Tyler A. Johnson

The marine sponge metabolite mycothiazole: A novel prototype mitochondrial complex I inhibitor

A natural product chemistry-based approach was applied to discover small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1). A Petrosaspongia mycofijiensis marine sponge extract yielded mycothiazole (1), a solid tumor selective compound with no known mechanism for its cell line-dependent cytotoxic activity. Compound 1 inhibited hypoxic HIF-1 signaling in tumor cells (IC(50) 1nM) that correlated with the suppression of hypoxia-stimulated tumor angiogenesis in vitro. However, 1 exhibited pronounced neurotoxicity in vitro. Mechanistic studies revealed that 1 selectively suppresses mitochondrial respiration at complex I (NADH-ubiquinone oxidoreductase). Unlike rotenone, MPP(+), annonaceous acetogenins, piericidin A, and other complex I inhibitors, mycothiazole is a mixed polyketide/peptide-derived compound with a central thiazole moiety. The exquisite potency and structural novelty of 1 suggest that it may serve as a valuable molecular probe for mitochondrial biology and HIF-mediated hypoxic signaling.

Mass Stranding of Marine Birds Caused by a Surfactant-Producing Red Tide

In November-December 2007 a widespread seabird mortality event occurred in Monterey Bay, California, USA, coincident with a massive red tide caused by the dinoflagellate Akashiwo sanguinea. Affected birds had a slimy yellow-green material on their feathers, which were saturated with water, and they were severely hypothermic. We determined that foam containing surfactant-like proteins, derived from organic matter of the red tide, coated their feathers and neutralized natural water repellency and insulation. No evidence of exposure to petroleum or other oils or biotoxins were found. This is the first documented case of its kind, but previous similar events may have gone undetected. The frequency and amplitude of red tides have increased in Monterey Bay since 2004, suggesting that impacts on wintering marine birds may continue or increase.

Azonazine, a novel dipeptide from a Hawaiian marine sediment-derived fungus, Aspergillus insulicola.

Azonazine, a unique hexacyclic dipeptide, was isolated from a Hawaiian marine sediment-derived fungus eventually identified as Aspergillus insulicola. Its absolute configuration, 2R,10R,11S,19R, was established using NMR, HRESIMS, and CD data plus insights derived from molecular models. A possible route for its biogenesis is proposed, and biological properties were explored against cancer cell lines and in an NFκB inhibition assay.

Natural Product Libraries to Accelerate the High Throughput Discovery of Therapeutic Leads

A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.

Structures and cytotoxicities of fascaplysin and related alkaloids from two marine phyla—Fascaplysinopsis sponges and Didemnum tunicates

The structural variations and bioactivity properties of the alkaloids in the fascaplysin (1) and the reticulatine (3) families were examined. Four organisms were analyzed consisting of two collections of the sponge Fascaplysinopsis reticulata and two collections of the tunicate Didemnum sp. Reported are the isolation of three new compounds: 3-bromofascaplysin (2), 14-bromoreticulatine (4), and 14-bromoreticulatate (6) along with reticulatate (5) previously known as a semi-synthetic product of 1. Compounds 1 and 5 showed selectivity in a cell based cytotoxicity assay.

Rapid enzyme-linked immunosorbent assay for detection of the algal toxin domoic acid

Abstract Domoic acid (DA) is a potent toxin produced by bloom-forming phytoplankton in the genus Pseudo-nitzschia, which is responsible for causing amnesic shellfish poisoning (ASP) in humans. ASP symptoms include vomiting, diarrhea, and in more severe cases confusion, loss of memory, disorientation, and even coma or death. This paper describes the development and validation of a rapid, sensitive, enzyme linked immunosorbent assay test kit for detecting DA using a monoclonal antibody. The assay gives equivalent results to those obtained using standard high performance liquid chromatography, fluorenylmethoxycarbonyl high performance liquid chromatography, or liquid chromatography—mass spectrometry methods. It has a linear range from 0.1–3 ppb and was used successfully to measure DA in razor clams, mussels, scallops, and phytoplankton. The assay requires approximately 1.5 h to complete and has a standard 96-well format where each strip of eight wells is removable and can be stored at 4°C until needed. The first two wells of each strip serve as an internal control eliminating the need to run a standard curve. This allows as few as 3 or as many as 36 duplicate samples to be run at a time enabling real-time sample processing and limiting degradation of DA, which can occur during storage. There was minimal cross-reactivity in this assay with glutamine, glutamic acid, kainic acid, epi- or iso-DA. This accurate, rapid, cost-effective, assay offers environmental managers and public health officials an effective tool for monitoring DA concentrations in environment samples.

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.

Assessing Pressurized Liquid Extraction for the High-Throughput Extraction of Marine-Sponge Derived Natural Products

In order to compare the utility of standard solvent partitioning (SSP) versus accelerated solvent extraction (ASE), a series of experiments were performed and evaluated. Overall yields, solvent consumption, processing time, and chemical stability of the fractions obtained by both methods were compared. Five marine sponges were selected for processing and analysis containing 12 structurally distinct, bioactive natural products. Extracts generated using SSP and ASE were assessed for chemical degradation using comparative LC MS-ELSD. The extraction efficiency (EE) of the ASE apparatus was 3 times greater than the SSP method on average, while the total extraction yields (TEY) were roughly equivalent. Furthermore, the ASE methodology required only 2 h to process each sample versus 80 h for SSP, and the LC MS-ELSD from extracts of both methods appeared comparable. These results demonstrate that ASE can serve as an effective high-throughput methodology for extracting marine organisms to streamline the discovery of novel and bioactive natural products.

Lipophilic stinging nettle extracts possess potent anti-inflammatory activity, are not cytotoxic and may be superior to traditional tinctures for treating inflammatory disorders.

Extracts of four plant portions (roots, stems, leaves and flowers) of Urtica dioica (the stinging nettle) were prepared using accelerated solvent extraction (ASE) involving water, hexanes, methanol and dichloromethane. The extracts were evaluated for their anti-inflammatory and cytotoxic activities in an NF-κB luciferase and MTT assay using macrophage immune (RAW264.7) cells. A standardized commercial ethanol extract of nettle leaves was also evaluated. The methanolic extract of the flowering portions displayed significant anti-inflammatory activity on par with a standard compound celastrol (1) but were moderately cytotoxic. Alternatively, the polar extracts (water, methanol, ethanol) of the roots, stems and leaves displayed moderate to weak anti-inflammatory activity, while the methanol and especially the water soluble extracts exhibited noticeable cytotoxicity. In contrast, the lipophilic dichloromethane extracts of the roots, stems and leaves exhibited potent anti-inflammatory effects greater than or equal to 1 with minimal cytotoxicity to RAW264.7 cells. Collectively these results suggest that using lipophilic extracts of stinging nettle may be more effective than traditional tinctures (water, methanol, ethanol) in clinical evaluations for the treatment of inflammatory disorders especially arthritis. A chemical investigation into the lipophilic extracts of stinging nettle to identify the bioactive compound(s) responsible for their observed anti-inflammatory activity is further warranted.

The Aignopsanes, a New Class of Sesquiterpenes from Selected Chemotypes of the Sponge Cacospongia mycofijiensis

A survey of individual specimens of northern Papua New Guinea derived Cacospongia mycofijiensis has yielded novel sesquiterpenes, aignopsanoic acid A (1), methyl aignopsanoate A (2), and isoaignopsanoic acid A (3). The structures and absolute configurations of 1-3 were established using NMR data, X-ray crystallography results, and an analysis of CD properties. Two of these metabolites, 1 and 2, were moderately active against Trypanosoma brucei, the parasite responsible for sleeping sickness.