Dentithecamides A-H, Diacylated Zoanthoxanthin Derivatives with PAX3-FOXO1 Inhibitory Activity from the Hydroid Dentitheca habereri.

Chemical investigation of the marine hydroid Dentitheca habereri led to the identification of eight new diacylated zoanthoxanthin alkaloids, named dentithecamides A-H (1-8), along with three previously reported analogues, zoamides B-D (9-11). The structures of compounds 1-11 were elucidated by spectroscopic and spectrometric analyses, including IR, HRESIMS, and NMR experiments, and by comparison with literature data. Compounds 1-11 are the first zoanthoxanthin alkaloids to be reported from a hydroid. Dentithecamides A (1) and B (2) along with zoamides B-D (9-11), which all share a conformationally mobile cycloheptadiene core, inhibited PAX3-FOXO1 regulated transcriptional activity and thus provided a structural framework for the potential development of more potent PAX3-FOXO1 inhibitors.

Denigrins and Dactylpyrroles, Arylpyrrole Alkaloids from a Dactylia sp. Marine Sponge.

Seven new arylpyrrole alkaloids (1-7), along with four known compounds, were isolated from an extract of a Dactylia sp. nov. marine sponge, and their structures were elucidated by interpretation of NMR and MS spectroscopic data. Denigrins D-G (1-4) have highly substituted pyrrole or pyrrolone rings in their core structures, while dactylpyrroles A-C (5-7) have tricyclic phenanthrene cores. Due to the proton-deficient nature of these scaffolds, key heteronuclear correlations from 1H-15N HMBC and LR-HSQMBC NMR experiments were used in the structure assignment of denigrin D (1). Dictyodendrin F (8), a previously described co-metabolite, inhibited transcription driven by the oncogenic PAX3-FOXO1 fusion gene with an IC50 value of 13 µM.

The Natural Product Butylcycloheptyl Prodiginine Binds Pre-miR-21, Inhibits Dicer-Mediated Processing of Pre-miR-21, and Blocks Cellular Proliferation.

Identification of RNA-interacting pharmacophores could provide chemical probes and, potentially, small molecules for RNA-based therapeutics. Using a high-throughput differential scanning fluorimetry assay, we identified small-molecule natural products with the capacity to bind the discrete stem-looped structure of pre-miR-21. The most potent compound identified was a prodiginine-type compound, butylcycloheptyl prodiginine (bPGN), with the ability to inhibit Dicer-mediated processing of pre-miR-21 in vitro and in cells. Time-dependent RT-qPCR, western blot, and transcriptomic analyses showed modulation of miR-21 expression and its target genes such as PDCD4 and PTEN upon treatment with bPGN, supporting on-target inhibition. Consequently, inhibition of cellular proliferation in HCT-116 colorectal cancer cells was also observed when treated with bPGN. The discovery that bPGN can bind and modulate the expression of regulatory RNAs such as miR-21 helps set the stage for further development of this class of natural product as a molecular probe or therapeutic agent against miRNA-dependent diseases.

Sensitization of renal carcinoma cells to TRAIL-induced apoptosis by rocaglamide and analogs.

Rocaglamide has been reported to sensitize several cell types to TRAIL-induced apoptosis. In recent years, advances in synthetic techniques have led to generation of novel rocaglamide analogs. However, these have not been extensively analyzed as TRAIL sensitizers, particularly in TRAIL-resistant renal cell carcinoma cells. Evaluation of rocaglamide and analogs identified 29 compounds that are able to sensitize TRAIL-resistant ACHN cells to TRAIL-induced, caspase-dependent apoptosis with sub-µM potency which correlated with their potency as protein synthesis inhibitors and with loss of cFLIP protein in the same cells. Rocaglamide alone induced cell cycle arrest, but not apoptosis. Rocaglates averaged 4-5-fold higher potency as TRAIL sensitizers than as protein synthesis inhibitors suggesting a potential window for maximizing TRAIL sensitization while minimizing effects of general protein synthesis inhibition. A wide range of other rocaglate effects (e.g. on JNK or RAF-MEK-ERK signaling, death receptor levels, ROS, ER stress, eIF4E phosphorylation) were assessed, but did not contribute to TRAIL sensitization. Other than a rapid loss of MCL-1, rocaglates had minimal effects on mitochondrial apoptotic pathway proteins. The identification of structurally diverse/mechanistically similar TRAIL sensitizing rocaglates provides insights into both rocaglate structure and function and potential further development for use in RCC-directed combination therapy.

Tricyclic guanidine alkaloids from the marine sponge Acanthella cavernosa that stabilize the tumor suppressor PDCD4.

A cell-based high-throughput screen that assessed the cellular stability of a tumor suppressor protein PDCD4 (Programmed cell death 4) was used to identify a new guanidine-containing marine alkaloid mirabilin K (3), as well as the known compounds mirabilin G (1) and netamine M (2). The structures of these tricyclic guanidine alkaloids were established from extensive spectroscopic analyses. Compounds 1 and 2 inhibited cellular degradation of PDCD4 with EC50 values of 1.8 µg/mL and 2.8 µg/mL, respectively. Mirabilin G (1) and netamine M (2) are the first marine natural products reported to stabilize PDCD4 under tumor promoting conditions.

Synergistic TRAIL sensitizers from Barleria alluaudii and Diospyros maritima.

Barleria alluaudii and Diospyros maritima were both investigated as part of an ongoing search for synergistic TRAIL (tumor necrosis factor-α-related apoptosis-inducing ligand) sensitizers. As a result of this study, two naphthoquinone epoxides, 2,3-epoxy-2,3-dihydrolapachol (1) and 2,3-epoxy-2,3-dihydro-8-hydroxylapachol (2), both not previously isolated from natural sources, and the known 2-methylanthraquinone (3) were identified from B. alluaudii. Time-dependent density functional theory (TD-DFT) calculations of electronic circular dichroism (ECD) spectra were utilized to establish the absolute configuration of 1 and 2. Additionally, five known naphthoquinone derivatives, maritinone (4), elliptinone (5), plumbagin (6), (+)-cis-isoshinanolone (7), and ethylidene-6,6'-biplumbagin (8), were isolated from D. maritima. Compounds 1, 2, and 4-6 showed varying levels of synergy with TRAIL. Maritinone (4) and elliptinone (5) showed the highest synergistic effect, with more than a 3-fold increase in activity observed with TRAIL than with compound alone.

Effects of cucurbitacins on cell morphology are associated with sensitization of renal carcinoma cells to TRAIL-induced apoptosis.

Cucurbitacins B and D were among the compounds identified as sensitizers of cancer cells to TRAIL-mediated apoptosis in a high-throughput screen. Therefore a series of cucurbitacins was further investigated for TRAIL sensitization and possible mechanisms of action. A total of six cucurbitacins promoted TRAIL-induced apoptosis (B, I, E, C, D, and K) and one (P) was inactive. Sensitization of renal adenocarcinoma cells to TRAIL was apparent after as little as 1-4 h pretreatment and did not require continued presence of cucurbitacin. Active cucurbitacins induced caspase-8 activation only after subsequent TRAIL addition and caspase activation was required for apoptosis suggesting amplified proximal signaling from TRAIL death receptors. Cucurbitacin-sensitized TRAIL-induced cytotoxicity was inhibited by N-acetyl cysteine. Structure-activity relationship analysis in comparison to published studies suggests that TRAIL-sensitizing and general cytotoxic activities of cucurbitacins may be decoupled. Cucurbitacins are reported to be inhibitors of STAT3 activation. However, their TRAIL-sensitizing activity is STAT3-independent. Treatment of renal carcinoma cells with active cucurbitacins produced rapid and dramatic changes in cell morphology and cytoskeletal organization (also prevented by NAC). Therefore, cucurbitacins may be useful as tools for investigating the molecular mechanism(s) of action of TRAIL sensitizers, particularly with regard to temporal aspects of sensitization and modulation of TRAIL signaling by cell morphology, and could form the basis for future therapeutic development in combination with TRAIL death receptor agonists.

Cryptocaryols A-H, α-pyrone-containing 1,3-polyols from Cryptocarya sp. implicated in stabilizing the tumor suppressor Pdcd4.

A high-throughput cell-based reporter assay designed to identify small-molecule stabilizers of the tumor suppressor Pdcd4 was used to screen extracts in the NCI Natural Products Repository. Bioassay-guided fractionation of an extract from a Papua New Guinea collection of the tropical tree Cryptocarya sp. provided a series of new 5,6-dihydro-α-pyrone-containing 1,3-polyols (1-8), named cryptocaryols A-H. Their structures were assigned from a combination of NMR, MS, and CD studies in conjunction with NMR database comparisons. Compounds 1-8 were found to rescue Pdcd4 from TPA-induced degradation with EC50 concentrations that ranged from 1.3 to 4.9 µM.

Clerodane diterpenes from Casearia arguta that act as synergistic TRAIL sensitizers.

Casearia arguta was investigated as part of the ongoing search for synergistic TRAIL (tumor necrosis factor-α-related apoptosis-inducing ligand) sensitizers. As a result of this study, argutins A-H, eight new highly oxygenated clerodane diterpenes, were isolated from the plant Casearia arguta collected in Guatemala. The modified Mosher ester method was utilized to establish the absolute configuration of argutins A and F. Each of the argutins showed varying levels of synergy with TRAIL. Argutin B showed the highest TRAIL sensitization; the synergistic effect of argutin B and TRAIL together was 3-fold greater than argutin B alone.

Development of a high-throughput cell-based reporter assay to identify stabilizers of tumor suppressor Pdcd4.

The novel tumor suppressor Pdcd4 affects tumorigenesis by inhibiting translation. Pdcd4 is phosphorylated and subsequently lost by proteasomal degradation in response to tumor-promoting conditions. Here, the authors describe the development of a reporter cell system to monitor the stability of Pdcd4. The phosphorylation-dependent degradation domain ("target") or an adjacent ("off-target") region of Pdcd4 was cloned into a luciferase expression system. The target constructs were responsive to Pdcd4 degrading conditions (e.g., TPA, p70(S6K1) overactivation), whereas the off-target constructs remained stable. The system was optimized for and shown to be reliable in a high-throughput compatible 384-well format. Screening of 15,275 pure compounds resulted in a hit rate of 0.30% (>50% inhibition of TPA-induced loss of signal, confirmed by reassay). Among the hits were inhibitors of previously identified critical signaling events for TPA-induced Pdcd4 degradation. One compound was identified to be nonspecific using the off-target control cell line. Screening of 135,678 natural product extracts yielded 42 confirmed, specific hits. Z' averaged 0.58 across 446 plates. Further characterization of active natural products and synthetic compounds is expected to identify novel Pdcd4 stabilizers that may be useful in targeting translation to prevent or treat cancers.

A cell-based high-throughput screen to identify synergistic TRAIL sensitizers.

We have developed a high-throughput screen (HTS) to search for novel molecules that can synergize with TRAIL, thus promoting apoptosis of ACHN renal tumor cells in a combinatorial fashion. The HTS detects synthetic compounds and pure natural products that can pre-sensitize the cancer cells to TRAIL-mediated apoptosis, yet have limited toxicity on their own. We have taken into account the individual effects of the single agents, versus the combination, and have identified hits that are synergistic, synergistic-toxic, or additive when combined with TRAIL in promoting tumor cell death. Preliminary mechanistic studies indicate that a subset of the synergistic TRAIL sensitizers act very rapidly to promote cleavage and activation of caspase-8 following TRAIL binding. Caspase-8 is an apical enzyme that initiates programmed cell death via the extrinsic apoptotic pathway. Thus, these TRAIL sensitizers may potentially reduce resistance of tumor cells to TRAIL-mediated apoptosis. Two representative sensitizers were found to increase levels of p53 but did not inhibit the proteasome, suggesting that early DNA damage-sensing pathways may be involved in their mechanisms of action.

Multiplexing siRNAs to compress RNAi-based screen size in human cells.

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study ( approximately 800 siRNAs, approximately 400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.