Clinical Efficacy and Safety of a Novel Antifungal, Fosmanogepix, in Patients with Candidemia Caused by Candida auris: Results from a Phase 2 Trial.

Fosmanogepix (FMGX), a novel antifungal available in intravenous (IV) and oral formulations, has broad-spectrum activity against pathogenic yeasts and molds, including fungi resistant to standard of care antifungals. This multicenter, open-label, single-arm study evaluated FMGX safety and efficacy for treatment of candidemia and/or invasive candidiasis caused by Candida auris. Eligible participants were ≥18 years, with established candidemia and/or invasive candidiasis caused by C. auris, (cultured within 120 h [for candidemia] or 168 h [for invasive candidiasis without candidemia] with accompanying clinical signs) and limited treatment options. Participants were treated with FMGX (≤42 days; loading dose: 1000 mg IV twice daily [Day 1], followed by 600 mg IV once daily [QD]). Switching to oral FMGX 800 mg QD was permitted from Day 4. Primary endpoint was treatment success (survival and clearance of C. auris from blood/tissue cultures without additional antifungals) at the end of the study treatment (EOST), assessed by an independent data review committee (DRC). Day 30 survival was a secondary endpoint. In vitro susceptibility of Candida isolates was assessed. Nine participants with candidemia (male:6, female:3; 21 to 76 years) in intensive care units in South Africa were enrolled; all received IV FMGX only. DRC-assessed treatment success at EOST and Day 30 survival were 89% (8/9). No treatment related adverse events or study drug discontinuations were reported. FMGX demonstrated potent in vitro activity against all C. auris isolates (MIC range: 0.008 to 0.015 µg/mL [CLSI]; 0.004-0.03 µg/mL [EUCAST]), with the lowest MICs compared to other antifungals tested. Thus, the results showed that FMGX was safe, well-tolerated, and efficacious in participants with candidemia caused by C. auris.

Safety and Pharmacokinetics of Intravenous and Oral Fosmanogepix, a First-in-Class Antifungal Agent, in Healthy Volunteers.

Fosmanogepix (FMGX, APX001), a first-in-class, intravenous (i.v.) and oral (p.o.) antifungal prodrug candidate is currently in clinical development for the treatment of invasive fungal infections. Manogepix (MGX, APX001A), the active moiety of FMGX, interferes with cell wall synthesis by targeting fungal glycosylphosphatidylinositol-anchored cell wall transfer protein 1, thereby causing loss of cell viability. Data from two phase 1, placebo-controlled, single-ascending dose (SAD) and multiple-ascending dose (MAD) studies evaluating safety, tolerability, and pharmacokinetics of FMGX (doses up to 1,000 mg, i.v. and p.o.) are presented. Eligible participants were healthy adults (aged 18 to 55 years) randomized to receive either FMGX or placebo. Across both phase 1 studies, 151 of 154 participants (aged 23 to 35 years; FMGX: 116, placebo: 38) completed the study. Administration of FMGX i.v. demonstrated linear- and dose-proportional pharmacokinetics of MGX in terms of geometric mean maximum concentration of drug in serum (Cmax) (SAD: 0.16 to 12.0 µg/mL, dose: 10 to 1,000 mg; MAD: 0.67 to 15.4 µg/mL, dose: 50 to 600 mg) and area under the concentration-time curve (AUC) (SAD: 4.05 to 400, MAD: 6.39 to 245 µg · h/mL). With single and repeat p.o., dose-proportional increases in Cmax (SAD: 1.30 to 6.41 µg/mL, dose: 100 to 500 mg; MAD: 6.18 to 21.3 µg/mL, dose: 500 to 1,000 mg) and AUC (SAD: 87.5 to 205, MAD: 50.8 to 326 µg · h/mL) were also observed, with high oral bioavailability (90.6% to 101.2%). Administration of FMGX p.o. under post cibum conditions improved tolerability versus ante cibum conditions. No severe treatment-emergent adverse events (TEAEs), serious AEs, or withdrawals due to a drug-related TEAEs were reported with single or multiple i.v. and p.o. doses. Preclinical target exposures were achieved and were not accompanied by any serious/unexpected concerns with generally safe and well-tolerated dose regimens.

Activities of Manogepix and Comparators against 1,435 Recent Fungal Isolates Collected during an International Surveillance Program (2020).

We evaluated the in vitro activity of manogepix and comparator agents against 1,435 contemporary fungal isolates collected worldwide from 73 medical centers in North America, Europe, the Asia-Pacific region, and Latin America during 2020. Of the isolates tested, 74.7% were Candida spp.; 3.7% were non-Candida yeasts, including 27 Cryptococcus neoformans var. grubii (1.9%); 17.1% were Aspergillus spp.; and 4.5% were other molds. All fungal isolates were tested by reference broth microdilution according to CLSI methods. Based on MIC90 values, manogepix (MIC50/MIC90, 0.008/0.06 mg/liter) was 16- to 64-fold more active than anidulafungin, micafungin, and fluconazole against Candida spp. isolates and the most active agent tested. Similarly, manogepix (MIC50/MIC90, 0.5/1 mg/liter) was ≥8-fold more active than anidulafungin, micafungin, and fluconazole against C. neoformans var. grubii. Based on minimum effective concentration for 90% of the isolates tested (MEC90) and MIC90 values, manogepix (MEC90, 0.03 mg/liter) was 16- to 64-fold more potent than itraconazole, posaconazole, and voriconazole (MIC90s, 0.5 to 2 mg/liter) against 246 Aspergillus spp. isolates. Aspergillus fumigatus isolates exhibited a wild-type (WT) phenotype for the mold-active triazoles, including itraconazole (87.0% WT) and voriconazole (96.4% WT). Manogepix was highly active against uncommon species of Candida, non-Candida yeasts, and rare molds, including 11 isolates of Candida auris (MIC50/MIC90, 0.004/0.015 mg/liter) and 12 isolates of Scedosporium spp. (MEC50/MEC90, 0.06/0.12 mg/liter). Additional studies are in progress to evaluate the clinical utility of the manogepix prodrug fosmanogepix in difficult-to-treat resistant fungal infections.

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.

Antifungal Activity of Gepinacin Scaffold Glycosylphosphatidylinositol Anchor Biosynthesis Inhibitors with Improved Metabolic Stability.

The glycosylphosphatidylinositol anchor biosynthesis inhibitor gepinacin demonstrates broad-spectrum antifungal activity and negligible mammalian toxicity in culture but is metabolically labile. The stability and bioactivity of 39 analogs were tested in vitro to identify LCUT-8, a stabilized lead with increased potency and promising single-dose pharmacokinetics. Unfortunately, no antifungal activity was seen at the maximum dosing achievable in a neutropenic rabbit model. Nevertheless, structure-activity relationships identified here suggest strategies to further improve compound potency, solubility, and stability.

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.

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.

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.

Fosmanogepix (APX001) Is Effective in the Treatment of Immunocompromised Mice Infected with Invasive Pulmonary Scedosporiosis or Disseminated Fusariosis.

There are limited treatment options for immunosuppressed patients with lethal invasive fungal infections due to Fusarium and Scedosporium Manogepix (MGX; APX001A) is a novel antifungal that targets the conserved Gwt1 enzyme required for localization of glycosylphosphatidylinositol-anchored mannoproteins in fungi. We evaluated the in vitro activity of MGX and the efficacy of the prodrug fosmanogepix (APX001) in immunosuppressed murine models of hematogenously disseminated fusariosis and pulmonary scedosporiosis. The MGX minimum effective concentration (MEC) for Scedosporium isolates was 0.03 µg/ml and ranged from 0.015 to 0.03 µg/ml for Fusarium isolates. In the scedosporiosis model, treatment of mice with 78 mg/kg and 104 mg/kg of body weight fosmanogepix, along with 1-aminobenzotriazole (ABT) to enhance the serum half-life of MGX, significantly increased median survival time versus placebo from 7 days to 13 and 11 days, respectively. Furthermore, administration of 104 mg/kg fosmanogepix resulted in an ∼2-log10 reduction in lung, kidney, or brain conidial equivalents/gram tissue (CE). Similarly, in the fusariosis model, 78 mg/kg and 104 mg/kg fosmanogepix plus ABT enhanced median survival time from 7 days to 12 and 10 days, respectively. A 2- to 3-log10 reduction in kidney and brain CE was observed. In both models, reduction in tissue fungal burden was corroborated with histopathological data, with target organs showing reduced or no abscesses in fosmanogepix-treated mice. Survival and tissue clearance were comparable to a clinically relevant high dose of liposomal amphotericin B (10 to 15 mg/kg). Our data support the continued development of fosmanogepix as a first-in-class treatment for infections caused by these rare molds.

In Vitro Activity of APX001A (Manogepix) and Comparator Agents against 1,706 Fungal Isolates Collected during an International Surveillance Program in 2017.

Current antifungal agents cover a majority of opportunistic fungal pathogens; however, breakthrough invasive fungal infections continue to occur and increasingly involve relatively uncommon yeasts and molds, which often exhibit decreased susceptibility. APX001A (manogepix) is a first-in-class small-molecule inhibitor of the conserved fungal Gwt1 protein. This enzyme is required for acylation of inositol during glycosylphosphatidylinositol anchor biosynthesis. APX001A is active against the major fungal pathogens, i.e., Candida (except Candida krusei), Aspergillus, and hard-to-treat molds, including Fusarium and Scedosporium In this study, we tested APX001A and comparators against 1,706 contemporary clinical fungal isolates collected in 2017 from 68 medical centers in North America (37.3%), Europe (43.4%), the Asia-Pacific region (12.7%), or Latin America (6.6%). Among the isolates tested, 78.5% were Candida spp., 3.9% were non-Candida yeasts, including 30 (1.8%) Cryptococcus neoformans var. grubii isolates, 14.7% were Aspergillus spp., and 2.9% were other molds. All isolates were tested by CLSI reference broth microdilution. APX001A (MIC50, 0.008 µg/ml; MIC90, 0.06 µg/ml) was the most active agent tested against Candida sp. isolates; corresponding anidulafungin, micafungin, and fluconazole MIC90 values were 16- to 64-fold higher. Similarly, APX001A (MIC50, 0.25 µg/ml; MIC90, 0.5 µg/ml) was ≥8-fold more active than anidulafungin, micafungin, and fluconazole against C. neoformans var. grubii Against Aspergillus spp., AXP001A (50% minimal effective concentration [MEC50], 0.015 µg/ml; MEC90, 0.03 µg/ml) was comparable in activity to anidulafungin and micafungin. Aspergillus isolates (>98%) exhibited a wild-type phenotype for the mold-active triazoles (itraconazole, posaconazole, and voriconazole). APX001A was highly active against uncommon species of Candida, non-Candida yeasts, and rare molds, including 11 isolates of Scedosporium spp. (MEC values, 0.015 to 0.06 µg/ml). APX001A demonstrated potent in vitro activity against recent fungal isolates, including echinocandin- and fluconazole-resistant strains. The extended spectrum of APX001A was also notable for its potency against many less common but antifungal-resistant strains. Further studies are in progress to evaluate the clinical utility of the methyl phosphate prodrug, APX001, in difficult-to-treat resistant fungal infections.

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 Is Effective in the Treatment of Murine Invasive Pulmonary Aspergillosis.

Invasive pulmonary aspergillosis (IPA) due to Aspergillus fumigatus is a serious fungal infection in the immunosuppressed patient population. Despite the introduction of new antifungal agents, mortality rates remain high, and new treatments are needed. The novel antifungal APX001A targets the conserved Gwt1 enzyme required for the localization of glycosylphosphatidylinositol-anchored mannoproteins in fungi. We evaluated the in vitro activity of APX001A against A. fumigatus and the in vivo activity of its prodrug APX001 in an immunosuppressed mouse model of IPA. APX001A inhibited the growth of A. fumigatus with a minimum effective concentration of 0.03 µg/ml. The use of 50 mg/kg 1-aminobenzotriazole (ABT), a suicide inhibitor of cytochrome P450 enzymes, enhanced APX001A exposures (area under the time-concentration curve [AUC]) 16- to 18-fold and enhanced serum half-life from ∼1 to 9 h, more closely mimicking human pharmacokinetics. We evaluated the efficacy of APX001 (with ABT) in treating murine IPA compared to posaconazole treatment. Treatment of mice with 78 mg/kg once daily (QD), 78 mg/kg twice daily, or 104 mg/kg QD APX001 significantly enhanced the median survival time and prolonged day 21 postinfection overall survival compared to the placebo. Furthermore, administration of APX001 resulted in a significant reduction in lung fungal burden (4.2 to 7.6 log10 conidial equivalents/g of tissue) versus the untreated control and resolved the infection, as judged by histopathological examination. The observed survival and tissue clearance were comparable to a clinically relevant posaconazole dose. These results warrant the continued development of APX001 as a broad-spectrum, first-in-class treatment of invasive fungal infections.

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.

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).

Efficacy of Delayed Therapy with Fosmanogepix (APX001) in a Murine Model of Candida auris Invasive Candidiasis.

The emerging pathogenic yeast Candida auris is associated with antifungal resistance and high mortality. The novel antifungal agent manogepix (APX001A) inhibits glycosylphosphatidylinositol-anchored protein maturation and has demonstrated activity against numerous pathogenic fungi, including C. auris Our objective was to evaluate the in vivo efficacy of fosmanogepix, the N-phosphonooxymethyl prodrug (APX001), following delayed initiation of therapy in a murine model of C. auris invasive candidiasis. Neutropenic mice were intravenously infected with a fluconazole-resistant clinical isolate of C. auris Twenty-four hours postinoculation, treatment began with vehicle control, fosmanogepix (104 and 130 mg/kg of body weight by intraperitoneal injection three times daily, or intraperitoneal 260 mg/kg twice daily), fluconazole (20 mg/kg by oral gavage once daily), or caspofungin (intraperitoneal 10 mg/kg once daily) and continued for 7 days. Fungal burden was assessed via colony count in the kidneys and brains on day 8 in the fungal burden arm and on day 21 as the mice became moribund in the survival arm. Significant improvements in survival were observed in each group administered fosmanogepix and caspofungin. Similarly, reductions in fungal burden were also observed in both the kidneys and brains of mice treated with the highest dose of fosmanogepix in the fungal burden arm and in each fosmanogepix group and with caspofungin in the survival arm. In contrast, no improvements in survival or reductions in fungal burden were observed in mice treated with fluconazole. These results demonstrate that fosmanogepix is effective in vivo against fluconazole-resistant C. auris even when therapy is delayed.

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.

A novel fungicide aminopyrifen inhibits GWT-1 protein in glycosylphosphatidylinositol-anchor biosynthesis in Neurospora crassa.

Aminopyrifen, 4-phenoxybenzyl 2-amino-6-methylnicotinate, strongly inhibited the mycelial growth of a wild-type Neurospora crassa strain on Vogel's minimal medium containing 1.2% sucrose, with a 0.001 mg/L concentration required for 50% growth inhibition. Similar to micafungin, an inhibitor of beta-1, 3-glucan synthetase, aminopyrifen further inhibited the growth of N. crassa deletion mutants of MAP kinase cascade genes, such as mak-1 and mak-2, than the wild-type strain, suggesting that aminopyrifen perturbs cell wall-related processes. Furthermore, we found that three chitin synthase gene mutants (chs-1, chs-5, and chs-7) were highly sensitive to both chemicals; however, aminopyrifen, but not micafungin, induced a swollen germ tube from the conidia of chs-5 and chs-7 mutants on Vogel's medium containing 1.2% sucrose. To elucidate the target protein of aminopyrifen, we isolated mutants resistant to aminopyrifen after UV treatment of conidia of the wild-type strain or the chs-5 strain. The resistance mutations were localized to the gwt-1 gene that encodes an acyltransferase, GWT-1, which participates in the biosynthesis of the glycosylphosphatidylinositol (GPI) precursor, and were found to result in S180F and V178A alterations in the protein. These results strongly suggest that aminopyrifen works as an inhibitor targeting GWT-1, a protein involved in GPI-anchor biosynthesis.

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.

GWT1 encoding an inositol acyltransferase homolog is required for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans.

The transcriptional expression of laccase, which has been confirmed to contribute to the virulence of Cryptococcus neoformans, is often repressed by a high concentration of glucose in many fungi, including C. neoformans. The underlying mechanism of the repression remains largely unknown. In this study, we found that a GWT1 gene that encodes a glycosylphosphatidylinositol (GPI) anchor biosynthesis-related protein is required for laccase repression by glucose in the basidiomycete C. neoformans. Disruption of GWT1 with the Agrobacterium tumefaciens-mediated T-DNA random insertional mutagenesis (ATMT) method resulted in constitutive expression of the laccase gene LAC1 and constant melanin formation. The loss of GWT1 also dramatically affected the cell membrane integrity and stress resistance. Our results revealed a GPI-dependent glucose repression mechanism in C. neoformans, and it may be helpful for understanding the virulence of C. neoformans.