Emily Hickey

Isolation, structure, and antibacterial activity of philipimycin, a thiazolyl peptide discovered from Actinoplanes philippinensis MA7347

Bacterial resistance to antibiotics, particularly to multiple drug resistant antibiotics, is becoming cause for significant concern. The only really viable course of action is to discover new antibiotics with novel mode of actions. Thiazolyl peptides are a class of natural products that are architecturally complex potent antibiotics but generally suffer from poor solubility and pharmaceutical properties. To discover new thiazolyl peptides potentially with better desired properties, we designed a highly specific assay with a pair of thiazomycin sensitive and resistant strains of Staphylococcus aureus, which led to the discovery of philipimycin, a new thiazolyl peptide glycoside. It was isolated along with an acid-catalyzed degradation product by bioassay-guided fractionation. Structure of both compounds was elucidated by extensive application of 2D NMR, 1D TOCSY, and HRESIFT-MS/MS. Both compounds showed strong antibacterial activities against gram-positive bacteria including MRSA and exhibited MIC values ranging from 0.015 to 1 microg/mL. Philipimycin was significantly more potent than the degradation product. Both compounds showed selective inhibition of protein synthesis, indicating that they targeted the ribosome. Philipimycin was effective in vivo in a mouse model of S. aureus infection exhibiting an ED50 value of 8.4 mg/kg. The docking studies of philipimycin suggested that a part of the molecule interacts with the ribosome and another part with Pro23, Pro22, and Pro26 of L11 protein, which helped in explaining the differential of activities between the sensitive and resistant strains. The design and execution of the bioassay, the isolation, structure, in vitro and in vivo antibacterial activity, and docking studies of philipimycin and its degradation product are described.

Isolation, structure elucidation, and biological activity of virgineone from Lachnum virgineum 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.

Antibacterial evaluations of thiazomycin- a potent thiazolyl peptide antibiotic from Amycolatopsis fastidiosa.

Thiazomycin is a novel thiazolyl peptide closely related to nocathiacin I. It was isolated from Amycolatopsis fastidiosa by chemical and biological screening. Thiazomycin showed highly potent bactericidal activity against Gram-positive pathogens (MIC range 0.002~0.064 μg/ml) and did not show cross-resistance to clinically relevant antibiotic classes such as β-lactams, vancomycin, oxazolidinone and quinolones. It was highly efficacious against Staphylococcus aureus infection in mice exhibiting an ED99 value of 0.15 mg/kg by subcutaneous administration. It inhibited bacterial growth by selective inhibition of protein synthesis and it was thought to interact with L11 protein and 23S rRNA of the 50S ribosome. Structurally, it possesses an oxazolidine ring in the amino-sugar residue that provides further opportunities for selective chemical modifications that are not feasible with other thiazolyl peptides. More importantly such a modification can potentially lead to semi-synthetic compounds that overcome problems that have hampered clinical development of this class of compounds. Despite its positive attributes, emergence of an unacceptable frequency of resistance poses significant challenges for further development of thiazomycin and this class of molecules for therapeutic use.

Side Chain Sar of Bicyclic Beta-Lactamase Inhibitors (Blis). 1. Discovery of a Class C Bli for Combination with Imipinem.

Bridged monobactam beta-lactamase inhibitors were prepared and evaluated as potential partners for combination with imipenem to overcome class C beta-lactamase mediated resistance. The (S)-azepine analog 2 was found to be effective in both in vitro and in vivo assays and was selected for preclinical development.

Efficacy of Caspofungin against Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans

ABSTRACT The echinocandin caspofungin is a potent inhibitor of the activity of 1,3-β-d-glucan synthase from Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans. In murine models of disseminated infection, caspofungin prolonged survival and reduced the kidney fungal burden. Caspofungin was at least as effective as amphotericin B against these filamentous fungi in vivo.

Inhibition of Spleen Tyrosine Kinase Attenuates Allergen-Mediated Airway Constriction

Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.

Nocathiacin analogs: Synthesis and antibacterial activity of novel water-soluble amides

Novel water-soluble amide analogs were synthesized from nocathiacin I (1) through the formation of the carboxylic acid intermediate followed by coupling to primary or secondary amines. Several compounds with potent antibacterial activity and adequate water solubility were identified. Of these, compound 19 was selected for more extensive evaluation because of its excellent in vitro antibacterial activity and in vivo efficacy, as well as clean off-target screening.

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.

In Vivo Efficacy of a Novel Oxazolidinone Compound in Two Mouse Models of Infection

ABSTRACT 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.

Efficacy of Caspofungin in a Juvenile Mouse Model of Central Nervous System Candidiasis

ABSTRACT 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 × 104 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.