Cedric J. Pearce

Romidepsin (Istodax, NSC 630176, FR901228, FK228, depsipeptide): a natural product recently approved for cutaneous T-cell lymphoma.

Romidepsin (Istodax), a selective inhibitor of histone deacetylases (HDACs), was approved for the treatment of cutaneous T-cell lymphoma in November 2009 by the US Food and Drug Administration. This unique natural product was discovered from cultures of Chromobacterium violaceum, a Gram-negative bacterium isolated from a Japanese soil sample. This bicyclic compound acts as a prodrug, its disulfide bridge being reduced by glutathione on uptake into the cell, allowing the free thiol groups to interact with Zn ions in the active site of class I and II HDAC enzymes. Due to the synthetic complexity of the compound, as well as the low yield from the producing organism, analogs are sought to create synthetically accessible alternatives. As a T-cell lymphoma drug, romidepsin offers a valuable new treatment for diseases with few effective therapies.

Fingolimod (FTY720): A Recently Approved Multiple Sclerosis Drug Based on a Fungal Secondary Metabolite

Fingolimod (Gilenya; FTY720), a synthetic compound based on the fungal secondary metabolite myriocin (ISP-I), is a potent immunosuppressant that was approved (September 2010) by the U.S. FDA as a new treatment for multiple sclerosis (MS). Fingolimod was synthesized by the research group of Tetsuro Fujita at Kyoto University in 1992 while investigating structure-activity relationships of derivatives of the fungal metabolite ISP-I, isolated from Isaria sinclairii. Fingolimod becomes active in vivo following phosphorylation by sphingosine kinase 2 to form fingolimod-phosphate, which binds to extracellular G protein-coupled receptors, sphingosine 1-phosphates, and prevents the release of lymphocytes from lymphoid tissue. Fingolimod is orally active, which is unique among current first-line MS therapies, and it has the potential to be used in the treatment of organ transplants and cancer. This review highlights the discovery and development of fingolimod, from an isolated lead natural product, through synthetic analogues, to an approved drug.

High-resolution MS, MS/MS, and UV database of fungal secondary metabolites as a dereplication protocol for bioactive natural products.

A major problem in the discovery of new biologically active compounds from natural products is the reisolation of known compounds. Such reisolations waste time and resources, distracting chemists from more promising leads. To address this problem, dereplication strategies are needed that enable crude extracts to be screened for the presence of known compounds before isolation efforts are initiated. In a project to identify anticancer drug leads from filamentous fungi, a significant dereplication challenge arises, as the taxonomy of the source materials is rarely known, and, thus, the literature cannot be probed to identify likely known compounds. An ultraperformance liquid chromatography-photodiode array-high-resolution tandem mass spectrometric (UPLC-PDA-HRMS-MS/MS) method was developed for dereplication of fungal secondary metabolites in crude culture extracts. A database was constructed by recording HRMS and MS/MS spectra of fungal metabolites, utilizing both positive- and negative-ionization modes. Additional details, such as UV-absorption maxima and retention times, were also recorded. Small-scale cultures that showed cytotoxic activities were dereplicated before engaging in the scale-up or purification processes. Using these methods, approximately 50% of the cytotoxic extracts could be eliminated from further study after the confident identification of known compounds. The specific attributes of this dereplication methodology include a focus on bioactive secondary metabolites from fungi, the use of a 10 min chromatographic method, and the inclusion of both HRMS and MS/MS data.

Discovery of anticancer agents of diverse natural origin.

A collaborative multidisciplinary research project is described in which new natural product anticancer drug leads are obtained from a diverse group of organisms, constituted by tropical plants, aquatic cyanobacteria, and filamentous fungi. Information is provided on how these organisms are collected and processed. The types of bioassays are indicated in which crude extracts of these acquisitions are tested. Progress made in the isolation of lead bioactive secondary metabolites from three tropical plants is discussed.

Resorcylic acid lactones with cytotoxic and NF-κB inhibitory activities and their structure-activity relationships.

As part of our ongoing investigation of filamentous fungi for anticancer leads, an active fungal extract was identified from the Mycosynthetix library (MSX 63935; related to Phoma sp.). The initial extract exhibited cytotoxic activity against the H460 (human non-small cell lung carcinoma) and SF268 (human astrocytoma) cell lines and was selected for further study. Bioactivity-directed fractionation yielded resorcylic acid lactones (RALs) 1 (a new natural product) and 3 (a new compound) and the known RALs zeaenol (2), (5E)-7-oxozeaenol (4), (5Z)-7-oxozeaenol (5), and LL-Z1640-1 (6). Reduction of (5E)-7-oxozeaenol (4) with sodium borohydride produced 3, which allowed assignment of the absolute configuration of 3. Other known resorcylic acid lactones (7-12) were purchased and assayed in parallel for cytotoxicity with isolated 1-6 to investigate structure-activity relationships in the series. Moreover, the isolated compounds (1-6) were examined for activity in a suite of biological assays, including antibacterial, mitochondria transmembrane potential, and NF-κB. In the latter assay, compounds 1 and 5 displayed sub-micromolar activities that were on par with the positive control, and as such, these compounds may serve as a lead scaffold for future medicinal chemistry studies.

Revisiting the enniatins: a review of their isolation, biosynthesis, structure determination and biological activities.

Enniatins are cyclohexadepsipeptides isolated largely from Fusarium species of fungi, although they have been isolated from other genera, such as Verticillium and Halosarpheia. They were first described over 60 years ago, and their range of biological activities, including antiinsectan, antifungal, antibiotic and cytotoxic, drives contemporary interest. To date, 29 enniatins have been isolated and characterized, either as a single compound or mixtures of inseparable homologs. Structurally, these depsipeptides are biosynthesized by a multifunctional enzyme, termed enniatin synthetase, and are composed of six residues that alternate between N-methyl amino acids and hydroxy acids. Their structure elucidation can be challenging, particularly for enniatins isolated as inseparable homologs; however, several strategies and tools have been utilized to solve these problems. Currently, there is one drug that has been developed from a mixture of enniatins, fusafungine, which is used as a topical treatment of upper respiratory tract infections by oral and/or nasal inhalation. Given the range of biological activities observed for this class of compounds, research on enniatins will likely continue. This review strives to digest the past studies, as well as, describe tools and techniques that can be utilized to overcome the challenges associated with the structure elucidation of mixtures of enniatin homologs.

Peptaibols from two unidentified fungi of the order Hypocreales with cytotoxic, antibiotic, and anthelmintic activities

As part of an ongoing investigation of filamentous fungi for anticancer leads, an active culture was identified from the Mycosynthetix library (MSX 70741, of the order Hypocreales, Ascomycota). The fungal extract exhibited cytotoxic activity against the H460 (human nonsmall cell lung carcinoma) cell line, and bioactivity‐directed fractionation yielded peptaibols 1–12 and harzianums A (13) and B (14). Structure elucidation of 1–12 was facilitated by high‐resolution MS/MS using higher‐energy collisional dissociation and by high field NMR (950 MHz). The absolute configuration was determined by Marfeys analysis of the individual amino acids; the time required for such analysis was decreased via the development of a 10‐min ultra performance liquid chromatography method. The isolated peptaibols (1–12), along with three other peptaibols isolated and elucidated from a different fungus (MSX 57715) of the same order (15–17), were examined for activity in a suite of biological assays, including those for cytotoxic, antibacterial, and anthelmintic activities. Copyright

Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community

Fungi are morphologically, ecologically, metabolically, and phylogenetically diverse. They are known to produce numerous bioactive molecules, which makes them very useful for natural products researchers in their pursuit of discovering new chemical diversity with agricultural, industrial, and pharmaceutical applications. Despite their importance in natural products chemistry, identification of fungi remains a daunting task for chemists, especially those who do not work with a trained mycologist. The purpose of this review is to update natural products researchers about the tools available for molecular identification of fungi. In particular, we discuss (1) problems of using morphology alone in the identification of fungi to the species level; (2) the three nuclear ribosomal genes most commonly used in fungal identification and the potential advantages and limitations of the ITS region, which is the official DNA barcoding marker for species-level identification of fungi; (3) how to use NCBI-BLAST search for DNA barcoding, with a cautionary note regarding its limitations; (4) the numerous curated molecular databases containing fungal sequences; (5) the various protein-coding genes used to augment or supplant ITS in species-level identification of certain fungal groups; and (6) methods used in the construction of phylogenetic trees from DNA sequences to facilitate fungal species identification. We recommend that, whenever possible, both morphology and molecular data be used for fungal identification. Our goal is that this review will provide a set of standardized procedures for the molecular identification of fungi that can be utilized by the natural products research community.

Cytotoxic epipolythiodioxopiperazine alkaloids from filamentous fungi of the Bionectriaceae

Bioactivity-directed fractionation of the organic extracts of two filamentous fungi of the Bionectriaceae, strains MSX 64546 and MSX 59553 from the Mycosynthetix library, led to the isolation of a new dimeric epipolythiodioxopiperazine alkaloid, verticillin H (1), along with six related analogs, Sch 52900 (2), verticillin A (3), gliocladicillin C (4), Sch 52901 (5), 11′-deoxyverticillin A (6) and gliocladicillin A (7). The structures of compounds 1–7 were determined by extensive NMR and HRMS analyses, as well as by comparisons to the literature. All compounds (1–7) were evaluated for cytotoxicity against a panel of human cancer cell lines, displaying IC50 values ranging from 1.2 μM to 10 nM. Compounds 1–5 were examined for activity in the NF-κB assay, where compounds 2 and 3 revealed activity in the sub-micromolar range. Additionally, compounds 1, 3 and 4 were tested for EGFR inhibition using an enzymatic assay, while compound 3 was examined against an overexpressing EGFR+ve cancer cell line.

Benzoquinones and Terphenyl Compounds As Phosphodiesterase-4B Inhibitors from a Fungus of the Order Chaetothyriales (MSX 47445)

Three bioactive compounds were isolated from an organic extract of an ascomycete fungus of the order Chaetothyriales (MSX 47445) using bioactivity-directed fractionation as part of a search for anticancer leads from filamentous fungi. Of these, two were benzoquinones [betulinan A (1) and betulinan C (3)], and the third was a terphenyl compound, BTH-II0204-207:A (2). The structures were elucidated using a set of spectroscopic and spectrometric techniques; the structure of the new compound (3) was confirmed via single-crystal X-ray diffraction. Compounds 1-3 were evaluated for cytotoxicity against a human cancer cell panel, for antimicrobial activity against Staphylococcus aureus and Candida albicans, and for phosphodiesterase (PDE4B2) inhibitory activities. The putative binding mode of 1-3 with PDE4B2 was examined using a validated docking protocol, and the binding and enzyme inhibitory activities were correlated.