Ibrexafungerp: An orally active ß-1,3-glucan synthesis inhibitor.

We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious ß-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. Further extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl aminoether of MK-5204 provided the best balance of these two key parameters, culminating in the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp displayed significantly improved oral efficacy in murine infection models, making it a superior candidate for clinical development as an oral treatment for Candida and Aspergillus infections.

MK-5204: An orally active ß-1,3-glucan synthesis inhibitor.

Our previously reported efforts to produce an orally active ß-1,3-glucan synthesis inhibitor through the semi-synthetic modification of enfumafungin focused on replacing the C2 acetoxy moiety with an aminotetrazole and the C3 glycoside with a N,N-dimethylaminoether moiety. This work details further optimization of the C2 heterocyclic substituent, which identified 3-carboxamide-1,2,4-triazole as a replacement for the aminotetrazole with comparable antifungal activity. Alkylation of either the carboxamidetriazole at C2 or the aminoether at C3 failed to significantly improve oral efficacy. However, replacement of the isopropyl alpha amino substituent with a t-butyl, improved oral exposure while maintaining antifungal activity. These two structural modifications produced MK-5204, which demonstrated broad spectrum activity against Candida species and robust oral efficacy in a murine model of disseminated Candidiasis without the N-dealkylation liability observed for the previous lead.

Molecular mechanisms of sulbactam antibacterial activity and resistance determinants in Acinetobacter baumannii.

Sulbactam is a class A ß-lactamase inhibitor with intrinsic whole-cell activity against certain bacterial species, including Acinetobacter baumannii. The clinical use of sulbactam for A. baumannii infections is of interest due to increasing multidrug resistance in this pathogen. However, the molecular drivers of its antibacterial activity and resistance determinants have yet to be precisely defined. Here we show that the antibacterial activities of sulbactam vary widely across contemporary A. baumannii clinical isolates and are mediated through inhibition of the penicillin-binding proteins (PBPs) PBP1 and PBP3, with very low frequency of resistance; the rare pbp3 mutants with high levels of resistance to sulbactam are attenuated in fitness. These results support further investigation of the potential clinical utility of sulbactam.

In vitro antibacterial activity of AZD0914, a new spiropyrimidinetrione DNA gyrase/topoisomerase inhibitor with potent activity against Gram-positive, fastidious Gram-Negative, and atypical bacteria.

AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, ß-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development.

Novel orally active inhibitors of ß-1,3-glucan synthesis derived from enfumafungin.

The clinical success of the echinocandins, which can only be administered parentally, has validated ß-1,3-glucan synthase (GS) as an antifungal target. Semi-synthetic modification of enfumafungin, a triterpene glycoside natural product, was performed with the aim of producing a new class of orally active GS inhibitors. Replacement of the C2 acetoxy moiety with various heterocycles did not improve GS or antifungal potency. However, replacement of the C3 glycoside with an aminoether moiety dramatically improved oral pharmacokinetic (PK) properties while maintaining GS and antifungal potency. Installing an aminotetrazole at C2 in conjunction with an N-alkylated aminoether at C3 produced derivatives with significantly improved GS and antifungal potency that exhibited robust oral efficacy in a murine model of disseminated candidiasis.

Synergy of streptogramin antibiotics occurs independently of their effects on translation.

Streptogramin antibiotics are divided into types A and B, which in combination can act synergistically. We compared the molecular interactions of the streptogramin combinations Synercid (type A, dalfopristin; type B, quinupristin) and NXL 103 (type A, flopristin; type B, linopristin) with the Escherichia coli 70S ribosome by X-ray crystallography. We further analyzed the activity of the streptogramin components individually and in combination. The streptogramin A and B components in Synercid and NXL 103 exhibit synergistic antimicrobial activity against certain pathogenic bacteria. However, in transcription-coupled translation assays, only combinations that include dalfopristin, the streptogramin A component of Synercid, show synergy. Notably, the diethylaminoethylsulfonyl group in dalfopristin reduces its activity but is the basis for synergy in transcription-coupled translation assays before its rapid hydrolysis from the depsipeptide core. Replacement of the diethylaminoethylsulfonyl group in dalfopristin by a nonhydrolyzable group may therefore be beneficial for synergy. The absence of general streptogramin synergy in transcription-coupled translation assays suggests that the synergistic antimicrobial activity of streptogramins can occur independently of the effects of streptogramin on translation.

Activity of avibactam against Enterobacter cloacae producing an extended-spectrum class C ß-lactamase enzyme.

BACKGROUND: Extended-spectrum AmpC (ESAC) ß-lactamase enzymes, which are either chromosomally encoded or plasmid encoded, have minor structural changes that broaden their substrate hydrolysis profile. The derepressed AmpC enzyme found once in Enterobacter cloacae CHE was shown to contain a six residue deletion in the H-10 helix in close proximity to the active site. Avibactam is a non-ß-lactam inhibitor of Ambler class A, class C and some class D ß-lactamases that is in clinical development with several ß-lactam agents. It has been shown to inhibit AmpC enzymes, but its microbiological activity against isolates carrying different ESAC enzymes is less well understood. METHODS: MICs were determined using the broth microdilution technique. RT-PCR analyses were performed to measure the level of ampC expression and whole genome sequencing was performed to enable sequence-based analyses. RESULTS: Structural analyses of avibactam bound to a representative AmpC ß-lactamase suggested that the H-10 helix deletion would impact the potency of the inhibitor. Under standard conditions, the ceftazidime/avibactam and ceftaroline/avibactam MIC values for E. cloacae CHE were 64 and 4 mg/L, respectively, representing a significant decrease in susceptibility over control E. cloacae isolates. However, use of higher avibactam concentrations restored the susceptibility of E. cloacae CHE in a dose-dependent manner. Comparison with other E. cloacae isolates carrying derepressed AmpC enzymes suggested that this difference in inhibition by avibactam was unrelated to the level of AmpC being produced. CONCLUSIONS: The E. cloacae CHE ESAC enzyme is inhibited less efficiently by avibactam than other E. cloacae AmpC proteins due to a subtle rearrangement of the binding site. Although the variants are not commonly observed, the different ESAC enzymes may be inhibited to varied extents by avibactam.

Isolation, structure, and biological activity of Phaeofungin, a cyclic lipodepsipeptide from a Phaeosphaeria sp. Using the Genome-Wide Candida albicans Fitness Test.

Phaeofungin (1), a new cyclic depsipeptide isolated from Phaeosphaeria sp., was discovered by application of reverse genetics technology, using the Candida albicans fitness test (CaFT). Phaeofungin is comprised of seven amino acids and a ß,γ-dihydroxy-γ-methylhexadecanoic acid arranged in a 25-membered cyclic depsipeptide. Five of the amino acids were assigned with d-configurations. The structure was elucidated by 2D-NMR and HRMS-MS analysis of the natural product and its hydrolyzed linear peptide. The absolute configuration of the amino acids was determined by Marfey's method by complete and partial hydrolysis of 1. The CaFT profile of the phaeofungin-containing extract overlapped with that of phomafungin (3), another structurally different cyclic lipodepsipeptide isolated from a Phoma sp. using the same approach. Comparative biological characterization further demonstrated that these two fungal lipodepsipeptides are functionally distinct. While phomafungin was potentiated by cyclosporin A (an inhibitor of the calcineurin pathway), phaeofungin was synergized with aureobasidin A (2) (an inhibitor of the sphingolipid biosynthesis) and to some extent caspofungin (an inhibitor of glucan synthase). Furthermore, phaeofungin caused ATP release in wild-type C. albicans strains but phomafungin did not. It showed modest antifungal activity against C. albicans (MIC 16-32 µg/mL) and better activity against Aspergillus fumigatus (MIC 8-16 µg/mL) and Trichophyton mentagrophytes (MIC 4 µg/mL). The linear peptide was inactive, suggesting that the macrocyclic depsipeptide ring is essential for target engagement and antifungal activity.

Synthesis and biological evaluation of antifungal derivatives of enfumafungin as orally bioavailable inhibitors of ß-1,3-glucan synthase.

Orally bioavailable inhibitors of ß-(1,3)-D-glucan synthase have been pursued as new, broad-spectrum fungicidal therapies suitable for treatment in immunocompromised patients. Toward this end, a collaborative medicinal chemistry program was established based on semisynthetic derivatization of the triterpenoid glycoside natural product enfumafungin in order to optimize in vivo antifungal activity and oral absorption properties. In the course of these studies, it was hypothesized that the pharmacokinetic properties of the semisynthetic enfumafungin analog 3 could be improved by tethering the alkyl groups proximal to the basic nitrogen of the C3-aminoether side chain into an azacyclic system, so as to preclude oxidative N-demethylation. The results of this research effort are disclosed herein.

Discovery of an Orally Available Diazabicyclooctane Inhibitor (ETX0282) of Class A, C, and D Serine ß-Lactamases.

Multidrug resistant Gram-negative bacterial infections are an increasing public health threat due to rapidly rising resistance toward ß-lactam antibiotics. The hydrolytic enzymes called ß-lactamases are responsible for a large proportion of the resistance phenotype. ß-Lactamase inhibitors (BLIs) can be administered in combination with ß-lactam antibiotics to negate the action of the ß-lactamases, thereby restoring activity of the ß-lactam. Newly developed BLIs offer some advantage over older BLIs in terms of enzymatic spectrum but are limited to the intravenous route of administration. Reported here is a novel, orally bioavailable diazabicyclooctane (DBO) ß-lactamase inhibitor. This new DBO, ETX1317, contains an endocyclic carbon-carbon double bond and a fluoroacetate activating group and exhibits broad spectrum activity against class A, C, and D serine ß-lactamases. The ester prodrug of ETX1317, ETX0282, is orally bioavailable and, in combination with cefpodoxime proxetil, is currently in development as an oral therapy for multidrug resistant and carbapenem-resistant Enterobacterales infections.

PAP inhibitor with in vivo efficacy identified by Candida albicans genetic profiling of natural products.

Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.

Isolation, structure and biological activity of phomafungin, a cyclic lipodepsipeptide from a widespread tropical Phoma sp.

We isolated a cyclic lipodepsipeptide, phomafungin, from a Phoma sp. The distinct antifungal activity of phomafungin in the crude extract was initially discovered by mechanistic profiling in the Candida albicans fitness test. The purified compound contains a 28 member ring consisting of eight amino acids and a beta-hydroxy-gamma-methyl-hexadecanoic acid, and displays a broad spectrum of antifungal activity against Candida spp., Aspergillus fumigatus and Trichophyton mentagrophytes with MIC of 2-8 microg/ml, and toxicity to mice at 25 mg/kg. The linear peptide derived from opening of the lactone ring was devoid of antifungal activity as well as toxicity. Phomafungin has been identified in a number of Phoma spp. collected from Africa and the Indian and Pacific Ocean islands.

Isolation, structure elucidation, and biological activity of virgineone from Lachnum Wirgineum using the genome-wide Candida albicans fitness test.

A glycosylated tetramic acid, virgineone (1), was isolated from saprotrophic Lachnum virgineum. The antifungal activity of the fermentation extract of L. virgineum was characterized in the Candida albicans fitness test as distinguishable from other natural products tested. Bioassay-guided fractionation yielded 1, a tyrosine-derived tetramic acid with a C-22 oxygenated chain and a beta-mannose. It displayed broad-spectrum antifungal activity against Candida spp. and Aspergillus fumigatus with a MIC of 4 and 16 microg/mL, respectively. Virgineone was also identified in a number of Lachnum strains collected from diverse geographies and habitats.

Development of an integrated semi-automated system for in vitro pharmacodynamic modelling.

OBJECTIVES: The aim of this study was to develop an integrated system for in vitro pharmacodynamic modelling of antimicrobials with greater flexibility, easier control and better accuracy than existing in vitro models. METHODS: Custom-made bottle caps, fittings, valve controllers and a modified bench-top shaking incubator were used. A temperature-controlled automated sample collector was built. Computer software was developed to manage experiments and to control the entire system including solenoid pinch valves, peristaltic pumps and the sample collector. The system was validated by pharmacokinetic simulations of linezolid 600 mg infusion. The antibacterial effect of linezolid against multiple Staphylococcus aureus strains was also studied in this system. RESULTS: An integrated semi-automated bench-top system was built and validated. The temperature-controlled automated sample collector allowed unattended collection and temporary storage of samples. The system software reduced the labour necessary for many tasks and also improved the timing accuracy for performing simultaneous actions in multiple parallel experiments. The system was able to simulate human pharmacokinetics of linezolid 600 mg intravenous infusion accurately. A pharmacodynamic study of linezolid against multiple S. aureus strains with a range of MICs showed that the required 24 h free drug AUC/MIC ratio was approximately 30 in order to keep the organism counts at the same level as their initial inoculum and was about > or = 68 in order to achieve > 2 log(10) cfu/mL reduction in the in vitro model. CONCLUSIONS: The integrated semi-automated bench-top system provided the ability to overcome many of the drawbacks of existing in vitro models. It can be used for various simple or complicated pharmacokinetic/pharmacodynamic studies efficiently and conveniently.

Effect of Susceptibility Testing Conditions on the In Vitro Antibacterial Activity of ETX0914.

The effect of various conditions including pH, inoculum, temperature, atmosphere, divalent cations, and several body fluids on the in vitro activity of the novel antibacterial spiropyrimidinetrione ETX0914 in standard susceptibility tests was investigated against several species. None of the parameters investigated affected the activity of ETX0914, with the exception of pH. Whereas the MIC values for ETX0914 with S. aureus, E. faecalis, and E. coli did not change when the pH of the growth medium was varied from 5 to 8, they did increase at least 8-fold at pH values above 8. This loss of activity can be attributed to the deprotonation of the molecule at elevated pH. The data suggest that routine susceptibility testing with ETX0914 should result in reproducible MIC values.

ETX2514 is a broad-spectrum ß-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii.

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of ß-lactamases, enzymes that inactivate ß-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new ß-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine ß-lactamase inhibitors that potently inhibit clinically relevant class A, C and D ß-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of ß-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.

4,5-Disubstituted 6-Aryloxy-1,3-dihydrobenzo[c][1,2]oxaboroles Are Broad-Spectrum Serine ß-Lactamase Inhibitors.

Bacterially expressed ß-lactamases are rapidly eroding the clinical utility of the important ß-lactam class of antibacterials, significantly impairing our ability to fight serious bacterial infections. This paper describes a study of oxaborole-derived ß-lactamase inhibitors in which crystal structures and computational modeling aided in the rational design of analogues with improved spectrum of activity against class A, C, and D enzymes. Crystal structures of two of these inhibitors covalently bound to two different serine ß-lactamases, class C Pseudomonas aeruginosa AmpC and class D OXA-10, are described herein. Improved physicochemical properties as well as increased activity against an array of ß-lactamases resulted in substantial restoration of susceptibility to ceftazidime in Escherichia coli and Klebsiella pneumoniae.

Caspofungin susceptibility in Aspergillus and non-Aspergillus molds: inhibition of glucan synthase and reduction of beta-D-1,3 glucan levels in culture.

Caspofungin inhibits synthesis of beta-D-1,3 glucan, essential to cell walls in Candida and Aspergillus spp., but activity against less common molds is largely uncharacterized. We demonstrate that caspofungin inhibits beta-D-1,3 glucan synthesis and reduces in vitro growth of clinical isolates from the genera Alternaria, Curvularia, Scedosporium, Acremonium, Bipolaris, and Trichoderma.

Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species.

Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual "prominent growth reduction" (MIC(2)) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode +/- one dilution range across all 17 laboratories. MIC(2) at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.