarily at risk are immunocompromised individuals. Up to 80% of human immunodeficiency virus-infected patients are thought to develop a fungal infection at some point in the course of their illness (21). Aspergillus fumigatus, originally viewed as a weak pathogen, is now a major cause of death at cancer centers, with the mortality rate for leukemia patients approaching 90% even with antifungal therapy (12). Neither the current standards, amphotericin B (AMB) and itraconazole (ITZ), nor new therapies, caspofungin (CAS) and voriconazole (VOR), have satisfactorily met the therapeutic needs of patients with aspergillosis. Clinical failures with AMB are common, but correlating such failures with resistance is often difficult (19). Mechanism-based toxicity has limited the usefulness of AMB (12); newer less-toxic formulations are now available but are expensive. Isolates resistant to ITZ have been obtained from both in vivo (5, 7, 8) and in vitro sources (14, 15). VOR-resistant isolates have been generated in the laboratory (16; E. K. Manavathu, I. Baskaran, G. J. Alangaden, and P. H. Chandrasekar, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. J-817, 2001), but there have been no reports of in vivo resistance. Finally, there are insufficient data available on CAS to assess development of resistance. Posaconazole (POS; SCH56592), a novel triazole currently in phase 3 clinical trials, is active against a broad spectrum of fungal pathogens including Aspergillus and Candida spp. (4, 10, 15). In vitro and in vivo testing has demonstrated that the drug is more effective than either ITZ or AMB against Aspergillus spp. (15, 25, 29) and that it is more effective than fluconazole (FLZ) against Candida spp. (4). In this study we characterized isolates of A. fumigatus, both clinical and in vitro laboratory-selected mutants, which exhibit a reduced susceptibility to POS. We demonstrate that resistance is caused by mutations in the cyp51A gene, which encodes the target protein for POS. (Part of this work was presented at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., September 2002.)

Six high-level evernimicin-resistant Enterococcus faecium isolates were identified among 304 avilamycin-resistant E . faecium isolates from animals and 404 stool samples from humans with diarrhea. All four animal isolates, and one of the human isolates, were able to transfer resistance to a susceptible E . faecium strain. The resulting transconjugants all tested positive for the presence of emtA , a gene encoding a methyltransferase previously linked with high-level evernimicin resistance. The four transconjugants derived from animal isolates all carried the same plasmid, while a differently sized plasmid was found in the isolate from humans. This study demonstrated a low incidence of high-level evernimicin resistance mediated by the emtA gene in different E . faecium isolates of animal and human origin.

Molybdenum is an essential component of the molybdopterin cofactor in nearly all species, including bacteria, plants, and animals, where it is located at the active center of a certain oxidoreductases including nitrate reductase, dimethyl sulfoxide (DMSO) reductase, trimethylamine-N-oxide (TMAO) reductase, and biotin sulfoxide reductase. Escherichia coli has evolved a regulatory scheme to coordinate molybdenum uptake, in the form of molybdate, which is utilized for cofactor synthesis and assembly into the mature molybdoenzymes. The key regulatory element in this scheme is the ModE protein, a molybdate-responsive transcription factor the structure of which was recently determined (14). ModE was first identified in E. coli as the negative regulator of the high-affinity molybdate uptake system, encoded by the modABCD operon (11, 23, 33). Utilizing a combination of in vivo and in vitro approaches, ModE was shown to bind the modA promoter in a molybdate-dependent fashion (2, 11, 23). ModE also binds and regulates expression of the moaADCDE and dmsABC operons, which encode enzymes involved in the first steps of molybdate assimilation into molybdopterin and the DMSO reductase, respectively (1, 20, 21, 23). Recently ModE was shown to play a minor role in regulating the hyc and nar operons in E. coli (29). Finally, ModE orthologues have been identified in a wide number of bacteria, including Azotobacter vinelandii, Rhodobacter capsulatus, Ralstonia eutropha (Alcaligenes eutrophus), and Thiosphaera pantotropha (Paracoccus denitrificans) (reviewed in reference 13). ModE has been shown to regulate various molybdate-associated operons in several of these cases (18, 24, 32). In this study, we examine the role of ModE in regulating the expression of the napFDAGHBC operon of E. coli. Sequence and biochemical analysis indicates this operon encodes a molybdenum-containing periplasmic nitrate reductase (10). Expression of the E. coli napFDAGHBC operon is positively regulated in response to anaerobiosis by Fnr and by the presence of nitrate and/or nitrite by NarP (5, 7, 26, 34). Here we show that in the absence of ModE, expression of the napF operon in response to both nitrate and nitrite is dramatically reduced. A ModE site was identified well upstream of the Fnr and NarP binding sites at the napF promoter, and we demonstrate that ModE binds this site in a molybdate-responsive manner to thereby coordinate enzyme synthesis with molybdate availability. We further demonstrate that by deleting the region containing the ModE binding site or by replacing modE+ with a molybdate-independent modE allele (modE*), induction of napF-lacZ expression in response to nitrate is rendered molybdate independent.

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro , suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Chemical mutagenesis of Staphylococcus aureus RN450 generated two strains that displayed a stable reduction (30- to 60-fold) in susceptibility to evernimicin. Cell-free translation reactions demonstrated that the resistance determinant was located in the ribosomal fraction. Compared to ribosomes isolated from a wild-type strain, ribosomes from the mutant strains displayed an 8- to 10-fold reduction in affinity for [ 14 C]evernimicin. In contrast, the mutants displayed no alteration in either binding affinity or in vitro susceptibility to erythromycin. Exponential cultures of the mutant strains accumulated significantly less [ 14 C]evernimicin than the wild-type strain, suggesting that accumulation is dependent on the high affinity that evernimicin displays for its binding site. Sequencing rplP (encodes ribosomal protein L16) in the mutant strains revealed a single base change in each strain, which resulted in a substitution of either cysteine or histidine for arginine at residue 51. Introduction of a multicopy plasmid carrying wild-type rplP into the mutant strains restored sensitivity to evernimicin, confirming that the alterations in rplP were responsible for the change in susceptibility. Overexpression of the mutant alleles in S. aureus RN450 had no effect on susceptibility to evernimicin, demonstrating that susceptibility is dominant over resistance.

The product of the Escherichia coli modE gene, ModE, is a member of a unique class of molybdate-responsive DNA binding proteins. Here we investigated the roles of the N- and C-terminal domains of ModE in mediating DNA binding and protein dimerization, respectively. Compared to the full-length protein, the N-terminal half of ModE has a greatly diminished capacity to bind the modA promoter in vitro and to repress expression from a modA-lacZ operon fusion in vivo. Fusing a protein dimerization domain, encoded by the C terminus of λ CI repressor protein, to the truncated ModE protein generated a ModE-CI fusion protein that not only displayed a greatly increased in vivo repressor activity but could also substitute for ModE at the moaA and dmsA promoters. In the reciprocal experiment, we restored repressor activity to a truncated CI protein by addition of the C-terminal domain of ModE, which is comprised of two MopI-like subdomains. By an in vivo competition assay, we also demonstrated that the CI-ModE chimeric protein retained the ability to interact with wild-type ModE. Finally, specific deletions within the ModE portion of the CI-ModE protein chimera abolished both in vivo repression and the ability to interact with wild-type ModE. Together, these data demonstrate that the N-terminal domain of ModE is sufficient to mediate DNA binding, although efficient binding requires that ModE form a dimer, a function that is supplied by the C-terminal MopI-like subdomains.