Discovery and preliminary SAR of bisbenzylisoquinoline alkaloids as inducers of C/EBPα.

A high throughput in vitro screen has been developed to identify substances that induce expression of C/EBPα in tumor cells. An extract of the fruit of Gyrocarpus jacquinii showed induction of C/EBPα activity that was attributed to the bisbenzylisoquinoline (BBIQ) alkaloid pheanthine (13) by dereplication analysis. The research project was broadened to assess the effect of other natural BBIQ structural types occurring outside the genus Gyrocarpus. Several of the 28 compounds assayed showed enhancement of C/EBPα induction in U937 cells. The results of this study should encourage future efforts toward obtaining and screening a larger set of both natural and synthetic analogs of this interesting group of alkaloids.

Histone deacetylase inhibitors from Burkholderia thailandensis.

Bioactivity-guided fractionation of an extract of Burkholderia thailandensis led to the isolation and identification of a new cytotoxic depsipeptide and its dimer. Both compounds potently inhibited the function of histone deacetylases 1 and 4. The monomer, spiruchostatin C (2), was tested side by side with the clinical depsipeptide FK228 (1, Istodax, romidepsin) in a murine hollow fiber assay consisting of 12 implanted tumor cell lines. Spiruchostatin C (2) showed good activity toward LOX IMVI melanoma cells and NCI-H522 non small cell lung cancer cells. Overall, however, FK228 (1) showed a superior in vivo antitumor profile in comparison to the new compound.

Isolation and characterization of minor analogues of silvestrol and other constituents from a large-scale re-collection of Aglaia foveolata.

Two new minor silvestrol analogues [2'''-episilvestrol (1) and 2''',5'''-diepisilvestrol (2)], together with a new 21-norbaccharane-type triterpene (3), two new 3,4-secodammarane triterpenes (4 and 5), and a new eudesmane sesquiterpene (6), as well as nine known compounds, were isolated from a large-scale re-collection of the CHCl(3)-soluble extract of the stem bark of Aglaia foveolata obtained in Kalimantan, Indonesia. The structures of the new compounds were established by interpretation of their spectroscopic data. All of the isolates were tested for cytotoxicity against HT-29 cells. The new silvestrol analogues, 1 and 2, were considerably less active as cytotoxic agents than silvestrol (7) and episilvestrol (5'''-episilvestrol) (8) against this cell line, showing the importance of the configuration at C-2''' in mediating such activity within this compound class. Several of the compounds isolated were also evaluated in a NF-κB (p65) inhibition assay.

High throughput extraction of plant, marine and fungal specimens for preservation of biologically active molecules.

The Developmental Therapeutics Program (DTP) of the U.S. National Cancer Institute (NCI), at its NCI-Frederick facility, has built perhaps the largest and most diverse natural products screening library in the world for drug discovery. Composed of plant, marine organism and microbial extracts, it currently contains in excess of 230,000 unique materials. From the inception of this program to identify new anticancer chemotherapeutics from natural products sources in 1987, two extracts have been sequentially prepared from each specimen: one produced by organic solvent extraction, which yields a complex material that contains non- to moderately polar small molecules, and a water-soluble extract, a milieu largely unexplored for useful drugs in earlier years, which contains polar small to medium-sized molecules. Plant specimens and microbial ferments are extracted by modified traditional methods, while the method developed to produce extracts from marine organisms is unique and very different from that used by marine natural products chemists previously, but again yields both an organic solvent soluble and a water soluble material for inclusion into the screening library. Details of high throughput extract production for preservation of biologically active molecules are presented.

Schweinfurthins I and J from Macaranga schweinfurthii.

Demand for the experimental antineoplastic agent schweinfurthin A, for developmental testing, prompted a re-collection of leaf material of Macaranga schweinfurthii from the original collection site in Cameroon. During chromatographic purification of the organic solvent extract, analytical UPLC-PDA-TOFMS of stilbene-enriched fractions revealed the presence of six known schweinfurthins and two previously unknown stilbenes. The structures of these new compounds, schweinfurthins I and J (1 and 2), were elucidated by 1D- and 2D-NMR techniques.

An inhibitor of CCL2-induced chemotaxis from the fungus Leptoxyphium sp.

A biological screen used to identify inhibitors of monocyte chemotactic protein-1 (CCL2)-induced chemotaxis was applied in the activity-guided fractionation of an extract from a fungus of the genus Leptoxyphium sp. Inhibition of CCL2-induced chemotaxis was traced to a new dichlorinated diketopiperazine, cyclo(13,15-dichloro-L-Pro-L-Tyr). A structure-activity relationship (SAR) study evaluating relative activities of cyclo(13,15-dichloro-L-Pro-L-Tyr) and a nonchlorinated homologue cyclo(L-Pro-L-Tyr) showed that the dichlorinated molecule was 10- to 20-fold more active than the nonchlorinated form, while no activity was observed for cyclo(D-N-methylLeu-L-Trp).

Overcoming problems of compound storage in DMSO: solvent and process alternatives.

The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various solvent combinations, and investigate resolubilization techniques to ensure proper drug solution for HTS. Recommendations are made on how to best maximize the probability that problem compounds will remain in solution, be accurately transferred during assay plate production, and, as a result, be accurately bioassayed at the specified molar concentration.

Cytotoxic and HIF-1alpha inhibitory compounds from Crossosoma bigelovii.

Cytotoxicity-guided fractionation of an organic solvent extract of the plant Crossosoma bigelovii led to the discovery of a new strophanthidin glycoside (1) and two new 2-methylchromone glycosides (2 and 3). Also isolated were the known chromones eugenin and noreugenin, the indole alkaloid ajmalicine, the dibenzylbutane lignan secoisolariciresinol, the dibenzylbutyrolactone lignan matairesinol, and the furanone 5-tetradec-5-enyldihydrofuran-2-one. Further investigation into the biological properties of strophanthidin glycosides revealed a connection between inhibition of HIF-1 activation and the glycosylation of the genin. This work is the first published study of the bioactive phytochemicals of the family Crossosomataceae.

Inhibitors of the NF-kappaB activation pathway from Cryptocarya rugulosa.

The nuclear factor-kappaB (NF-kappaB) signaling pathway is constitutively active in many types of cancers and is a potential therapeutic target. Using a cell-based assay for stability of inhibitor of kappa B (IkappaB), a critical regulator of NF-kappaB activity, we found that an organic solvent extract of the plant Cryptocarya rugulosa inhibited constitutive NF-kappaB activity in human lymphoma cell lines. The active components were identified as rugulactone, a new alpha-pyrone (1), and the known cryptocaryone (2). Rugulactone was the more active compound, exhibiting up to 5-fold induction of IkappaB at 25 microg/mL; maximal activity was observed with 10 h exposure of test cells to 1 or 2.

Separation and SAR study of HIF-1alpha inhibitory tubulosines from Alangium cf. longiflorum.

A crude organic solvent extract of Alangium cf. longiflorum exhibited potent inhibition of hypoxia-induced HIF-1 transcriptional activity in human U251 glioma cells. Dereplication and bioactivity-guided fractionation, including Sephadex LH-20 and chiral HPLC chromatographies, led to the isolation of tubulosine ( 1), 9-desmethyltubulosine ( 2), and isotubulosine ( 3). Structures were verified by complete (1)H and (13)C assignments using 1D- and 2D-NMR techniques. Tubulosine strongly inhibited HIF-1 transcriptional activity, isotubulosine was devoid of activity, and 9-desmethyltubulosine possessed 6-fold less potency than tubulosine.

Identification of a new natural camptothecin analogue in targeted screening for HIF-1alpha inhibitors.

Screening to detect compounds that inhibit the HIF-1alpha transcriptional activation pathway identified an extract of Ophiorrhiza trichocarpon for investigation. A high throughput dereplication strategy was employed, involving chromatography with spectral data acquisition supported by bioactivity testing and literature referencing, which led to rapid identification of camptothecin (1) and three analogues (2 - 4) as the active compounds. 9,10-Methylenedioxy-(20S)-camptothecin (4) was found for the first time from a plant.

Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions.

Kaposi's sarcoma (KS) and its causative agent, Kaposi's sarcoma associated herpesvirus (KSHV/HHV-8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the "oncoweed" hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV-associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the "oncoweed" hypothesis by demonstrating basic biological plausibility.

Tropane aromatic ester alkaloids from a large-scale re-collection of Erythroxylum pervillei stem bark obtained in Madagascar.

Fractionation by pH zone-refining countercurrent chromatography of an extract of the stem bark of Erythroxylum pervillei, obtained on a kilogram scale in southern Madagascar, led to the isolation and characterization of four tropane aromatic ester alkaloids as minor constituents, namely, pervilleines G (5) and H (6) and cis-pervilleines B (7) and F (8). Their structures were determined by spectroscopic data interpretation.

Antifungal flavonoids from Hildegardia barteri.

A new isoflavan, (3R)-6,2'-dihydroxy-7-methoxy-4',5'-methylenedioxyisoflavan, hildegardiol (1), and two known flavonoids, 2-hydroxymaackiain (2) and farrerol (3), were isolated from the antifungal root extract of Hildegardia barteri. The pterocarpan 2 was largely responsible for the observed antifungal activity.

Aspirochlorine class compounds from Aspergillus flavus inhibit azole-resistant Candida albicans.

Dereplication of the antifungal extracts of Aspergillus flavus indicated that the primary antifungal compound present was the known aspirochlorine (1). Preparative isolation work resulted in the identification of the new compounds tetrathioaspirochlorine (2) and cyclo(D-N-methyl-Leu-L-Trp) (3).

A novel antimicrobial indolizinium alkaloid from Aniba panurensis.

Activity-guided fractionation of an Aniba panurensis organic solvent extract has led to the isolation of the novel alkaloid 6,8-didec-(1Z)-enyl-5,7-dimethyl-2,3-dihydro-1H-indolizinium, as the trifluoroacetic acid salt (1). Its structure was determined by NMR and mass spectrometry. Bioassays performed in vitro demonstrated toxicity of compound 1 to a drug-resistant strain of Candida albicans.

Tannic acid is an inhibitor of CXCL12 (SDF-1alpha)/CXCR4 with antiangiogenic activity.

PURPOSE: Increasing evidence suggests that interaction between the chemoattractant CXCL12/stromal cell-derived factor-1alpha and its receptor CXCR4 plays a pivotal role in the metastasis of various tumors. Our previous studies showed that multi-component Chinese herbal medicines inhibited the effects of CXCL12/CXCR4. As a result of sequential chromatographic fractionation of one herbal medicine ingredient, Lianqiao (fruit of Forsythia suspensa), we observed that tannins were, at least in part, responsible for this activity. The aim of this study was to assess the anti-CXCL12/CXCR4 activity of a commercial tannic acid and evaluate its potential to inhibit tumor cell migration and angiogenesis in vitro. EXPERIMENTAL DESIGN: The inhibitory effect of tannic acid on CXCL12/CXCR4 was measured by chemotaxis assay, ligand binding assay, and fluorescence-activated cell sorter analysis. The antiangiogenic effect of tannic acid was assessed by in vitro endothelial cell tube formation. RESULTS: Tannic acid, at nontoxic concentrations, specifically inhibited CXCL12-induced human monocyte migration (IC(50), 7.5 micro g/ml) but did not inhibit CCL2-, CCL3-, CCL5-, formylmethionylleucylphenylalanine (fMLP)-, or C5a-induced migration. The compound markedly blocked CXCL12 binding to THP-1 cells (IC(50), 0.36 micro g/ml). Tannic acid also inhibited CXCL12-induced, but not epidermal growth factor-induced, migration of MDA 231 breast tumor cells. Additionally, 0.5 micro g/ml of tannic acid selectively inhibited CXCL12-mediated, but not basic fibroblast growth factor- or endothelial cell growth supplement-mediated, bovine aorta endothelial cell capillary tube formation. CONCLUSION: These studies indicate that tannic acid is a novel selective CXCL12/CXCR4 antagonist and consequently may provide a mechanistic basis for the reported antitumor and anti-inflammatory properties of tannic acid.

Identification of HIV-1 nucleocapsid protein: nucleic acid antagonists with cellular anti-HIV activity.

The crucial functions of HIV-1 nucleocapsid-p7 protein (NC-p7) at different stages of HIV replication are dependent on its nucleic acid binding properties. In this study, a search has been made to identify antagonists of the interaction between NC-p7 and d(TG)(4). A chemical library of approximately 2000 small molecules (the NCI Diversity Set) was screened, of the 26 active inhibitors that were identified, five contained a xanthenyl ring structure. Further analysis of 63 structurally related compounds led to the identification of 2,3,4,5-tetrachloro-6-(4('),5('),6(')-trihydroxy-3(')-oxo-3H-xanthen-9(')-yl)benzoic acid, which binds to NC-p7 stoichiometrically. This compound exerted a significant anti-HIV activity in vitro with an IC(50) of 16.6+/-4.3 microM (means+/-SD). Synthetic variants lacking the two hydroxyls at positions 4(') and 5(') in the xanthenyl ring system failed to bind NC-p7 and showed significantly less protection against HIV infection. Molecular modeling predicts that these hydroxyl groups would bind to the amide nitrogen of Gly(35) with other contacts at the carbonyl oxygens of Gly(40) and Lys(33).