Global SLAM-seq for accurate mRNA decay determination and identification of NMD targets.

Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5% of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives, instead of steady-state RNA level changes. With SLAM-seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2% of our candidates being identified in previous studies. This indicates that SLAM-seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.

Truncating the spliceosomal 'rope protein' Prp45 results in Htz1 dependent phenotypes.

Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1-169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1-330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1-169), to the chromatin environment.

Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity.

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.

Crotofolane Diterpenoids and Other Constituents Isolated from Croton kilwae.

Six new crotofolane diterpenoids (1-6) and 13 known compounds (7-19) were isolated from the MeOH-CH2Cl2 (1:1, v/v) extracts of the leaves and stem bark of Croton kilwae. The structures of the new compounds were elucidated by extensive analysis of spectroscopic and mass spectrometric data. The structure of crotokilwaepoxide A (1) was confirmed by single-crystal X-ray diffraction, allowing for the determination of its absolute configuration. The crude extracts and the isolated compounds were investigated for antiviral activity against respiratory syncytial virus (RSV) and human rhinovirus type-2 (HRV-2) in HEp-2 and HeLa cells, respectively, for antibacterial activity against the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli, and for antimalarial activity against the Plasmodium falciparum Dd2 strain. ent-3ß,19-Dihydroxykaur-16-ene (7) and ayanin (16) displayed anti-RSV activities with IC50 values of 10.2 and 6.1 µM, respectively, while exhibiting only modest cytotoxic effects on HEp-2 cells that resulted in selectivity indices of 4.9 and 16.4. Compounds 2 and 5 exhibited modest anti-HRV-2 activity (IC50 of 44.6 µM for both compounds), while compound 16 inhibited HRV-2 with an IC50 value of 1.8 µM. Compounds 1-3 showed promising antiplasmodial activities (80-100% inhibition) at a 50 µM concentration.

Post-transcriptional regulation during stress.

To remain competitive, cells exposed to stress of varying duration, rapidity of onset, and intensity, have to balance their expenditure on growth and proliferation versus stress protection. To a large degree dependent on the time scale of stress exposure, the different levels of gene expression control: transcriptional, post-transcriptional, and post-translational, will be engaged in stress responses. The post-transcriptional level is appropriate for minute-scale responses to transient stress, and for recovery upon return to normal conditions. The turnover rate, translational activity, covalent modifications, and subcellular localisation of RNA species are regulated under stress by multiple cellular pathways. The interplay between these pathways is required to achieve the appropriate signalling intensity and prevent undue triggering of stress-activated pathways at low stress levels, avoid overshoot, and down-regulate the response in a timely fashion. As much of our understanding of post-transcriptional regulation has been gained in yeast, this review is written with a yeast bias, but attempts to generalise to other eukaryotes. It summarises aspects of how post-transcriptional events in eukaryotes mitigate short-term environmental stresses, and how different pathways interact to optimise the stress response under shifting external conditions.

Oxygenated Cyclohexene Derivatives from the Stem and Root Barks of Uvaria pandensis.

Five new cyclohexene derivatives, dipandensin A and B (1 and 2) and pandensenols A-C (3-5), and 16 known secondary metabolites (6-21) were isolated from the methanol-soluble extracts of the stem and root barks of Uvaria pandensis. The structures were characterized by NMR spectroscopic and mass spectrometric analyses, and that of 6-methoxyzeylenol (6) was further confirmed by single-crystal X-ray crystallography, which also established its absolute configuration. The isolated metabolites were evaluated for antibacterial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus epidermidis and the Gram-negative bacteria Enterococcus raffinosus, Escherichia coli, Paraburkholderia caledonica, Pectobacterium carotovorum, and Pseudomonas putida, as well as for cytotoxicity against the MCF-7 human breast cancer cell line. A mixture of uvaretin (20) and isouvaretin (21) exhibited significant antibacterial activity against B. subtilis (EC50 8.7 µM) and S. epidermidis (IC50 7.9 µM). (8'α,9'ß-Dihydroxy)-3-farnesylindole (12) showed strong inhibitory activity (EC50 9.8 µM) against B. subtilis, comparable to the clinical reference ampicillin (EC50 17.9 µM). None of the compounds showed relevant cytotoxicity against the MCF-7 human breast cancer cell line.

Biflavanones, Chalconoids, and Flavonoid Analogues from the Stem Bark of Ochna holstii.

Two new biflavanones (1 and 2), three new bichalconoids (3-5), and 11 known flavonoid analogues (6-16) were isolated from the stem bark extract (CH3OH-CH2Cl2, 7:3, v/v) of Ochna holstii. The structures of the isolated metabolites were elucidated by NMR spectroscopic and mass spectrometric analyses. The crude extract and the isolated metabolites were evaluated for antibacterial activity against Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative) as well as for cytotoxicity against the MCF-7 human breast cancer cell line. The crude extract and holstiinone A (1) exhibited moderate antibacterial activity against B. subtilis with MIC values of 9.1 µg/mL and 14 µM, respectively. The crude extract and lophirone F (14) showed cytotoxicity against MCF-7 with EC50 values of 11 µg/mL and 24 µM, respectively. The other isolated metabolites showed no significant antibacterial activities (MIC > 250 µM) and cytotoxicities (EC50 ≥ 350 µM).

The Lsm1-7/Pat1 complex binds to stress-activated mRNAs and modulates the response to hyperosmotic shock.

RNA-binding proteins (RBPs) establish the cellular fate of a transcript, but an understanding of these processes has been limited by a lack of identified specific interactions between RNA and protein molecules. Using MS2 RNA tagging, we have purified proteins associated with individual mRNA species induced by osmotic stress, STL1 and GPD1. We found members of the Lsm1-7/Pat1 RBP complex to preferentially bind these mRNAs, relative to the non-stress induced mRNAs, HYP2 and ASH1. To assess the functional importance, we mutated components of the Lsm1-7/Pat1 RBP complex and analyzed the impact on expression of osmostress gene products. We observed a defect in global translation inhibition under osmotic stress in pat1 and lsm1 mutants, which correlated with an abnormally high association of both non-stress and stress-induced mRNAs to translationally active polysomes. Additionally, for stress-induced proteins normally triggered only by moderate or high osmostress, in the mutants the protein levels rose high already at weak hyperosmosis. Analysis of ribosome passage on mRNAs through co-translational decay from the 5' end (5P-Seq) showed increased ribosome accumulation in lsm1 and pat1 mutants upstream of the start codon. This effect was particularly strong for mRNAs induced under osmostress. Thus, our results indicate that, in addition to its role in degradation, the Lsm1-7/Pat1 complex acts as a selective translational repressor, having stronger effect over the translation initiation of heavily expressed mRNAs. Binding of the Lsm1-7/Pat1p complex to osmostress-induced mRNAs mitigates their translation, suppressing it in conditions of weak or no stress, and avoiding a hyperresponse when triggered.

Computational Chemogenomics Drug Repositioning Strategy Enables the Discovery of Epirubicin as a New Repurposed Hit for Plasmodium falciparum and P. vivax.

Widespread resistance against antimalarial drugs thwarts current efforts for controlling the disease and urges the discovery of new effective treatments. Drug repositioning is increasingly becoming an attractive strategy since it can reduce costs, risks, and time-to-market. Herein, we have used this strategy to identify novel antimalarial hits. We used a comparative in silico chemogenomics approach to select Plasmodium falciparum and Plasmodium vivax proteins as potential drug targets and analyzed them using a computer-assisted drug repositioning pipeline to identify approved drugs with potential antimalarial activity. Among the seven drugs identified as promising antimalarial candidates, the anthracycline epirubicin was selected for further experimental validation. Epirubicin was shown to be potent in vitro against sensitive and multidrug-resistant P. falciparum strains and P. vivax field isolates in the nanomolar range, as well as being effective against an in vivo murine model of Plasmodium yoelii Transmission-blocking activity was observed for epirubicin in vitro and in vivo Finally, using yeast-based haploinsufficiency chemical genomic profiling, we aimed to get insights into the mechanism of action of epirubicin. Beyond the target predicted in silico (a DNA gyrase in the apicoplast), functional assays suggested a GlcNac-1-P-transferase (GPT) enzyme as a potential target. Docking calculations predicted the binding mode of epirubicin with DNA gyrase and GPT proteins. Epirubicin is originally an antitumoral agent and presents associated toxicity. However, its antiplasmodial activity against not only P. falciparum but also P. vivax in different stages of the parasite life cycle supports the use of this drug as a scaffold for hit-to-lead optimization in malaria drug discovery.

The fission yeast FHIT homolog affects checkpoint control of proliferation and is regulated by mitochondrial electron transport.

Genetic analysis has strongly implicated human FHIT (Fragile Histidine Triad) as a tumor suppressor gene, being mutated in a large proportion of early-stage cancers. The functions of the FHIT protein have, however, remained elusive. Here, we investigated aph1+ , the fission yeast homolog of FHIT, for functions related to checkpoint control and oxidative metabolism. In sublethal concentrations of DNA damaging agents, aph1Δ mutants grew with a substantially shorter lag phase. In aph1Δ mutants carrying a hypomorphic allele of cds1 (the fission yeast homolog of Chk2), in addition, increased chromosome fragmentation and missegregation were found. We also found that under hypoxia or impaired electron transport function, the Aph1 protein level was strongly depressed. Previously, FHIT has been linked to regulation of the human 9-1-1 checkpoint complex constituted by Hus1, Rad1, and Rad9. In Schizosaccharomyces pombe, the levels of all three 9-1-1 proteins are all downregulated by hypoxia in similarity with Aph1. Moreover, deletion of the aph1+ gene reduced the Rad1 protein level, indicating a direct relationship between these two proteins. We conclude that the fission yeast FHIT homolog has a role in modulating DNA damage checkpoint function, possibly through an effect on the 9-1-1 complex, and that this effect may be critical under conditions of limiting oxidative metabolism and reoxygenation.

Oxygenated Cyclohexene Derivatives and Other Constituents from the Roots of Monanthotaxis trichocarpa.

Three new oxygenated cyclohexene derivatives, trichocarpeols A (1), B (2), and C (3), along with nine known secondary metabolites, were isolated from the methanolic root extract of Monanthotaxis trichocarpa. They were identified by NMR spectroscopic and mass spectrometric analyses, and the structure of trichocarpeol A (1) was confirmed by single-crystal X-ray diffraction. Out of the 12 isolated natural products, uvaretin (4) showed activity against the Gram-positive bacterium Bacillus subtilis with a MIC value of 18 µM. None of the isolated metabolites was active against the Gram-negative Escherichia coli at a ∼5 mM (2000 µg/mL) concentration. Whereas 4 showed cytotoxicity at EC50 10.2 µM against the MCF-7 human breast cancer cell line, the other compounds were inactive or not tested.

The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters.

The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription.

Stress-induced inhibition of translation independently of eIF2α phosphorylation.

Exposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis. Phosphorylation of eIF2α has been taken as a readout for downregulation of translation, but the role of eIF2α phosphorylation in the downregulation of general translation has not been much investigated. We show here that UV-induced global inhibition of translation in fission yeast cells is independent of eIF2α phosphorylation and the eIF2α kinase general control nonderepressible-2 protein (Gcn2). Also, in budding yeast and mammalian cells, the UV-induced translational depression is largely independent of GCN2 and eIF2α phosphorylation. Furthermore, exposure of fission yeast cells to oxidative stress generated by hydrogen peroxide induced an inhibition of translation that is also independent of Gcn2 and of eIF2α phosphorylation. Our findings show that stress-induced translational inhibition occurs through an unknown mechanism that is likely to be conserved through evolution.

Stress granule-defective mutants deregulate stress responsive transcripts.

To reduce expression of gene products not required under stress conditions, eukaryotic cells form large and complex cytoplasmic aggregates of RNA and proteins (stress granules; SGs), where transcripts are kept translationally inert. The overall composition of SGs, as well as their assembly requirements and regulation through stress-activated signaling pathways remain largely unknown. We have performed a genome-wide screen of S. cerevisiae gene deletion mutants for defects in SG formation upon glucose starvation stress. The screen revealed numerous genes not previously implicated in SG formation. Most mutants with strong phenotypes are equally SG defective when challenged with other stresses, but a considerable fraction is stress-specific. Proteins associated with SG defects are enriched in low-complexity regions, indicating that multiple weak macromolecule interactions are responsible for the structural integrity of SGs. Certain SG-defective mutants, but not all, display an enhanced heat-induced mutation rate. We found several mutations affecting the Ran GTPase, regulating nucleocytoplasmic transport of RNA and proteins, to confer SG defects. Unexpectedly, we found stress-regulated transcripts to reach more extreme levels in mutants unable to form SGs: stress-induced mRNAs accumulate to higher levels than in the wild-type, whereas stress-repressed mRNAs are reduced further in such mutants. Our findings are consistent with the view that, not only are SGs being regulated by stress signaling pathways, but SGs also modulate the extent of stress responses. We speculate that nucleocytoplasmic shuttling of RNA-binding proteins is required for gene expression regulation during stress, and that SGs modulate this traffic. The absence of SGs thus leads the cell to excessive, and potentially deleterious, reactions to stress.

Ancient evolutionary trade-offs between yeast ploidy states.

The number of chromosome sets contained within the nucleus of eukaryotic organisms is a fundamental yet evolutionarily poorly characterized genetic variable of life. Here, we mapped the impact of ploidy on the mitotic fitness of baker's yeast and its never domesticated relative Saccharomyces paradoxus across wide swaths of their natural genotypic and phenotypic space. Surprisingly, environment-specific influences of ploidy on reproduction were found to be the rule rather than the exception. These ploidy-environment interactions were well conserved across the 2 billion generations separating the two species, suggesting that they are the products of strong selection. Previous hypotheses of generalizable advantages of haploidy or diploidy in ecological contexts imposing nutrient restriction, toxin exposure, and elevated mutational loads were rejected in favor of more fine-grained models of the interplay between ecology and ploidy. On a molecular level, cell size and mating type locus composition had equal, but limited, explanatory power, each explaining 12.5%-17% of ploidy-environment interactions. The mechanism of the cell size-based superior reproductive efficiency of haploids during Li(+) exposure was traced to the Li(+) exporter ENA. Removal of the Ena transporters, forcing dependence on the Nha1 extrusion system, completely altered the effects of ploidy on Li(+) tolerance and evoked a strong diploid superiority, demonstrating how genetic variation at a single locus can completely reverse the relative merits of haploidy and diploidy. Taken together, our findings unmasked a dynamic interplay between ploidy and ecology that was of unpredicted evolutionary importance and had multiple molecular roots.

Caffeine stabilizes Cdc25 independently of Rad3 in Schizosaccharomyces pombe contributing to checkpoint override.

Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both Schizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S. pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S. pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25.

Analysis of stress granule assembly in Schizosaccharomyces pombe.

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.

Selective inhibition of RET mediated cell proliferation in vitro by the kinase inhibitor SPP86.

BACKGROUND: The RET tyrosine kinase receptor has emerged as a target in thyroid and endocrine resistant breast cancer. We previously reported the synthesis of kinase inhibitors with potent activity against RET. Herein, we have further investigated the effect of the lead compound SPP86 on RET mediated signaling and proliferation. Based on these observations, we hypothesized that SPP86 may be useful for studying the cellular activity of RET. METHODS: We compared the effects of SPP86 on RET-induced signaling and proliferation in thyroid cancer cell lines expressing RET-PTC1 (TPC1), or the activating mutations BRAFV600E (8505C) and RASG13R (C643). The effect of SPP86 on RET- induced phosphatidylinositide 3-kinases (PI3K)/Akt and MAPK pathway signaling and cell proliferation in MCF7 breast cancer cells was also investigated. RESULTS: SPP86 inhibited MAPK signaling and proliferation in RET/PTC1 expressing TPC1 but not 8505C or C643 cells. In TPC1 cells, the inhibition of RET phosphorylation required co-exposure to SPP86 and the focal adhesion kinase (FAK) inhibitor PF573228. In MCF7 cells, SPP86 inhibited RET- induced phosphatidylinositide 3-kinases (PI3K)/Akt and MAPK signaling and estrogen receptorα (ERα) phosphorylation, and inhibited proliferation to a similar degree as tamoxifen. Interestingly, SPP86 and PF573228 inhibited RET/PTC1 and GDNF- RET induced activation of Akt and MAPK signaling to a similar degree. CONCLUSION: SPP86 selectively inhibits RET downstream signaling in RET/PTC1 but not BRAFV600E or RASG13R expressing cells, indicating that downstream kinases were not affected. SPP86 also inhibited RET signaling in MCF7 breast cancer cells. Additionally, RET- FAK crosstalk may play a key role in facilitating PTC1/RET and GDNF- RET induced activation of Akt and MAPK signaling in TPC1 and MCF7 cells.

Flemingins G-O, cytotoxic and antioxidant constituents of the leaves of Flemingia grahamiana.

The known flemingins A-C (1-3) and nine new chalcones, named flemingins G-O (4-12), along with deoxyhomoflemingin (13) and emodin (14) were isolated from a leaf extract of Flemingia grahamiana. The isolated chalcones were found to have a geranyl substituent modified into a chromene ring possessing a residual chain, as shown by spectroscopic methods. The leaf extract showed an IC50 value of 5.9 µg/mL in a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The chalcones flemingins A, B, C, G, and H were active in the DPPH radical scavenging assay (ED50 4.4-8.9 µM), while flemingins A and C showed cytotoxicity against MCF-7 human breast cancer cells (IC50 8.9 and 7.6 µM, respectively).