Unlike normal tissues, cancers experience profound alterations in protein homeostasis. Powerful innate adaptive mechanisms, especially the transcriptional response regulated by Heat Shock Factor 1 (HSF1), are activated in cancers to enable survival under these stressful conditions. Natural products that further tax these stress responses can overwhelm the ability to cope and could provide leads for the development of new, broadly effective anticancer drugs. To identify compounds that drive the HSF1-dependent stress response, we evaluated over 80,000 natural and synthetic compounds as well as partially purified natural product extracts using a reporter cell line optimized for high-throughput screening. Surprisingly, many of the strongly active compounds identified were natural products representing five diverse chemical classes (limonoids, curvularins, withanolides, celastraloids and colletofragarones). All of these compounds share the same chemical motif, an α,β-unsaturated carbonyl functionality, with strong potential for thiol-reactivity. Despite the lack of a priori mechanistic requirements in our primary phenotypic screen, this motif was found to be necessary albeit not sufficient, for both heat-shock activation and inhibition of glioma tumor cell growth. Within the withanolide class, a promising therapeutic index for the compound withaferin A was demonstrated in vivo using a stringent orthotopic human glioma xenograft model in mice. Our findings reveal that diverse organisms elaborate structurally complex thiol-reactive metabolites that act on the stress responses of heterologous organisms including humans. From a chemical biology perspective, they define a robust approach for discovering candidate compounds that target the malignant phenotype by disrupting protein homeostasis.

To understand the relationship between the structure and the remarkably diverse bioactivities reported for withanolides, we obtained withaferin A (WA; 1) and 36 analogues (2-37) and compared their cytotoxicity to cytoprotective heat-shock-inducing activity (HSA). By analyzing structure-activity relationships for the series, we found that the ring A enone is essential for both bioactivities. Acetylation of 27-OH of 4-epi-WA (28) to 33 enhanced both activities, whereas introduction of β-OH to WA at C-12 (29) and C-15 (30) decreased both activities. Introduction of β-OAc to 4,27-diacetyl-WA (16) at C-15 (37) decreased HSA without affecting cytotoxicity, but at C-12 (36), it had minimal effect. Importantly, acetylation of 27-OH, yielding 15 from 1, 16 from 14, and 35 from 34, enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can be modified to enhance HSA selectively, thereby assisting development of natural product-inspired drugs to combat protein aggregation-associated diseases by stimulating cellular defense mechanisms.

d 10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strain Aspergillus terreus AH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (A. terreus CURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted in Saccharomyces cerevisiae by heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta-or hexaketide starters in the 14-membered rings of resorcylic acid lactones. Tetraketide N-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides.

The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen-and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.actinomycetes | GlnR | carbon sources utilization | ABC transport system | nitrogen metabolism

Nitrilotriacetate (NTA), a synthetic chelating agent, has been used for various radionuclide processing and decontamination procedures. NTA has been codisposed with radionuclides and heavy metals to soils and subsurface sediments (4, 27, 32), where it can greatly increase the mobility of these metals in the environment by forming soluble complexes with them. Microbial degradation of NTA may ultimately aid in immobilizing these radionuclides. Several NTA-degrading bacteria have been isolated (2,7,13,18,23,42). An NTA monooxygenase (NTA-Mo) of Chelatobacter heintzii ATCC 29600 has been identified (16) (47). NTA oxidation is proposed to be catalyzed by a heterodimer of components A (cA) and B (cB), but it is unclear how these two components interact with each other or what the function of each component is (44). In order to provide additional information on the function of cA and cB and to facilitate understanding natural attenuation or engineered bioremediation of NTA in the environment through the use of specific PCR primers or gene probes, we cloned, sequenced, and analyzed a gene cluster involved in NTA degradation. On the basis of DNA sequence and activity analysis, the two components were shown to be two separate enzymes, an monooxygenase that oxidized NTA at the expense of reduced flavin mononucleotide (FMNH 2 ) and O 2 and an NADH:flavin mononucleotide (FMN) oxidoreductase that uses NADH to reduce FMN to FMNH 2 .(A preliminary account of this work was presented at the 1995 American Society for Microbiology General Meeting [46], and the DNA sequence and gene organization have been published in a master's degree thesis [46]). MATERIALS AND METHODSBacterial strains and plasmids. The plasmids used or constructed in this study are listed in Table 1. C. heintzii ATCC 29600 was obtained from the American Type Culture Collection (Rockville, Md.) and was cultured with using a mineral salt medium with NTA as the sole carbon source (42). Escherichia coli HB101 was used as the host for pRK311, strain DH5␣ was used as the host for pBS, and strain JM105 was used as the host for pTrc99A. E. coli strains were routinely grown at 37ЊC in Luria-Bertani (LB) medium or on LB agar (37). Tetracycline and ampicillin (Sigma, St. Louis, Mo.) were used at 25 and 100 g/ml, respectively.Gene cloning. The two components of NTA-Mo were purified according to the method of Uetz et al. (44), subjected to discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (24), and then electroblotted onto a polyvinylidene difluoride membrane (28, 31) for N-terminal amino acid sequence analysis on an ABI 470 protein sequencer at the Department of Biochemistry and Biophysics, Washington State University.Oligonucleotides were 5Ј end labeled with 32 P by polynucleotide kinase (37). C. heintzii ATCC 29600 genomic DNA was isolated by a combination of largescale CsCl gradient preparation and hexadecyltrimethyl ammonium bromide and phenol extraction (3, 37). The PolarPlex Chemiluminescent Kit of Millipore (Bedford, Mass.) was used to ...

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Prostate cancer (PC) is the second most prevalent cancer among men in Western societies, and those who develop metastatic castration-resistant PC (CRPC) invariably succumb to the disease. The need for effective treatments for CRPC is a pressing concern, especially due to limited durable responses with currently employed therapies. Here, we demonstrate the successful application of a high-throughput gene-expression profiling assay directly targeting genes of the androgen receptor pathway to screen a natural products library leading to the identification of 17β-hydroxywithanolides 1-5, of which physachenolide D (5) exhibited potent and selective in vitro activity against two PC cell lines, LNCaP and PC-3. Epoxidation of 5 afforded physachenolide C (6) with higher potency and stability. Structure-activity relationships for withanolides as potential anti-PC agents are presented together with in vivo efficacy studies on compound 6, suggesting that 17β-hydroxywithanolides are promising candidates for further development as CRPC therapeutics.