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.

In Vitro and In Vivo Characterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase.

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, and in vivo characterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli and displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergence in vitro at concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activity in vitro and in vivo.

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro ß-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the ß-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore ß-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

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.

Selective small-molecule inhibition of an RNA structural element.

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

Isolation, structure elucidation and antibacterial activity of a new tetramic acid, ascosetin.

The ever-increasing bacterial resistance to clinical antibiotics is making many drugs ineffective and creating significant treatment gaps. This can be only circumvented by the discovery of antibiotics with new mechanisms of action. We report here the identification of a new tetramic acid, ascosetin, from an Ascomycete using the Staphylococcus aureus fitness test screening method. The structure was elucidated by spectroscopic methods including 2D NMR and HRMS. Relative stereochemistry was determined by ROESY and absolute configuration was deduced by comparative CD spectroscopy. Ascosetin inhibited bacterial growth with 2-16 µg ml(-1) MIC values against Gram-positive strains including methicillin-resistant S. aureus. It also inhibited the growth of Haemophilus influenzae with a MIC value of 8 µg ml(-1). It inhibited DNA, RNA, protein and lipid synthesis with similar IC50 values, suggesting a lack of specificity; however, it produced neither bacterial membrane nor red blood cell lysis. It showed selectivity for bacterial growth inhibition compared with fungal but not mammalian cells. The isolation, structure and biological activity of ascosetin have been detailed here.

Discovery of wall teichoic acid inhibitors as potential anti-MRSA ß-lactam combination agents.

Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore ß-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA ß-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies.

Restoring methicillin-resistant Staphylococcus aureus susceptibility to ß-lactam antibiotics.

Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to ß-lactam antibiotics paired with ß-lactamase inhibitors. We have adapted a Staphylococcus aureus antisense knockdown strategy to genetically identify the cell division Z ring components-FtsA, FtsZ, and FtsW-as ß-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA). We demonstrate that the FtsZ-specific inhibitor PC190723 acts synergistically with ß-lactam antibiotics in vitro and in vivo and that this combination is efficacious in a murine model of MRSA infection. Fluorescence microscopy localization studies reveal that synergy between these agents is likely to be elicited by the concomitant delocalization of their cognate drug targets (FtsZ and PBP2) in MRSA treated with PC190723. A 2.0 Å crystal structure of S. aureus FtsZ in complex with PC190723 identifies the compound binding site, which corresponds to the predominant location of mutations conferring resistance to PC190723 (PC190723(R)). Although structural studies suggested that these drug resistance mutations may be difficult to combat through chemical modification of PC190723, combining PC190723 with the ß-lactam antibiotic imipenem markedly reduced the spontaneous frequency of PC190723(R) mutants. Multiple MRSA PC190723(R) FtsZ mutants also displayed attenuated virulence and restored susceptibility to ß-lactam antibiotics in vitro and in a mouse model of imipenem efficacy. Collectively, these data support a target-based approach to rationally develop synergistic combination agents that mitigate drug resistance and effectively treat MRSA infections.

Efficacy of caspofungin in a juvenile mouse model of central nervous system candidiasis.

Neonatal candidiasis is an increasingly common occurrence causing significant morbidity and mortality and a higher risk of dissemination to the central nervous system (CNS) than that seen with older patients. The current understanding of optimal antifungal therapy in this setting is limited. We have developed a model of disseminated candidiasis with CNS involvement in juvenile mice to assess the efficacy of the echinocandin caspofungin relative to amphotericin B (AmB). Juvenile mice were inoculated intravenously with 5.64 × 10(4) CFU of Candida albicans MY1055. Treatment with caspofungin at 1, 2, 4, and 8 mg/kg of body weight/day, AmB at 1 mg/kg/day, or a vehicle control (VC) was initiated 30 h after infection and continued for 7 days. Pharmacokinetic parameters for caspofungin were also determined. Culture and histology showed evidence of disseminated candidiasis with multifocal encephalitis at the start of antifungal therapy. Survival was 100% in all treated groups, while mortality was 100% in the VC by day 11 after infection. By day 5, all mice in the caspofungin treatment (four doses) groups showed reductions in kidney and brain burden relative to the VC, while AmB treatment reduced kidney burden but gave no reduction of brain fungal burden. Systemic levels of caspofungin were similar in infected and uninfected mice, while brain levels were higher in infected animals. In this juvenile mouse model, caspofungin demonstrated dose-dependent activity, equivalent to or better than that of AmB at 1 mg/kg, against disseminated candidiasis with CNS involvement.

In vivo efficacy of a novel oxazolidinone compound in two mouse models of infection.

A novel oxazolidinone, AM 7359, was evaluated in two mouse models of Staphylococcus aureus infection. AM 7359 and linezolid were equally efficacious in a methicillin-susceptible S. aureus organ burden model and a methicillin-resistant S. aureus localized infection model. However, AM 7359 was eightfold more efficacious than linezolid against a linezolid- and methicillin-resistant S. aureus strain in this localized (thigh) infection model.