Efficacy of APX2039 in a Rabbit Model of Cryptococcal Meningitis.

Cryptococcal Meningitis (CM) is uniformly fatal if not treated, and treatment options are limited. We previously reported on the activity of APX2096, the prodrug of the novel Gwt1 inhibitor APX2039, in a mouse model of CM. Here, we investigated the efficacy of APX2039 in mouse and rabbit models of CM. In the mouse model, the controls had a mean lung fungal burden of 5.95 log10 CFU/g, whereas those in the fluconazole-, amphotericin B-, and APX2039-treated mice were 3.56, 4.59, and 1.50 log10 CFU/g, respectively. In the brain, the control mean fungal burden was 7.97 log10 CFU/g, while the burdens were 4.64, 7.16, and 1.44 log10 CFU/g for treatment with fluconazole, amphotericin B, and APX2039, respectively. In the rabbit model of CM, the oral administration of APX2039 at 50 mg/kg of body weight twice a day (BID) resulted in a rapid decrease in the cerebrospinal fluid (CSF) fungal burden, and the burden was below the limit of detection by day 10 postinfection. The effective fungicidal activity (EFA) was -0.66 log10 CFU/mL/day, decreasing from an average of 4.75 log10 CFU/mL to 0 CFU/mL, over 8 days of therapy, comparing favorably with good clinical outcomes in humans associated with reductions of the CSF fungal burden of -0.4 log10 CFU/mL/day, and, remarkably, 2-fold the EFA of amphotericin B deoxycholate in this model (-0.33 log10 CFU/mL/day). A total drug exposure of the area under the concentration-time curve from 0 to 24 h (AUC0-24) of 25 to 50 mg · h/L of APX2039 resulted in near-maximal antifungal activity. These data support the further preclinical and clinical evaluation of APX2039 as a new oral fungicidal monotherapy for the treatment of CM. IMPORTANCE Cryptococcal meningitis (CM) is a fungal disease with significant global morbidity and mortality. The gepix Gwt1 inhibitors are a new class of antifungal drugs. Here, we demonstrated the efficacy of APX2039, the second member of the gepix class, in rabbit and mouse models of cryptococcal meningitis. We also analyzed the drug levels in the blood and cerebrospinal fluid in the highly predictive rabbit model and built a mathematical model to describe the behavior of the drug with respect to the elimination of the fungal pathogen. We demonstrated that the oral administration of APX2039 resulted in a rapid decrease in the CSF fungal burden, with an effective fungicidal activity of -0.66 log10 CFU/mL/day, comparing favorably with good clinical outcomes in humans associated with reductions of -0.4 log10 CFU/mL/day. The drug APX2039 had good penetration of the central nervous system and is an excellent candidate for future clinical testing in humans for the treatment of CM.

GR-2397: Review of the Novel Siderophore-like Antifungal Agent for the Treatment of Invasive Aspergillosis.

GR-2397 (previously VL-2397, ASP2397) is a first-in-class antifungal agent for the treatment of invasive aspergillosis. This siderophore-like molecule resembles ferrichrome; however, it is differentiated by three amino acid changes and an aluminum rather than iron chelate. GR-2397 is transported into fungal cells via the Sit1 transporter, which is not found in humans, leading to fungal specificity. Although the precise mechanism of action is currently unknown, GR-2397 is active against Aspergillus spp. including azole-resistant strains, Fusarium solani, and Candida glabrata in addition to other organisms. Efficacy has been demonstrated in several animal models of invasive aspergillosis, including a 24 h delayed-treatment model where rapid fungicidal activity was observed. Phase 1 single- and multiple-ascending intravenous dose studies showed that GR-2397 was safe and well-tolerated in humans. No signs of GR-2397 accumulation were observed following IV infusions of 300, 600, and 1200 mg every 24 h (q24h) for 7 days. The favorable safety, tolerability and drug-drug interaction profile, along with good tissue distribution, support further development of GR-2397 as a new treatment option for patients with invasive aspergillosis. This systematic review summarizes the published findings of GR-2397.

The Combination Treatment of Fosmanogepix and Liposomal Amphotericin B Is Superior to Monotherapy in Treating Experimental Invasive Mold Infections.

Invasive pulmonary aspergillosis (IPA), invasive mucormycosis (IM), and invasive fusariosis (IF) are associated with high mortality and morbidity. Fosmanogepix (FMGX) is a first-in-class antifungal in clinical development with demonstrated broad-spectrum activity in animal models of infections. We sought to evaluate the benefit of combination therapy of FMGX plus liposomal amphotericin B (L-AMB) in severe delayed-treatment models of murine IPA, IM, and IF. While FMGX was equally as effective as L-AMB in prolonging the survival of mice infected with IPA, IM, or IF, combination therapy was superior to monotherapy in all three models. These findings were validated by greater reductions in the tissue fungal burdens (determined by quantitative PCR) of target organs in all three models versus the burdens in infected vehicle-treated (placebo) or monotherapy-treated mice. In general, histopathological examination of target organs corroborated the findings for fungal tissue burdens among all treatment arms. Our results show that treatment with the combination of FMGX plus L-AMB demonstrated high survival rates and fungal burden reductions in severe animal models of invasive mold infections, at drug exposures in mice similar to those achieved clinically. These encouraging results warrant further investigation of the FMGX-plus-L-AMB combination treatment for severely ill patients with IPA, IM, and IF.

In Vitro Activity of APX2041, a New Gwt1 Inhibitor, and In Vivo Efficacy of the Prodrug APX2104 against Aspergillus fumigatus.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a deadly infection for which new antifungal therapies are needed. Here, we demonstrate the efficacy of a Gwt1 inhibitor, APX2041, and its prodrug, APX2104, against A. fumigatus. The wild-type, azole-resistant, and echinocandin-resistant A. fumigatus strains were equally susceptible to APX2041 in vitro. APX2104 treatment in vivo significantly prolonged survival of neutropenic mice challenged with the wild-type and azole-resistant strains, revealing APX2104 as a potentially promising therapeutic against IA.

Therapeutic Potential of Fosmanogepix (APX001) for Intra-abdominal Candidiasis: from Lesion Penetration to Efficacy in a Mouse Model.

Intra-abdominal candidiasis (IAC) is one of the most common yet underappreciated forms of invasive candidiasis. IAC is difficult to treat, and therapeutic failure and drug-resistant breakthrough infections are common in some institutions despite the use of echinocandins as first-line agents. Fosmanogepix (FMGX, formerly APX001) is a first-in-class antifungal prodrug that can be administered both intravenously and orally. FMGX is currently in phase 2 clinical development for the treatment of life-threatening invasive fungal infections. To explore the pharmacological properties and therapeutic potential of FMGX for IAC, we evaluated both drug penetration and efficacy of the active moiety manogepix (MGX, formerly APX001A) in liver tissues in a clinically relevant IAC mouse model infected with Candida albicans Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) and laser capture microdissection (LCM)-directed absolute drug quantitation were employed to evaluate drug penetration into liver abscess lesions both spatially and quantitatively. The partitioning of MGX into lesions occurred slowly after a single dose; however, robust accumulation in the lesion was achieved after 3 days of repeated dosing. Associated with this drug penetration pattern, reduction in fungal burden and clearance in the liver were observed in mice receiving the multiday FMGX regimen. In comparison, administration of micafungin resulted in marginal reduction in fungal burden at the end of 4 days of treatment. These results suggest that FMGX is a promising candidate for the treatment of IAC.

Efficacy and Pharmacokinetics of Fosmanogepix (APX001) in the Treatment of Candida Endophthalmitis and Hematogenous Meningoencephalitis in Nonneutropenic Rabbits.

Candida endophthalmitis is a serious sight-threatening complication of candidemia that may occur before or during antifungal therapy. Hematogenous Candida meningoencephalitis (HCME) is also a serious manifestation of disseminated candidiasis in premature infants, immunosuppressed children, and immunocompromised adults. We evaluated the antifungal efficacy and pharmacokinetics of the prodrug fosmanogepix (APX001) in a rabbit model of endophthalmitis/HCME. Manogepix (APX001A), the active moiety of prodrug fosmanogepix, inhibits the fungal enzyme Gwt1 and is highly active in vitro and in vivo against Candida spp., Aspergillus spp., and other fungal pathogens. Plasma pharmacokinetics of manogepix after oral administration of fosmanogepix on day 6 at 25, 50, and 100 mg/kg resulted in maximum concentration of drug in plasma (Cmax) of 3.96 ± 0.41, 4.14 ± 1.1, and 11.5 ± 1.1 µg/ml, respectively, and area under the concentration-time curve from 0 to 12 h (AUC0-12) of 15.8 ± 3.1, 30.8 ± 5.0, 95.9 ± 14 µg·h/ml, respectively. Manogepix penetrated the aqueous humor, vitreous, and choroid with liquid-to-plasma ratios ranging from 0.19 to 0.52, 0.09 to 0.12, and 0.02 to 0.04, respectively. These concentrations correlated with a significant decrease in Candida albicans burden in vitreous (>101 to 103 log CFU/g) and choroid (>101 to 103 log CFU/g) (P ≤ 0.05 and P ≤ 0.001, respectively). The aqueous humor had no detectable C. albicans in treatment and control groups. The tissue/plasma concentration ratios of manogepix in meninges, cerebrum, cerebellum, and spinal cord were approximately 1:1, which correlated with a >102 to 104 decline of C. albicans in tissue versus control (P ≤ 0.05). Serum and cerebrospinal fluid (CSF) (1→3)-ß-d-glucan levels demonstrated significant declines in response to fosmanogepix treatment. These findings provide an experimental foundation for fosmanogepix in treatment of Candida endophthalmitis and HCME and derisk the clinical trials of candidemia and invasive candidiasis.

Fosmanogepix: A Review of the First-in-Class Broad Spectrum Agent for the Treatment of Invasive Fungal Infections.

Fosmanogepix is a first-in-class antifungal currently in Phase 2 clinical trials for the treatment of invasive fungal infections caused by Candida, Aspergillus and rare molds. Fosmanogepix is the N-phosphonooxymethylene prodrug of manogepix, an inhibitor of the fungal enzyme Gwt1. Manogepix demonstrates broad spectrum in vitro activity against yeasts and molds, including difficult to treat pathogens. Because of its novel mechanism of action, manogepix retains potency against many resistant strains including echinocandin-resistant Candida and azole-resistant Aspergillus. Manogepix is also active against pathogens that demonstrate intrinsic resistance to other drug classes, such as Scedosporium, Lomentospora prolificans, and Fusarium with variable activity against Mucorales. Fosmanogepix demonstrates significant in vivo efficacy in mouse and rabbit disseminated infection models due to C. albicans, C. glabrata, C. auris, C. tropicalis, Coccidioides immitis, and F. solani as well as pulmonary infection models of A. fumigatus, A. flavus, S. prolificans, S. apiospermum and Rhizopus arrhizus. Clinical trials demonstrated high oral bioavailability (>90%), enabling switching between fosmanogepix intravenous and oral formulations without compromising blood levels. Favorable drug-drug interaction, tolerability, and wide tissue distribution profiles are observed making fosmanogepix an attractive option for the treatment of invasive fungal infections. This systematic review summarizes the findings of published data on fosmanogepix.

In Vitro Activity of Manogepix against Multidrug-Resistant and Panresistant Candida auris from the New York Outbreak.

An ongoing Candida auris outbreak in the New York metropolitan area is the largest recorded to date in North America. Laboratory surveillance revealed NY C. auris isolates are resistant to fluconazole, with variable resistance to other currently used broad-spectrum antifungal drugs, and that several isolates are panresistant. Thus, there is an urgent need for new drugs with a novel mechanism of action to combat the resistance challenge. Manogepix (MGX) is a first-in-class agent that targets the fungal Gwt1 enzyme. The prodrug fosmanogepix is currently in phase 2 clinical development for the treatment of fungal infections. We evaluated the susceptibility of 200 New York C. auris isolates to MGX and 10 comparator drugs using CLSI methodology. MGX demonstrated lower MICs than comparators (MIC50 and MIC90, 0.03 mg/liter; range, 0.004 to 0.06 mg/liter). The local epidemiological cutoff value (ECV) for MGX indicated all C. auris isolates were within the population of wild-type (WT) strains; 0.06 mg/liter defines the upper limit of wild type (UL-WT). MGX was 8- to 32-fold more active than the echinocandins, 16- to 64-fold more active than the azoles, and 64-fold more active than amphotericin B. No differences were found in the MGX or comparators' MIC50, MIC90, or geometric mean (GM) values when subsets of clinical, surveillance, and environmental isolates were evaluated. The range of MGX MIC values for six C. auris panresistant isolates was 0.008 to 0.015 mg/liter, and the median and mode MIC values were 0.015 mg/liter, demonstrating that MGX retains activity against these isolates. These data support further clinical evaluation of fosmanogepix for the treatment of C. auris infections, including highly resistant isolates.

Fosmanogepix (APX001) Is Effective in the Treatment of Pulmonary Murine Mucormycosis Due to Rhizopus arrhizus.

Mucormycosis is a life-threatening infection with high mortality that occurs predominantly in immunocompromised patients. Manogepix (MGX) is a novel antifungal that targets Gwt1, a protein involved in an early step in the conserved glycosylphosphotidyl inositol (GPI) posttranslational modification pathway of surface proteins in eukaryotic cells. Inhibition of fungal inositol acylation by MGX results in pleiotropic effects, including inhibition of maturation of GPI-anchored proteins necessary for growth and virulence. MGX has been previously shown to have in vitro activity against some strains of Mucorales. Here, we assessed the in vivo activity of the prodrug fosmanogepix, currently in clinical development for the treatment of invasive fungal infections, against two Rhizopus arrhizus strains with high (4.0 µg/ml) and low (0.25 µg/ml) minimum effective concentration (MEC) values. In both invasive pulmonary infection models, treatment of mice with 78 mg/kg or 104 mg/kg fosmanogepix, along with 1-aminobenzotriazole to enhance the serum half-life of MGX in mice, significantly increased median survival time and prolonged overall survival by day 21 postinfection compared to placebo. In addition, administration of fosmanogepix resulted in a 1 to 2 log reduction in both lung and brain fungal burden. For the 104 mg/kg fosmanogepix dose, tissue clearance and survival were comparable to clinically relevant doses of isavuconazole (ISA), which is FDA approved for the treatment of mucormycosis. These results support continued development of fosmanogepix as a first-in-class treatment for invasive mucormycosis.

Enhanced Efflux Pump Expression in Candida Mutants Results in Decreased Manogepix Susceptibility.

Manogepix is a broad-spectrum antifungal agent that inhibits glycosylphosphatidylinositol (GPI) anchor biosynthesis. Using whole-genome sequencing, we characterized two efflux-mediated mechanisms in the fungal pathogens Candida albicans and Candida parapsilosis that resulted in decreased manogepix susceptibility. In C. albicans, a gain-of-function mutation in the transcription factor gene ZCF29 activated expression of ATP-binding cassette transporter genes CDR11 and SNQ2 In C. parapsilosis, a mitochondrial deletion activated expression of the major facilitator superfamily transporter gene MDR1.

Synthesis of analogs of the Gwt1 inhibitor manogepix (APX001A) and in vitro evaluation against Cryptococcus spp.

Fosmanogepix (APX001) is a first-in-class prodrug molecule that is currently in Phase 2 clinical trials for invasive fungal infections. The active moiety manogepix (APX001A) inhibits the novel fungal protein Gwt1. Gwt1 catalyzes an early step in the GPI anchor biosynthesis pathway. Here we describe the synthesis and evaluation of 292 new and 24 previously described analogs that were synthesized using a series of advanced intermediates to allow for rapid analoging. Several compounds demonstrated significantly (8- to 32-fold) improved antifungal activity against both Cryptococcus neoformans and C. gattii as compared to manogepix. Further in vitro characterization identified three analogs with a similar preliminary safety and in vitro profile to manogepix and superior activity against Cryptococcus spp.

Galactomannan Is a Biomarker of Fosmanogepix (APX001) Efficacy in Treating Experimental Invasive Pulmonary Aspergillosis.

Galactomannan (GM) detection in biological samples has been shown to predict therapeutic response by azoles and polyenes. In a murine invasive pulmonary aspergillosis model, fosmanogepix or posaconazole treatment resulted in an ∼6- to 7-log reduction in conidial equivalents (CE)/g lung tissue after 96 h versus placebo. Changes in GM levels in BAL fluid and serum mirrored reductions in lung CE, with significant decreases seen after 96 h or 72 h for fosmanogepix or posaconazole, respectively (P < 0.02).

Evaluation of Resistance Development to the Gwt1 Inhibitor Manogepix (APX001A) in Candida Species.

Manogepix (MGX) targets the conserved fungal Gwt1 enzyme required for acylation of inositol early in the glycosylphosphatidylinositol biosynthesis pathway. The prodrug fosmanogepix is currently in clinical development for the treatment of invasive fungal infections. We determined that the median frequencies of spontaneous mutations conferring reduced susceptibility to MGX in Candida albicans, C. glabrata, and C. parapsilosis ranged from 3 × 10-8 to <1.85 × 10-8 Serial passage on agar identified mutants of C. albicans and C. parapsilosis with reduced susceptibility to MGX; however, this methodology did not result in C. glabrata mutants with reduced susceptibility. Similarly, serial passage in broth resulted in ≤2-fold changes in population MIC values for C. tropicalis, C. auris, and C. glabrata A spontaneous V163A mutation in the Gwt1 protein of C. glabrata and a corresponding C. albicans heterozygous V162A mutant were obtained. A C. glabrata V163A Gwt1 mutant generated using CRISPR, along with V162A and V168A mutants expressed in C. albicans and Saccharomyces cerevisiae Gwt1, respectively, all demonstrated reduced susceptibility to MGX versus control strains, suggesting the importance of this valine residue to MGX binding across different species. Cross-resistance to the three major classes of antifungals was evaluated, but no changes in susceptibility to amphotericin B or caspofungin were observed in any mutant. No change was observed in fluconazole susceptibility, with the exception of a single non-Gwt1 mutant, where a 4-fold increase in the fluconazole MIC was observed. MGX demonstrated a relatively low potential for resistance development, consistent with other approved antifungal agents and those in clinical development.

APX001 and Other Gwt1 Inhibitor Prodrugs Are Effective in Experimental Coccidioides immitis Pneumonia.

Coccidioidomycosis is a systemic fungal infection caused by the inhalation of the arthroconidia of either of two closely related dimorphic fungi, Coccidioides immitis and C. posadasii, that are endemic in the southwestern United States and other areas in the Western Hemisphere. Chronic cavitary pulmonary infections and extrapulmonary sites of infection are very difficult to treat and often require lifelong azole therapy. APX001A is the first in a new class of broad-spectrum antifungal agents that inhibit Gwt1, an enzyme which is required for cell wall localization of glycosylphosphatidylinositol (GPI)-anchored mannoproteins in fungi. APX001A and several analogs were highly active against clinical isolates of Coccidioides, inhibiting hyphal growth at low nanogram/ml concentrations. APX001 is the N-phosphonooxymethyl prodrug of APX001A, currently in clinical trials for the treatment of invasive fungal infections. Mice were treated orally once daily with 26 mg/kg/day of APX001 and the prodrug analog APX2097, 2 h after administration of the pan-cytochrome P450 inhibitor 1-aminobenzotriazole, which was used to enhance drug half-life and exposures to more closely mimic human pharmacokinetics of APX001A. Five days of treatment reduced lung colony counts by nearly 3 logs and prevented dissemination, similar to the efficacy of fluconazole dosed orally at 25 mg/kg twice daily. In a survival experiment, both APX001- and APX2097-treated mice survived significantly longer than control and fluconazole-treated mice. APX001 and other members of this new class of antifungal agents may offer great promise as effective therapies for coccidioidomycosis.

Significantly Improved Pharmacokinetics Enhances In Vivo Efficacy of APX001 against Echinocandin- and Multidrug-Resistant Candida Isolates in a Mouse Model of Invasive Candidiasis.

APX001 is a first-in-class, intravenous and orally available, broad-spectrum antifungal agent in clinical development for the treatment of life-threatening invasive fungal infections. The half-life of APX001A, the active moiety of APX001, is significantly shorter in mice than in humans (1.4 to 2.75 h in mice versus 2 to 2.5 days in humans), making the exploration of efficacy in mouse models difficult. After pretreatment with 1-aminobenzotriazole (ABT), a nonspecific cytochrome P450 inhibitor, greatly increased plasma APX001A exposure was observed in mice of different strains and of both genders. As a consequence, 26 mg/kg APX001 plus ABT sterilized kidneys in mice infected with Candida albicans, while APX001 alone at the same dose resulted in a modest burden reduction of only 0.2 log10 CFU/g, relative to the vehicle control. In the presence of ABT, 2 days of once-daily dosing with APX001 at 26 mg/kg also demonstrated significant in vivo efficacy in the treatment of Candida glabrata infections in mice. Potent kidney burden reduction was achieved in mice infected with susceptible, echinocandin-resistant, or multidrug-resistant strains. In contrast, the standard of care (micafungin) was ineffective in treating infections caused by the resistant C. glabrata isolates.

In Vitro and In Vivo Evaluation of APX001A/APX001 and Other Gwt1 Inhibitors against Cryptococcus.

Cryptococcal meningitis (CM), caused primarily by Cryptococcus neoformans, is uniformly fatal if not treated. Treatment options are limited, especially in resource-poor geographical regions, and mortality rates remain high despite current therapies. Here we evaluated the in vitro and in vivo activity of several compounds, including APX001A and its prodrug, APX001, currently in clinical development for the treatment of invasive fungal infections. These compounds target the conserved Gwt1 enzyme that is required for the localization of glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins in fungi. The Gwt1 inhibitors had low MIC values, ranging from 0.004 µg/ml to 0.5 µg/ml, against both C. neoformans and C. gattii APX001A and APX2020 demonstrated in vitro synergy with fluconazole (fractional inhibitory concentration index, 0.37 for both). In a CM model, APX001 and fluconazole each alone reduced the fungal burden in brain tissue (0.78 and 1.04 log10 CFU/g, respectively), whereas the combination resulted in a reduction of 3.52 log10 CFU/g brain tissue. Efficacy, as measured by a reduction in the brain and lung tissue fungal burden, was also observed for another Gwt1 inhibitor prodrug, APX2096, where dose-dependent reductions in the fungal burden ranged from 5.91 to 1.79 log10 CFU/g lung tissue and from 7.00 and 0.92 log10 CFU/g brain tissue, representing the nearly complete or complete sterilization of lung and brain tissue at the higher doses. These data support the further clinical evaluation of this new class of antifungal agents for the treatment of CM.

Anthracimycin activity against contemporary methicillin-resistant Staphylococcus aureus.

Anthracimycin is a recently discovered novel marine-derived compound with activity against Bacillus anthracis. We tested anthracimycin against an expanded panel of Staphylococcus aureus strains in vitro and in vivo. All strains of S. aureus tested, including methicillin-susceptible, methicillin-resistant (MRSA) and vancomycin-resistant strains of S. aureus, were susceptible to anthracimycin at MIC values of ⩽0.25 mg l(-1). Although its postantibiotic effects were minimal, anthracimycin exhibited potent and rapid bactericidal activity, with a >4-log kill of USA300 MRSA within 3 h at five times its MIC. At concentrations significantly below the MIC, anthracimycin slowed MRSA growth and potentiated the bactericidal activity of the human cathelicidin, LL-37. The bactericidal activity of anthracimycin was somewhat mitigated in the presence of 20% human serum, and the compound was minimally toxic to human cells, with an IC50 (inhibitory concentration 50)=70 mg l(-1) against human carcinoma cells. At concentrations near the MIC, anthracimycin inhibited S. aureus nucleic acid synthesis as determined by optimized macromolecular synthesis methodology, with inhibition of DNA and RNA synthesis occurring in the absence of DNA intercalation. Anthracimycin at a single dose of 1 or 10 mg kg(-1) was able to protect mice from MRSA-induced mortality in a murine peritonitis model of infection. Anthracimycin provides an interesting new scaffold for future development of a novel MRSA antibiotic.

Structure-based design of new dihydrofolate reductase antibacterial agents: 7-(benzimidazol-1-yl)-2,4-diaminoquinazolines.

A new series of dihydrofolate reductase (DHFR) inhibitors, the 7-(benzimidazol-1-yl)-2,4-diaminoquinazolines, were designed and optimized for antibacterial potency and enzyme selectivity. The most potent inhibitors in this series contained a five-membered heterocycle at the 2-position of the benzimidazole, leading to highly potent and selective compounds that exploit the differences in the size of a binding pocket adjacent to the NADPH cofactor between the bacterial and human DHFR enzymes. Typical of these compounds is 7-((2-thiazol-2-yl)benzimidazol-1-yl)-2,4 diaminoquinazoline, which is a potent inhibitor of S. aureus DHFR (Ki = 0.002 nM) with 46700-fold selectivity over human DHFR. This compound also has high antibacterial potency on Gram-positive bacteria with an MIC versus wild type S. aureus of 0.0125 µg/mL and a MIC versus trimethoprim-resistant S. aureus of 0.25 µg/mL. In vivo efficacy versus a S. aureus septicemia was demonstrated, highlighting the potential of this new series.

Tedizolid for the management of human infections: in vitro characteristics.

The emerging antibiotic resistance of Gram-positive pathogens represents a significant challenge to the management of human infections. The novel oxazolidinone tedizolid demonstrates antimicrobial activity across a broad range of Gram-positive pathogens and greater potency than linezolid against wild-type and drug-resistant pathogens, including linezolid-resistant Staphylococcus aureus strains possessing mutations in chromosomal genes encoding 23S rRNA or ribosomal proteins L3 or L4. Strains harboring such mutations are also selected for much less frequently with tedizolid than with linezolid. In addition, tedizolid has a significant potency advantage over linezolid-resistant strains carrying the horizontally transferable cfr gene. Methylation of A2503 of 23S rRNA by the Cfr methyltransferase confers resistance to linezolid (and a variety of other 50S ribosomal subunit-targeted antibiotics) but not to tedizolid because of structural differences in A-ring C5 substituents between the 2 drugs. The greater potency and improved resistance profile of tedizolid provides the microbiologic basis for considering this molecule as an alternative to linezolid for the treatment of serious infections caused by Gram-positive pathogens.

Genetic environment and stability of cfr in methicillin-resistant Staphylococcus aureus CM05.

The Cfr methyltransferase confers resistance to many 50S ribosomal subunit-targeted antibiotics, including linezolid (LZD), via methylation of the 23S rRNA base A2503 in the peptidyl transferase center. Methicillin-resistant Staphylococcus aureus strain CM05 is the first clinical isolate documented to carry cfr. While cfr is typically plasmid borne, in CM05 it is located on the chromosome and is coexpressed with ermB as part of the mlr operon. Here we evaluated the chromosomal locus, association with mobile genetic elements, and stability of the cfr insertion region in CM05. The cfr-containing mlr operon is located within a 15.5-kb plasmid-like insertion into 23S rRNA allele 4. The region surrounding the cfr gene has a high degree of sequence similarity to the broad-host-range toxin/antitoxin multidrug resistance plasmid pSM19035, including a second ermB gene downstream of the mlr locus and istAS-istBS. Analysis of several individual CM05 colonies revealed two distinct populations for which LZD MICs were either 8 or 2 µg/ml. In the LZD(s) colonies (designated CM05Δ), a recombination event involving the two ermB genes had occurred, resulting in the deletion of cfr and the 3' flanking region (cfr-istAS-istBS-ermB). The fitness advantage of CM05Δ over CM05 (though not likely due to the cfr deletion itself) results in the predominance of CM05Δ in the absence of selective pressure. Minicircles resulting from the ermB recombination event and the novel association of cfr with the pSM19035 plasmid system support the potential for the continued dissemination of cfr.