Short term analysis services, SOLO

Issued as Final report, Project no. G-36-62

Secondary Metabolites from the Marine Sponge Genus Phyllospongia.

Phyllospongia, one of the most common marine sponges in tropical and subtropical oceans, has been shown to be a prolific producer of natural products with a broad spectrum of biological activities. This review for the first time provides a comprehensive overview of secondary metabolites produced by Phyllospongia spp. over the 37 years from 1980 to 2016

Chemically diverse microtubule stabilizing agents initiate distinct mitotic defects and dysregulated expression of key mitotic kinases.

Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent than paclitaxel or laulimalide, which is consistent with the distinct defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer. This suggests a common mechanism of aster formation. However, taccalonolide AJ also causes pericentrin accumulation on every spindle aster. The presence of pericentrin at every spindle aster initiated by taccalonolide AJ might facilitate the maintenance and stability of the highly focused asters formed by this stabilizer. Laulimalide and paclitaxel cause completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Delineating how diverse microtubule stabilizers affect mitotic signaling pathways could identify key proteins involved in modulating sensitivity and resistance to the antimitotic actions of these compounds

In Vivo Evaluation of (-)-Zampanolide Demonstrates Potent and Persistent Antitumor Efficacy When Targeted to the Tumor Site.

Microtubule-stabilizing agents (MSAs) are a class of compounds used in the treatment of triple-negative breast cancer (TNBC), a subtype of breast cancer where chemotherapy remains the standard-of-care for patients. Taxanes like paclitaxel and docetaxel have demonstrated efficacy against TNBC in the clinic, however new classes of MSAs need to be identified due to the rise of taxane resistance in patients. (-)-Zampanolide is a covalent microtubule stabilizer that can circumvent taxane resistance in vitro but has not been evaluated for in vivo antitumor efficacy. Here, we determine that (-)-zampanolide has similar potency and efficacy to paclitaxel in TNBC cell lines, but is significantly more persistent due to its covalent binding. We also provide the first reported in vivo antitumor evaluation of (-)-zampanolide where we determine that it has potent and persistent antitumor efficacy when delivered intratumorally. Future work on zampanolide to further evaluate its pharmacophore and determine ways to improve its systemic therapeutic window would make this compound a potential candidate for clinical development through its ability to circumvent taxane-resistance mechanisms

The potential of achiral sponge-derived and synthetic bromoindoles as selective cytotoxins against PANC-1 tumor cells.

Our quest to isolate and characterize natural products with in vitro solid tumor selectivity is driven by access to repositories of Indo-Pacific sponge extracts. In this project an extract of a species of Haplosclerida sponge obtained from the US NCI Natural Products Repository displayed, by in vitro disk diffusion assay (DDA) and I

The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

BACKGROUND: Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied. RESULTS: Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers. CONCLUSION: (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs

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

Scale Up Isolation of Aaptamine for In Vivo Evaluation Indicates Its Neurobiological Activity is Linked to the Delta Opioid Receptor

Opioid receptors belong to the large superfamily of seven transmembrane-spanning (7TM) G protein-coupled receptors (GPCRs). As a class, GPCRs are of fundamental physiological importance mediating the actions of the majority of known neurotransmitters and hormones. The Mu, Delta, and Kappa (MOP, DOP, KOP) opioid receptors are particularly intriguing members of this receptor family as they are the targets involved in many neurobiological diseases such as addiction, pain, stress, anxiety, and depression. Recently we discovered that the aaptamine class of marine sponge derived natural products exhibit selective agonist activity in vitro for the DOP versus MOP receptor. Our findings may explain reports by others that aaptamine demonstrates in vivo anti-depressant effects in mouse models using the Porsolt Forced Swim Test. This project involved the extraction of the sponge Aaptos aaptos (a source of 1), establishing a scale up purification procedure to provide sufficient amounts of 1 (30 mg) for a follow up in vivo evaluation and ultimately confirmation of the structure of 1 using LC-MS and 1H NMR. The results our purification scheme, chemical analysis and in vivo evaluation of 1 using the Marble burying test in rodents are reported here in and suggest that the in vivo anti-depressant effects of 1 are linked directly to its agonist effects on the DOP receptor.https://scholar.dominican.edu/ug-student-posters/1008/thumbnail.jp

Scale up isolation of aaptamine for in vivo evaluation indicates its neurobiological activity is linked to the delta opioid receptor

Opioid receptors belong to the large superfamily of seven transmembrane-spanning (7TM) G protein-coupled receptors (GPCRs). As a class, GPCRs are of fundamental physiological importance mediating the actions of the majority of known neurotransmitters and hormones. The Mu (µ), Delta (δ) and Kappa (MOR, DOR, KOR) opioid receptors are particularly intriguing members of this receptor family as they are the targets involved in many neurobiological diseases such as addiction, pain, stress, anxiety, and depression. To date few marine natural products have been investigated for their neurobiological activities.1 One noteworthy example involves ziconotide (1) from the cone snail Conus magnus.2 Compound 1 was the first marine natural product approved by the FDA and is used for the treatment of pain, marketed under the trade name Prialt® (2004).3 More recently Hamman reported that aaptamine (2) is the first marine natural product to show in vivo anti-depressant activity, however no mechanism of action was proposed.1,4 During a separate collaborative screening project we profiled 96 sponge-derived extracts and discovered that demethyl–aaptamine (3) and demethyl (oxy)–aaptamine (4) were selective DOR agonists using an LC-MS based library of an active methanolic extract coll. no. 92553 FM as shown in Fig. 1. We speculated that the in vivo activity for 2 could thus be linked to the DOR target and to test this hypothesis we conducted the following experiments below.https://scholar.dominican.edu/ug-student-posters/1074/thumbnail.jp

Small-molecule antagonists of the oncogenic Tcf/β-catenin protein complex

AbstractKey molecular lesions in colorectal and other cancers cause β-catenin-dependent transactivation of T cell factor (Tcf)-dependent genes. Disruption of this signal represents an opportunity for rational cancer therapy. To identify compounds that inhibit association between Tcf4 and β-catenin, we screened libraries of natural compounds in a high-throughput assay for immunoenzymatic detection of the protein-protein interaction. Selected compounds disrupt Tcf/β-catenin complexes in several independent in vitro assays and potently antagonize cellular effects of β-catenin-dependent activities, including reporter gene activation, c-myc or cyclin D1 expression, cell proliferation, and duplication of the Xenopus embryonic dorsal axis. These compounds thus meet predicted criteria for disrupting Tcf/β-catenin complexes and define a general standard to establish mechanism-based activity of small molecule inhibitors of this pathogenic protein-protein interaction