Covering: 1985 to 2001.This paper describes a fifteen year journey from concept to clinical discovery and development of the first in class caspofungin acetate (CANCIDAS®) a parenteral antifungal agent. Caspofungin is a semisynthetic derivative of pneumocandin B0, a naturally occurring, lipophilic cyclic peptide isolated from the fungus, Glarea lozoyensis. While the echinocandins had been previously studied for antifungal activity by several organizations, the class was dropped for a variety of reasons. Merck subsequently initiated a research program leading to the discovery and development of caspofungin. The multitude of challenges that ensued during the discovery and development process and which were successfully resolved by multi-disciplinary teams constitute the content of this article. The article consists of five sections that describe the discovery and development of caspofungin in chronological order: (i) discovery of the natural product pneumocandin B0 from fungal fermentations, (ii) fermentation development to improve the titer of pneumocandin B0 to make it commercially viable, (iii) semisynthetic modification by medicinal chemistry to successfully improve the properties of pneumocandin B0 leading to the discovery of caspofungin, (iv) development of commercial semisynthesis and purification and formulation development to improve stability and (v) clinical development and approval of CANCIDAS® as an antifungal drug which subsequently saved thousands of lives.
Many volatile organic compounds (VOCs) are found in indoor environment as products of microbial metabolism. In damp indoor environments, fungi are associated with poor air quality. Some epidemiological studies have suggested that microbial VOCs have a negative impact on human health. Our study was designed to provide a reductionist approach toward studying fungal VOC-mediated toxicity using the inexpensive model organism, Drosophila melanogaster, and pure chemical standards of several important fungal VOCs. Low concentrations of the following known fungal VOCs, 0.1% of 1-octen-3-ol and 0.5% of 2-octanone; 2,5 dimethylfuran; 3-octanol; and trans-2-octenal, caused locomotory defects and changes in green fluorescent protein (GFP)- and antigen-labeled dopaminergic neurons in adult D. melanogaster. Locomotory defects could be partially rescued with L-DOPA. Ingestion of the antioxidant, vitamin E, improved the survival span and delayed the VOC-mediated changes in dopaminergic neurons, indicating that the VOC-mediated toxicity was due, in part, to generation of reactive oxygen species.
Zalerion arboricola ATCC20868 produces pneumocandin Ao (L-67 1 ,329), a cyclic hexapeptide with a dimethylmyristic acid side chain. This compoundhas anti-candida and anti-pneumocystis activities. We were interested in looking for other related compounds produced by this organism. To facilitate this search, a simple medium (S2) composed of D-mannitol, peptonized milk, lactic acid, glycine, KH2PO4and trace elements, which supported the production ofa number of such compounds, was designed. For the isolation of mutants, either spores or growing mycelia were treated with TV-nitroso-TV-methylurethaneor Af-methyl-Af'-nitro-TV-nitrosoguanidine and survivors were screened for changes in the product spectrum. From approximately 1,500 survivors tested, 5 mutants were isolated. Mutants ATCC20957, 74030, 20958 and 20988 exclusively produce various pneumocandins other than Ao. These compoundswere active against Candida and Pneumocystis carinii. The yield of Ao was found to be increased 2.5-fold over that of the parent in the fifth mutant, MF5415. Further mediumstudies indicated that the addition of soybean oil to S2 mediumimproved the yields.
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.
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