KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Synthesis and Biological Evaluation of New Madecassic Acid Derivatives Targeting ERK Cascade Signaling.

In the present study, a series of novel madecassic acid derivatives was synthesized and screened against the National Cancer Institute's 60 human cancer cell line panel. Among them, compounds 5, 12, and 17 displayed potent and highly differential antiproliferative activity against 80% of the tumor cells harboring the B-RafV600E mutation within the nanomolar range. Structure-activity analysis revealed that a 5-membered A ring containing an α,ß-unsaturated aldehyde substituted at C-23 with a 2-furoyl group seems to be crucial to produce this particular growth inhibition signature. In silico analysis of the cytotoxicity pattern of these compounds identified two highly correlated clinically approved drugs with known B-RafV600E inhibitory activity. Follow-up analysis revealed inhibition of the ERK signaling pathway through the reduction of cellular Raf protein levels is a key mechanism of action of these compounds. In particular, 17 was the most potent compound in suppressing tumor growth of B-RafV600E-mutant cell lines and displayed the highest reduction of Raf protein levels among the tested compounds. Taken together, this study revealed that modifications of madecassic acid structure can provide molecules with potent anticancer activity against cell lines harboring the clinically relevant B-RafV600E mutation, with compound 17 identified as a promising lead for the development of new anticancer drugs.

Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box.

Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.

Macrophilone A: Structure Elucidation, Total Synthesis, and Functional Evaluation of a Biologically Active Iminoquinone from the Marine Hydroid Macrorhynchia philippina.

A previously uncharacterized pyrroloiminoquinone natural product, macrophilone A, was isolated from the stinging hydroid Macrorhynchia philippina. The structure was assigned utilizing long-range NMR couplings and DFT calculations and proved by a concise, five-step total synthesis. Macrophilone A and a synthetic analogue displayed potent biological activity, including increased intracellular reactive oxygen species levels and submicromolar cytotoxicity toward lung adenocarcinoma cells.

Characterization and Synthesis of Eudistidine C, a Bioactive Marine Alkaloid with an Intriguing Molecular Scaffold.

An extract of Eudistoma sp. provided eudistidine C (1), a heterocyclic alkaloid with a novel molecular framework. Eudistidine C (1) is a racemic natural product composed of a tetracyclic core structure further elaborated with a p-methoxyphenyl group and a phenol-substituted aminoimidazole moiety. This compound presented significant structure elucidation challenges due to the large number of heteroatoms and fully substituted carbons. These issues were mitigated by application of a new NMR pulse sequence (LR-HSQMBC) optimized to detect four- and five-bond heteronuclear correlations and the use of computer-assisted structure elucidation software. Synthesis of eudistidine C (1) was accomplished in high yield by treating eudistidine A (2) with 4(2-amino-1H-imidazol-5-yl)phenol (4) in DMSO. Synthesis of eudistidine C (1) confirmed the proposed structure and provided material for further biological characterization. Treatment of 2 with various nitrogen heterocycles and electron-rich arenes provided a series of analogues (5-10) of eudistidine C. Chiral-phase HPLC resolution of epimeric eudistidine C provided (+)-(R)-eudistidine C (1a) and (-)-(S)-eudistidine C (1b). The absolute configuration of these enantiomers was assigned by ECD analysis. (-)-(S)-Eudistidine C (1b) modestly inhibited interaction between the protein binding domains of HIF-1α and p300. Compounds 1, 2, and 6-10 exhibited significant antimalarial activity against Plasmodium falciparum.

Screening and Biological Effects of Marine Pyrroloiminoquinone Alkaloids: Potential Inhibitors of the HIF-1α/p300 Interaction.

Inhibition of the hypoxia-inducible factor 1α (HIF-1α) pathway by disrupting its association with the transcriptional coactivator p300 inhibits angiogenesis and tumor development. Development of HIF-1α/p300 inhibitors has been hampered by preclinical toxicity; therefore, we aimed to identify novel HIF-1α/p300 inhibitors. Using a cell-free assay designed to test compounds that block HIF-1α/p300 binding, 170 298 crude natural product extracts and prefractionated samples were screened, identifying 25 active extracts. One of these extracts, originating from the marine sponge Latrunculia sp., afforded six pyrroloiminoquinone alkaloids that were identified as positive hits (IC50 values: 1-35 µM). Luciferase assays confirmed inhibition of HIF-1α transcriptional activity by discorhabdin B (1) and its dimer (2), 3-dihydrodiscorhabdin C (3), makaluvamine F (5), discorhabdin H (8), discorhabdin L (9), and discorhabdin W (11) in HCT 116 colon cancer cells (0.1-10 µM, p < 0.05). Except for 11, all of these compounds also reduced HIF-1α transcriptional activity in LNCaP prostate cancer cells (0.1-10 µM, p < 0.05). These effects occurred at noncytotoxic concentrations (<50% cell death) under hypoxic conditions. At the downstream HIF-1α target level, compound 8 (0.5 µM) significantly decreased VEGF secretion in LNCaP cells (p < 0.05). In COLO 205 colon cancer cells no activity was shown in the luciferase or cytotoxicity assays. Pyrroloiminoquinone alkaloids are a novel class of HIF-1α inhibitors, which interrupt the protein-protein interaction between HIF-1α and p300 and consequently reduce HIF-related transcription.

Englerin A Inhibits EWS-FLI1 DNA Binding in Ewing Sarcoma Cells.

High-throughput screening of extracts from plants, marine, and micro-organisms led to the identification of the extract from the plant Phyllanthus engleri as the most potent inhibitor of EWS-FLI1 induced luciferase reporter expression. Testing of compounds isolated from this extract in turn led to the identification of Englerin A (EA) as the active constituent of the extract. EA induced both necrosis and apoptosis in Ewing cells subsequent to a G2M accumulation of cells in the cell cycle. It also impacted clonogenic survival and anchorage-independent proliferation while also decreasing the proportion of chemotherapy-resistant cells identified by high ALDH activity. EA also caused a sustained increase in cytosolic calcium levels. EA appears to exert its effect on Ewing cells through a decrease in phosphorylation of EWS-FLI1 and its ability to bind DNA. This effect is mediated, at least in part, through a decrease in the levels of the calcium-dependent protein kinase PKC-ßI after a transient up-regulation.

Stelliosphaerols A and B, Sesquiterpene-Polyol Conjugates from an Ecuadorian Fungal Endophyte.

Endophytic fungi are plant tissue-associated fungi that represent a rich resource of unexplored biological and chemical diversity. As part of an ongoing effort to characterize Amazon rainforest-derived endophytes, numerous fungi were isolated and cultured from plants collected in the Yasuní National Park in Ecuador. Of these samples, phylogenetic and morphological data revealed a previously undescribed fungus in the order Pleosporales that was cultured from the tropical tree Duroia hirsuta. Extracts from this fungal isolate displayed activity against Staphylococcus aureus and were thus subjected to detailed chemical studies. Two compounds with modest antibacterial activity were isolated, and their structures were elucidated using a combination of NMR spectroscopic analysis, LC-MS studies, and chemical degradation. These efforts led to the identification of stelliosphaerols A (1) and B (2), new sesquiterpene-polyol conjugates that are responsible, at least in part, for the S. aureus inhibitory activity of the fungal extract.

Deguelins, Natural Product Modulators of NF1-Defective Astrocytoma Cell Growth Identified by High-Throughput Screening of Partially Purified Natural Product Extracts.

A high-throughput screening assay for modulators of Trp53/NF1 mutant astrocytoma cell growth was adapted for use with natural product extracts and applied to a novel collection of prefractionated/partially purified extracts. Screening 68 427 samples identified active fractions from 95 unique extracts, including the terrestrial plant Millettia ichthyotona. Only three of these extracts showed activity in the crude extract form, thus demonstrating the utility of a partial purification approach for natural product screening. The NF1 screening assay was used to guide purification of active compounds from the M. ichthyotona extract, which yielded the two rotenones deguelin (1) and dehydrodeguelin (2). The deguelins have been reported to affect growth of a number of cancer cell lines. They potently inhibited growth of only one of a panel of NF1/Trp53 mutant murine astrocytoma cell lines, possibly related to epigenetic factors, but had no effect on the growth of normal astrocytes. These results suggest the potential utility of deguelins as tools for further investigating NF1 astrocytoma cell growth. These bioprobes were identified only as a result of screening partially purified natural product extracts.

Stellettapeptins A and B, HIV-inhibitory cyclic depsipeptides from the marine sponge Stelletta sp.

Two new HIV-inhibitory depsipeptides, stellettapeptins A (1) and B (2), were isolated from an extract of the marine sponge Stelletta sp., collected from northwestern Australia. Structures of these cyclic nonribosomal peptides were elucidated on the basis of extensive NMR data analysis, and chemical degradation and derivatization studies. Stellettapeptins contain numerous nonproteinogenic amino acid residues and they are the first peptides reported to contain a 3-hydroxy-6,8-dimethylnon-4-(Z)-enoic acid moiety. Compounds 1 and 2 potently inhibit infection of human T-lymphoblastoid cells by HIV-1RF with EC50 values of 23 and 27 nM, respectively.

Structural Elucidation and Synthesis of Eudistidine A: An Unusual Polycyclic Marine Alkaloid that Blocks Interaction of the Protein Binding Domains of p300 and HIF-1α.

Low oxygen environments are a hallmark of solid tumors, and transcription of many hypoxia-responsive genes needed for survival under these conditions is regulated by the transcription factor HIF-1 (hypoxia-inducible factor 1). Activation of HIF-1 requires binding of its α-subunit (HIF-1α) to the transcriptional coactivator protein p300. Inhibition of the p300/HIF-1α interaction can suppress HIF-1 activity. A screen for inhibitors of the protein binding domains of p300 (CH1) and HIF-1α (C-TAD) identified an extract of the marine ascidian Eudistoma sp. as active. Novel heterocyclic alkaloids eudistidines A (1) and B (2) were isolated from the extract, and their structures assigned by spectroscopic analyses. They contain an unprecedented tetracyclic core composed of two pyrimidine rings fused with an imidazole ring. Eudistidine A (1) was synthesized in a concise four-step sequence featuring a condensation/cyclization reaction cascade between 4-(2-aminophenyl)pyrimidin-2-amine (3) and 4-methoxy-phenylglyoxal (4), while eudistidine B (2) was synthesized in a similar fashion with glyoxylic acid (5) in place of 4. Naturally occurring eudistidine A (1) effectively inhibited CH1/C-TAD binding with an IC50 of 75 µM, and synthetic 1 had similar activity. The eudistidine A (1) scaffold, which can be synthesized in a concise, scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1α interactions and the role these proteins play in tumor response to low oxygen conditions. The unique structural scaffolds and functional group arrays often found in natural products make these secondary metabolites a rich source of new compounds that can disrupt critical protein-protein binding events.

Engineering soya bean seeds as a scalable platform to produce cyanovirin-N, a non-ARV microbicide against HIV.

There is an urgent need to provide effective anti-HIV microbicides to resource-poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin-N (rCV-N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV-N was isolated with a yield of 350 µg/g of dry seed weight. The observed amino acid sequence of rCV-N matched the expected sequence of native CV-N, as did the mass of rCV-N (11 009 Da). Purified rCV-N from soya is active in anti-HIV assays with an EC50 of 0.82-2.7 nM (compared to 0.45-1.8 nM for E. coli-produced CV-N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV-N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti-HIV microbicide development.

Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus.

BACKGROUND: The lectin griffithsin (GRFT) is a potent antiviral agent capable of prevention and treatment of infections caused by a number of enveloped viruses and is currently under development as an anti-HIV microbicide. In addition to its broad antiviral activity, GRFT is stable at high temperature and at a broad pH range, displays little toxicity and immunogenicity, and is amenable to large-scale manufacturing. Native GRFT is a domain-swapped homodimer that binds to viral envelope glycoproteins and has displayed mid-picomolar activity in cell-based anti-HIV assays. Previously, we have engineered and analyzed several monomeric forms of this lectin (mGRFT) with anti-HIV EC50 values ranging up to 323 nM. Based on our previous analysis of mGRFT, we hypothesized that the orientation and spacing of the carbohydrate binding domains GRFT were key to its antiviral activity. RESULTS: Here we present data on engineered tandem repeats of mGRFT (mGRFT tandemers) with antiviral activity at concentrations as low as one picomolar in whole-cell anti-HIV assays. mGRFT tandemers were analyzed thermodynamically, both individually and in complex with HIV-1 gp120. We also demonstrate by dynamic light scattering and cryo-electron microscopy that mGRFT tandemers do not aggregate HIV virions. This establishes that, although the intra-virion crosslinking of HIV envelope glycoproteins is likely integral to their activity, the antiviral activity of these lectins is not due to virus aggregation caused by inter-virion crosslinking. CONCLUSIONS: The engineered tandemer constructs of mGRFT may provide novel and powerful agents for prevention of infection by HIV and other enveloped viruses.

Inhibition of the HIF1α-p300 interaction by quinone- and indandione-mediated ejection of structural Zn(II).

Protein-protein interactions between the hypoxia inducible factor (HIF) and the transcriptional coactivators p300/CBP are potential cancer targets due to their role in the hypoxic response. A natural product based screen led to the identification of indandione and benzoquinone derivatives that reduce the tight interaction between a HIF-1α fragment and the CH1 domain of p300. The indandione derivatives were shown to fragment to give ninhydrin, which was identified as the active species. Both the naphthoquinones and ninhydrin were observed to induce Zn(II) ejection from p300 and the catalytic domain of the histone demethylase KDM4A. Together with previous reports on the effects of related compounds on HIF-1α and other systems, the results suggest that care should be taken in interpreting biological results obtained with highly electrophilic/thiol modifying compounds.

Steroidal alkaloids from the marine sponge Corticium niger that inhibit growth of human colon carcinoma cells.

Bioinformatic analysis of data from the NCI-60 cell cytotoxicity screen revealed a subset of extracts that showed selective cytotoxic activity toward human colon carcinoma cell lines. Bioassay-guided fractionation of a colon cancer selective extract from a Philippines collection of the marine sponge Corticium niger provided two new steroidal alkaloids, plakinamines N (1) and O (2), along with two known compounds of the plakinamine class (3, 4). The structures of these compounds were elucidated by interpretation of combined MS and NMR spectroscopic data. Plakinamines N (1), O (2), and J (4) were tested for antiproliferative activity in the NCI-60 screen, and they showed enhanced inhibitory effects against all of the colon cell lines with mean GI50 values of 11.5, 2.4, and 1.4 µM, respectively.

The cyanobacterial lectin scytovirin displays potent in vitro and in vivo activity against Zaire Ebola virus.

The cyanobacterial lectin scytovirin (SVN) binds with high affinity to mannose-rich oligosaccharides on the envelope glycoprotein (GP) of a number of viruses, blocking entry into target cells. In this study, we assessed the ability of SVN to bind to the envelope GP of Zaire Ebola virus (ZEBOV) and inhibit its replication. SVN interacted specifically with the protein's mucin-rich domain. In cell culture, it inhibited ZEBOV replication with a 50% virus-inhibitory concentration (EC50) of 50 nM, and was also active against the Angola strain of the related Marburg virus (MARV), with a similar EC50. Injected subcutaneously in mice, SVN reached a peak plasma level of 100 nm in 45 min, but was cleared within 4h. When ZEBOV-infected mice were given 30 mg/kg/day of SVN by subcutaneous injection every 6h, beginning the day before virus challenge, 9 of 10 animals survived the infection, while all infected, untreated mice died. When treatment was begun one hour or one day after challenge, 70-90% of mice survived. Quantitation of infectious virus and viral RNA in samples of serum, liver and spleen collected on days 2 and 5 postinfection showed a trend toward lower titers in treated than control mice, with a significant decrease in liver titers on day 2. Our findings provide further evidence of the potential of natural lectins as therapeutic agents for viral infections.

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.

Characterization of pomiferin triacetate as a novel mTOR and translation inhibitor.

Deregulation of the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-70kDa ribosomal protein S6 kinase 1 (p70(S6K)) pathway is commonly observed in many tumors. This pathway controls proliferation, survival, and translation, and its overactivation is associated with poor prognosis for tumor-associated survival. Current efforts focus on the development of novel inhibitors of this pathway. In a cell-based high-throughput screening assay of 15,272 pure natural compounds, we identified pomiferin triacetate as a potent stabilizer of the tumor suppressor programmed cell death 4 (Pdcd4). Mechanistically, pomiferin triacetate appeared as a general inhibitor of the PI3K-Akt-mTOR-p70(S6K) cascade. Interference with this pathway occurred downstream of Akt but upstream of p70(S6K). Specifically, mTOR kinase emerged as the molecular target of pomiferin triacetate, with similar activities against mTOR complexes 1 and 2. In an in vitro mTOR kinase assay pomiferin triacetate dose-dependently inhibited mTOR with an IC50 of 6.2 µM. Molecular docking studies supported the interaction of the inhibitor with the catalytic site of mTOR. Importantly, pomiferin triacetate appeared to be highly selective for mTOR compared to a panel of 17 lipid and 50 protein kinases tested. As a consequence of the mTOR inhibition, pomiferin triacetate efficiently attenuated translation. In summary, pomiferin triacetate emerged as a novel and highly specific mTOR inhibitor with strong translation inhibitory effects. Thus, it might be an interesting lead structure for the development of mTOR- and translation-targeted anti-tumor therapies.

Compounds from Simarouba berteroana which inhibit proliferation of NF1-defective cancer cells.

A neurofibromatosis type 1 (NF1) based bioassay-guided phytochemical investigation on Simarouba berteroana led to the isolation of one new canthin-6-one-9-methoxy-5-O-ß-D-glucopyranoside (1), seven known canthine alkaloids (2-8), two known quassinoids (9-10) and a known neo-lignan (11). The structures of all compounds were established by HRMS, 1D- and 2D-NMR analysis and comparison with previously reported data. Most of the compounds inhibited the proliferation of an Nf1- and p53-deficient mouse glioma cell line at non-cytotoxic concentrations.

Identification of compounds by high-content screening that induce cytoplasmic to nuclear localization of a fluorescent estrogen receptor α chimera and exhibit agonist or antagonist activity in vitro.

We have completed a robust high-content imaging screen for novel estrogen receptor α (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen was very robust, with Z' values >0.7 and coefficients of variation (CV) <5%. The screen utilized a stably transfected green fluorescent protein-tagged glucocorticoid/estrogen receptor (GFP-GRER) chimera, which consisted of the N-terminus of the glucocorticoid receptor fused to the human ERα ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands and translocated to the nucleus in response to stimulation with ERα agonists and antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. We screened 224,891 samples from our synthetic, pure natural product libraries, prefractionated natural product extracts library, and crude natural product extracts library, which produced a 0.003% hit rate. In addition to identifying several known ER ligands, five compounds were discovered that elicited significant activity in the screen. Transactivation potential studies demonstrated that two hit compounds behave as agonists, while three compounds elicited antagonist activity in MCF-7 cells.