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Supplementary Table 1 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Supplementary Figure 1 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Supplementary Figure Legends from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Supplementary Table 2 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Supplementary Figure 2 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Supplementary Figure 7 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Supplementary Figure 4 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Supplementary Figure 6 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Supplementary Figure 3 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

There are several human genes that may encode proteins whose functions remain unknown. To find clues to their functions, we used the mutant yeast defective in Mad2, a component of the spindle checkpoint complex. Phenotypes that were provoked by the expression of a human C18orf26 protein in the mutant yeast encouraged further characterization of this protein in human cells. This protein was designated dynAP (dynactin-associated protein) because of its interaction with dynactin subunits that comprised a microtubule-based motor protein complex. The dynAP is a transmembrane protein localizing to Golgi apparatus and plasma membrane in a microtubule-dependent manner. This protein was expressed in half of human cancer cell lines but barely in normal human fibroblasts tested. The SV40-transformed fibroblasts expressed dynAP. Importantly, the expression of dynAP activated Akt (also known as protein kinase B) by promoting Ser473 phosphorylation required for the full activation, whereas knockdown of dynAP abolished this activation. The ergosterol-related compounds identified by the yeast cell–based high-throughput screen abrogated activation of Akt and induced apoptosis in a dynAP-dependent manner. We propose a possible advantage of dynAP expression in cancer cells; the survival of cancer cells that express dynAP is supported by dynAP-induced activation of Akt, sustaining high rates of proliferation. The inactivation of dynAP by the selected compounds nullifies this advantage, and thereby, the apoptotic machinery is allowed to operate. Taken together, dynAP can be a new target for cancer therapy, and the selected chemicals are useful for developing a new class of anticancer drugs. Mol Cancer Ther; 9(11); 2934–42. ©2010 AACR.

Supplementary Figure 5 from A Novel Human Dynactin-Associated Protein, dynAP, Promotes Activation of Akt, and Ergosterol-Related Compounds Induce dynAP-Dependent Apoptosis of Human Cancer Cells

Nitrogen-nitrogen bond-containing functional groups are rare, but they are found in a considerably wide class of natural products. Recent clarifications of the biosynthetic routes for such functional groups shed light onto overlooked biosynthetic genes distributed across the bacterial kingdom, highlighting the presence of yet-to-be identified natural products with peculiar functional groups. Here, the genome-mining approach targeting a unique hydrazine-forming gene led to the discovery of actinopyridazinones A (

A key intermediate in scopranone biosynthesis, prescopranone, accumulated in the mycelium of Streptomyces avermitilis SUKA carrying the biosynthetic gene cluster for scopranone lacking the sprT encoding the monooxygenase. The structure of prescopranone was elucidated by NMR and other spectral data. Prescopranone consists of a 2-pyranone ring with two atypical scoop-like moieties (1-ethyl-1-propenyl and 2-ethylbutyl groups), which was deduced as a product of the modular polyketide syntheses encoded by sprA, sprB, and sprC. Prescopranone inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity in a BMP receptor mutant cell line.

Supplementary Figures 1-4 from Disabling c-Myc in Childhood Medulloblastoma and Atypical Teratoid/Rhabdoid Tumor Cells by the Potent G-Quadruplex Interactive Agent S2T1-6OTD

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex–forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a dose- and time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC50, 0.25–0.39 μmol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1-6OTD resulted in a time-dependent (mainly c-Myc–dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors. Mol Cancer Ther; 9(1); 167–79

Supplementary Methods, Figure Legends 1-4 from Disabling c-Myc in Childhood Medulloblastoma and Atypical Teratoid/Rhabdoid Tumor Cells by the Potent G-Quadruplex Interactive Agent S2T1-6OTD

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Purpose: Glioma stem cells (GSC) are a critical therapeutic target of glioblastoma multiforme (GBM).Experimental Design: The effects of a G-quadruplex ligand, telomestatin, were evaluated using patient-derived GSCs, non-stem tumor cells (non-GSC), and normal fetal neural precursors in vitro and in vivo. The molecular targets of telomestatin were determined by immunofluorescence in situ hybridization (iFISH) and cDNA microarray. The data were then validated by in vitro and in vivo functional assays, as well as by immunohistochemistry against 90 clinical samples.Results: Telomestatin impaired the maintenance of GSC stem cell state by inducing apoptosis in vitro and in vivo. The migration potential of GSCs was also impaired by telomestatin treatment. In contrast, both normal neural precursors and non-GSCs were relatively resistant to telomestatin. Treatment of GSC-derived mouse intracranial tumors reduced tumor sizes in vivo without a noticeable cell death in normal brains. iFISH revealed both telomeric and non-telomeric DNA damage by telomestatin in GSCs but not in non-GSCs. cDNA microarray identified a proto-oncogene, c-Myb, as a novel molecular target of telomestatin in GSCs, and pharmacodynamic analysis in telomestatin-treated tumor-bearing mouse brains showed a reduction of c-Myb in tumors in vivo. Knockdown of c-Myb phenocopied telomestatin-treated GSCs both in vitro and in vivo, and restoring c-Myb by overexpression partially rescued the phenotype. Finally, c-Myb expression was markedly elevated in surgical specimens of GBMs compared with normal tissues.Conclusions: These data indicate that telomestatin potently eradicates GSCs through telomere disruption and c-Myb inhibition, and this study suggests a novel GSC-directed therapeutic strategy for GBMs. Clin Cancer Res; 18(5); 1268–80. ©2012 AACR.

Supplementary Methods, Figures 1-4, Tables 1-7 from Chemical Genomics Identifies the Unfolded Protein Response as a Target for Selective Cancer Cell Killing during Glucose Deprivation

Glucose deprivation, a cell condition that occurs in solid tumors, activates the unfolded protein response (UPR). A key feature of the UPR is the transcription program activation, which allows the cell to survive under stress conditions. Here, we show that the UPR transcription program is disrupted by the antidiabetic biguanides metformin, buformin, and phenformin depending on cellular glucose availability. These drugs inhibit production of the UPR transcription activators XBP1 and ATF4 and induce massive cell death during glucose deprivation as did the antitumor macrocyclic compound versipelostatin. Gene expression profiling shows remarkable similarity in the modes of action of biguanides and versipelostatin determined by the broad range of glucose deprivation-inducible genes. Importantly, during glucose deprivation, most of the biguanide suppression genes overlap with the genes induced by tunicamycin, a chemical UPR inducer. Gene expression profiling also identifies drug-driven signatures as a tool for discovering pharmacologic UPR modulators. Our findings show that disrupting the UPR during glucose deprivation could be an attractive approach for selective cancer cell killing and could provide a chemical genomic basis for developing UPR-targeting drugs against solid tumors. [Cancer Res 2009;69(10):4225–34]

Streptomyces graminofaciens A-8890 produces two macrolide antibiotics, FD-891 and virustomycin A, both of which show significant biological activities. In this study, we identified the virustomycin A biosynthetic gene cluster, which encodes type I polyketide synthases (PKSs), ethylmalonyl-CoA biosynthetic enzymes, methoxymalony-acyl carrier protein biosynthetic enzymes, and post-PKS modification enzymes. Next, we demonstrated that the acyltransferase domain can be exchanged between the Vsm PKSs and the PKSs involved in FD-891 biosynthesis (Gfs PKSs), without encountering supply problems of the unique extender units. We exchanged the malonyltransferase domain in the loading module of Gfs PKS with the ethylmalonyltransferase domain and the methoxymalonyltransferase domain of Vsm PKSs. Consequently, the expected two-carbon elongated analog 26-ethyl-FD-891 was successfully produced with a titer comparable to FD-891 production by the wild type; however, exchange with the methoxymalonyltransferase domain did not produce any FD-891 analogs. Furthermore, 26-ethyl-FD-891 showed potent cytotoxic activity against HeLa cells, like natural FD-891.

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The diazo group is an important functional group that can confer biological activity to natural products owing to its high reactivity. Recent studies have revealed that diazo groups are synthesized from amino groups using nitrous acid in secondary metabolites of actinomycetes. However, genome database analysis indicated that there are still many diazo group-biosynthesizing enzymes for unknown biosynthetic pathways. Here, we discovered an avenalumic acid biosynthesis gene cluster in Streptomyces sp. RI-77 by genome mining of enzymes involved in diazo group formation. Through heterologous expression, the gene cluster was revealed to direct avenalumic acid (AVA) biosynthesis via 3-aminoavenalumic acid (3-AAA). In vitro enzyme assays showed that AvaA6 and AvaA7 catalyzed the diazotization of 3-AAA using nitrous acid and substitution of the diazo group for hydride to synthesize AVA, respectively. This study revealed an unprecedented pathway for amino group removal via diazotization.

Recent progress in three-dimensional (3D) cell culture systems has attracted much attention in the fields of basic life science and drug development. Newly established methods include 3D co-culture, spheroid culture, and organoid culture; these methods enable more human tissue-like culture and have largely replaced traditional two-dimensional (2D) monolayer culture. By combining 3D culture methods with high-content imaging analysis, it is possible to obtain diverse and convincing data even during initial screening (which requires rapid and easy operating procedures). Until recently, 3D culture methods were considered expensive, time-consuming, complex, and unstable. However, by exploiting the self-assembling nature of cells and adding several technical improvements, we have developed several phenotypic screenings aimed at discovering anticancer compounds.

Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides . F moA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It use s α - methyl- l -serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography ( apo -form, with adenylyl-imidodiphosphate and α -methyl- l -seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α -methyl- l -serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activit y. The collective data support a proposed catalytic cycle of FmoA3.

Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 ∆saverm3882 ∆saverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. KEY POINTS: • Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq. • Selection validity was evaluated by an engineered S. avermitilis expression system. • Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds.

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A-D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.

Using genome mining approach, we identified a novel biosynthetic gene cluster containing trans-AT type PKS genes from Streptomyces versipellis 4083-SVS6. A bacterial artificial chromosome (BAC) clone, pKU503JL68_PN1_P10-C12, accommodating the entire biosynthetic gene cluster was obtained from a BAC library. Heterologous expression of the biosynthetic gene cluster in Streptomyces lividans TK23 led to the production of a novel polyene compound, JBIR-159. We report herein the biosynthetic gene cluster for JBIR-159, and the heterologous expression, isolation, structure determination and a brief biological activity.

We have previously identified receptor tyrosine kinase‐like orphan receptor 1 (ROR1) as a direct transcriptional target of TTF‐1/NKX2‐1, a lineage‐survival oncogene in lung adenocarcinoma. ROR1 sustains prosurvival signaling from multiple receptor tyrosine kinases including epidermal growth factor receptor, MET, and insulin‐like growth factor 1 receptor in part by maintaining the caveolae structure as a scaffold protein of cavin‐1 and caveolin‐1. In this study, a high throughput screening of the natural product library containing 2560 compounds was undertaken using a cell‐based FluoPPI assay detecting ROR1‐cavin‐1 interaction. As a result, geldanamycin (GA), a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Geldanamycin, as well as two GA derivatives tested in the clinic, 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) and 17‐dimethylaminoethylamino‐17‐demethoxygeldanamycin (17‐DMAG), decreased ROR1 protein expression. We found that ROR1 physically interacted with HSP90α, but not with other HSP90 paralogs, HSP90β or GRP94. Geldanamycin in turn destabilized and degraded ROR1 protein in a dose‐ and time‐dependent manner through the ubiquitin/proteasome pathway, resulting in a significant suppression of cell proliferation in lung adenocarcinoma cell lines, for which the kinase domain of ROR1, but not its kinase activity or N‐glycosylation, was required. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1‐positive lung adenocarcinoma., High throughput screening of the natural product library was carried out using a cell‐based FluoPPI assay detecting ROR1‐cavin‐1 interaction. As a result, geldanamycin, a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1‐positive lung adenocarcinoma.

A novel methymycin analog, 12-ketomethymycin N-oxide, was produced by the heterologous expression of the pikromycin/methymycin biosynthetic gene cluster of Streptomyces sp. AM4900 together with 12-ketomethymycin, which was only isolated by the biotransformation of the synthetic intermediate before. Their structures were determined by the spectroscopic data and the chemical derivatization. 12-Ketomethymycin showed a weak cytotoxicity against SKOV-3 and Jurkat cells, although its N-oxide analog did not show any activity. Both showed no antibacterial activities against Escherichia coli and Micrococcus luteus.

We report a complete genome sequence of a novel bacterial isolate, strain TBR-22, belonging to the class