Joseph Media

Evolved diversification of a modular natural product pathway: Apratoxins F and G, two cytotoxic cyclic depsipeptides from a palmyra collection of Lyngbya bouillonii

A collection of Lyngbya bouillonii from Palmyra Atoll in the Central Pacific, a site several thousand kilometers distant from all previous collections of this chemically prolific species of cyanobacterium, was found to contain two new cancer cell cytotoxins of the apratoxin family. The structures of the new compounds, apratoxins F and G, were determined by 1D and 2D NMR techniques in combination with mass spectrometric methods. Stereochemistry was explored by using chromatographic analyses of the hydrolytically released fragments in combination with NMR and optical rotation comparisons with known members of the apratoxin family. Apratoxins F and G add fresh insights into the SAR of this family because they incorporate an N‐methyl alanine residue at a position where all prior apratoxins have possessed a proline unit, yet they retain high potency as cytotoxins to H‐460 cancer cells with IC50 values of 2 and 14 nM, respectively. Additional assays using zone inhibition of cancer cells and clonogenic cells give a comparison of the activities of apratoxin F to apratoxin A. Additionally, the clonogenic studies in combination with maximum tolerated dose (MTD) studies provided insights as to dosing schedules that should be used for in vivo studies, and preliminary in vivo evaluation validated the predicted in vivo efficacy for apratoxin A. These new apratoxins are illustrative of a mechanism (the modification of an NRPS adenylation domain specificity pocket) for evolving a biosynthetic pathway so as to diversify the suite of expressed secondary metabolites.

Desmethoxymajusculamide C, a cyanobacterial depsipeptide with potent cytotoxicity in both cyclic and ring-opened forms

Cytotoxicity-guided fractionation of the organic extract from a Fijian Lyngbya majuscula led to the discovery of desmethoxymajusculamide C (DMMC) as the active metabolite. Spectroscopic analysis including 1D and 2D NMR, MS/MS, and chemical degradation and derivatization protocols were used to assign the planar structure and stereoconfiguration of this new cyclic depsipeptide. DMMC demonstrated potent and selective anti-solid tumor activity with an IC(50) = 20 nM against the HCT-116 human colon carcinoma cell line via disruption of cellular microfilament networks. A linear form of DMMC was generated by base hydrolysis, and the amino acid sequence was confirmed by mass spectrometry. Linearized DMMC was also evaluated in the biological assays and found to maintain potent actin depolymerization characteristics while displaying solid tumor selectivity equivalent to DMMC in the disk diffusion assay. A clonogenic assay assessing cytotoxicity to HCT-116 cells as a function of exposure duration showed that greater than 24 h of constant drug treatment was required to yield significant cell killing. Therapeutic studies with HCT-116 bearing SCID mice demonstrated efficacy at the highest dose used (%T/C = 60% at 0.62 mg/kg daily for 5 days).

Utilizing DART Mass Spectrometry to Pinpoint Halogenated Metabolites from a Marine Invertebrate-Derived Fungus

Prenylated indole alkaloids are a diverse group of fungal secondary metabolites and represent an important biosynthetic class. In this study we have identified new halogenated prenyl-indole alkaloids from an invertebrate-derived Malbranchea graminicola strain. Using direct analysis in real time (DART) mass spectrometry, these compounds were initially detected from hyphae of the fungus grown on agar plates, without the need for any organic extraction. Subsequently, the metabolites were isolated from liquid culture in artificial seawater. The structures of two novel chlorinated metabolites, named (-)-spiromalbramide and (+)-isomalbrancheamide B, provide additional insights into the assembly of the malbrancheamide compound family. Remarkably, two new brominated analogues, (+)-malbrancheamide C and (+)-isomalbrancheamide C, were produced by enriching the growth medium with bromine salts.

Interrogating the bioactive pharmacophore of the latrunculin chemotype by investigating the metabolites of two taxonomically unrelated sponges

This study involved a campaign to isolate and study additional latrunculin analogues from two taxonomically unrelated sponges, Cacospongia mycofijiensis and Negombata magnifica. A total of 13 latrunculin analogues were obtained by four different ways, reisolation (1-4), our repository (5, 6), new derivatives (7-12), and a synthetic analogue (7a). The structures of the new metabolites were elucidated on the basis of a combination of comprehensive 1D and 2D NMR analysis, application of DFT calculations, and the preparation of acetonide derivative 7a. The cytotoxicities against both murine and human cancer cell lines observed for 1, 2, 7, 7a, 8, 9, and 12 were significant, and the IC(50) range was 0.5-10 microM. Among the cytotoxic derivatives, compound 9 did not exhibit microfilament-disrupting activity at 5 microM. The implications of this observation and the value of further therapeutic study on key latrunculin derivatives are discussed.

Biostructural Features of Additional Jasplakinolide (Jaspamide) Analogues

The cyclodepsipeptide jasplakinolide (1) (aka jaspamide), isolated previously from the marine sponge Jaspis splendens, is a unique cytotoxin and molecular probe that operates through stabilization of filamentous actin (F-actin). We have recently disclosed that two analogues of 1, jasplakinolides B (3) and E, were referred to the National Cancer Institutes (NCI) Biological Evaluation Committee, and the objective of this study was to reinvestigate a Fijian collection of J. splendens in an effort to find jasplakinolide congeners with similar biological properties. The current efforts have afforded six known jasplakinolide analogues (4-7, 9, 10), two structures requiring revision (8 and 14), and four new congeners of 1 (11-13, 15) including open-chain derivatives and structures with modified β-tyrosine residues. Compounds were evaluated for biological activity in the NCIs 60 cell line screen and in a microfilament disruption assay in both HCT-116 and HeLa cells. These two phenotypic screens provide evidence that each cytotoxic analogue, including jasplakinolide B (3), operates by modification of microfilaments. The new structure jasplakinolide V (13) has also been selected for study by the NCIs Biological Evaluation Committee. In addition, the results of a clonogenic dose-response study on jasplakinolide are presented.

Resveratrol Decreases Noise-Induced Cyclooxygenase-2 Expression in the Rat Cochlea

Objective Our previous studies have demonstrated the efficacy of resveratrol, a grape constituent noted for its antioxidant and anti-inflammatory properties, in reducing temporary threshold shifts and decreasing cochlear hair cell damage following noise exposure. This study was designed to identify the potential protective mechanism of resveratrol by measuring its effect on cyclooxygenase-2 (COX-2) protein expression and reactive oxygen species (ROS) formation following noise exposure. Study Design Controlled animal intervention study. Setting Otology Laboratory, Henry Ford Health System. Subjects and Methods Twenty-two healthy male Fischer 344 rats (2-3 months old) were exposed to acoustic trauma of variable duration with or without intervention. An additional 20 healthy male rats were used to study COX-2 expression at different time points during and following treatment of 24 hours of noise exposure. Cochlear harvest was performed at various time intervals for measurement of COX-2 protein expression via Western blot analysis and immunostaining. Peripheral blood was also obtained for ROS analysis using flow cytometry. Results Acoustic trauma exposure resulted in a progressive up-regulation of COX-2 protein expression, commencing at 8 hours and peaking at 32 hours. Similarly, ROS production increased after noise exposure. However, treatment with resveratrol reduced noise-induced COX-2 expression as well as ROS formation in the blood as compared with the controls. Conclusion COX-2 levels are induced dramatically following noise exposure. This increased expression may be a potential mechanism of noise-induced hearing loss (NIHL) and a possible mechanism of resveratrol’s ability to mitigate NIHL by its ability to reduce COX-2 expression.

Immunomodulatory Activity of Synthetic Triterpenoids: Inhibition of Lymphocyte Proliferation, Cell-Mediated Cytotoxicity, and Cytokine Gene Expression Through Suppression of NF-κB

Synthetic oleanane triterpenoids (CDDO, CDDO-Im and CDDO-Me) are potent anti-inflammatory agents, but have not been investigated for effects on T cell-mediated immune responses. Here we demonstrate that CDDOs have profound immunosuppressive effects on T cell proliferation, development of IL-2 activated LAK cells and cytotoxic T lymphocytes (CTLs), and expression of cytokines at concentrations of 1.25 μM to 0.078 μM. Treatment with CDDO-Me also inhibited the generation of allo-reactive T cell responses in vivo. The suppression of these cell-mediated immune responses by CDDO-Me was associated with the inhibition of NF-κB transcription factor.

Development and Validation of a Simple Method for the Detection of Fascaplysin in Plasma

Fascaplysin is a cytotoxic natural product isolated from a variety of Indo-Pacific marine organisms, primarily Fascaplysinopsis sponges and Didemnum tunicates. Positive xenograft studies involving this alkaloid structural class have indicated that fascaplysin may serve as an important lead compound for preclinical development. This study was undertaken as a prelude to a full pharmacokinetics and therapeutic assessment of fascaplysin. We describe here a simple plasma preparation and a rapid HPLC method for the detection of fascaplysin in mice. The method was validated by parameters including good linear correlation, a limit of quantification of 107.1 μg/mL, and a good precision with a coefficient of variation of 0.92% for 10 μg/mL. This method provides excellent sensitivity and visualization of fascaplysin as a single peak allowing for rapid analysis of plasma samples involving absorption, distribution, and metabolism studies. A preliminary pharmacokinetics study in C57Bl/6 mice using 20.6 mg/kg fascaplysin yielded a biphasic curve with T½α=16.7 min, T½β=11.7 h, and C0 of 17.1 μg/mL.

Inhibition of macromolecular synthesis by cryptophycin-52.

Cryptophycin (CP)-52, a synthetic analog of CP-1, possesses potent and selective antiproliferative activity against human solid tumors both in vitro and in vivo. Based on an algorithm developed in this laboratory using HCT-116 human colon adenocarcinoma cells, CP-52 exhibited a time- and concentration-dependent antiproliferative effect in the in vitro clonogenic assay. Inhibition of both DNA and RNA synthesis was observed in the absence of any effect on protein synthesis following a 24-h exposure to CP-52, at a time when proliferating cells were arrested in the G2/M phase of the cell cycle. In summary, we interpret these data to indicate that the selective inhibition of DNA synthesis may be a major causative factor responsible for the antiproliferative activity of CP-52 and subsequent G2/M arrest.