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Zearalenone and radicicol are resorcylic acid lactones produced by numerous plant pathogenic fungi. Zearalenone is a non-steroidal estrogen mimic that can cause serious reproductive issues in livestock that consume contaminated feed. Radicicol is a potent inhibitor of the molecular chaperone Hsp90, which, in plants, has an important role in coordinating the host's immune response during infection. Here, we describe the identification and characterization of a soil-borne strain of the Gram-positive bacterium Aeromicrobium sp. capable of hydrolyzing the macrolide ring of resorcylic acid lactones, including zearalenone and radicicol. Proteomic analysis of biochemically enriched fractions from the isolated and cultured bacterium identified an α/β-hydrolase responsible for this activity. A recombinantly expressed and purified form of the hydrolase (termed RALH) was active against both zearalenone and radicicol. Interpretation of high-resolution mass spectrometry and NMR data confirmed the structures of the enzymatic products as the previously reported non-toxic metabolite hydrolyzed zearalenone and hydrolyzed radicicol. Hydrolyzed radicicol was demonstrated to no longer inhibit the ATPase activity of the Saccharomyces cerevisiae Hsp90 homolog in vitro. Enzymatic degradation of resorcylic acid lactones will enable insight into their biological functions. [ABSTRACT FROM AUTHOR]

Zearalenone and radicicol are resorcylic acid lactones produced by numerous plant pathogenic fungi. Zearalenone is a non-steroidal estrogen mimic that can cause serious reproductive issues in livestock that consume contaminated feed. Radicicol is a potent inhibitor of the molecular chaperone Hsp90, which, in plants, has an important role in coordinating the host’s immune response during infection. Here, we describe the identification and characterization of a soil-borne strain of the Gram-positive bacterium Aeromicrobium sp. capable of hydrolyzing the macrolide ring of resorcylic acid lactones, including zearalenone and radicicol. Proteomic analysis of biochemically enriched fractions from the isolated and cultured bacterium identified an α/β-hydrolase responsible for this activity. A recombinantly expressed and purified form of the hydrolase (termed RALH) was active against both zearalenone and radicicol. Interpretation of high-resolution mass spectrometry and NMR data confirmed the structures of the enzymatic products as the previously reported non-toxic metabolite hydrolyzed zearalenone and hydrolyzed radicicol. Hydrolyzed radicicol was demonstrated to no longer inhibit the ATPase activity of the Saccharomyces cerevisiae Hsp90 homolog in vitro. Enzymatic degradation of resorcylic acid lactones will enable insight into their biological functions.

The Fusarium solani species complex (FSSC) constitutes at least 77 phylogenetically distinct species including several agriculturally important and clinically relevant opportunistic pathogens. As with other Fusaria, they have been well documented to produce many secondary metabolites—compounds that are not required for the fungus to grow or develop but may be beneficial to the organism. An analysis of ten genomes from fungi within the terminal clade (clade 3) of the FSSC revealed each genome encoded 35 (F. cucurbitcola) to 48 (F. tenucristatum) secondary metabolite biosynthetic gene clusters (BGCs). A total of seventy-four different BGCs were identified from the ten FSSC genomes including seven polyketide synthases (PKS), thirteen nonribosomal peptide synthetases (NRPS), two terpene synthase BGCs, and a single dimethylallytryptophan synthase (DMATS) BGC conserved in all the genomes. Some of the clusters that were shared included those responsible for producing naphthoquinones such as fusarubins, a red pigmented compound, squalestatin, and the siderophores malonichrome, ferricrocin, and triacetylfusarinine. Eight novel NRPS and five novel PKS BGCs were identified, while BGCs predicted to produce radicicol, gibberellin, and fusaoctaxin were identified, which have not previously described in members of the FSSC. The diversity of the secondary metabolite repertoire of the FSSC may contribute to the expansive host range of these fungi and their ability to colonize broad habitats. [ABSTRACT FROM AUTHOR]

The yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and have acquired traits from the combined parental genomes such as ability to ferment a range of sugars at low temperatures and to produce aromatic flavour compounds, allowing for the production of lager beers with crisp, clean flavours. The polyploid strains are sterile and have reached an evolutionary bottleneck for genetic variation. Here we describe an accelerated evolution approach to obtain lager yeasts with enhanced flavour profiles. As the relative expression of orthologous alleles is a significant contributor to the transcriptome during fermentation, we aimed to induce genetic variation by altering the S. cerevisiae to S. eubayanus chromosome ratio. Aneuploidy was induced through the temporary inhibition of the cell's stress response and strains with increased production of aromatic amino acids via the Shikimate pathway were selected by resistance to amino acid analogues. Genomic changes such as gross chromosomal rearrangements, chromosome loss and chromosome gain were detected in the characterised mutants, as were single‐nucleotide polymorphisms in ARO4, encoding for DAHP synthase, the catalytic enzyme in the first step of the Shikimate pathway. Transcriptome analysis confirmed the upregulation of genes encoding enzymes in the Ehrlich pathway and the concomitant increase in the production of higher alcohols and esters such as 2‐phenylethanol, 2‐phenylethyl acetate, tryptophol, and tyrosol. We propose that the polyploid nature of S. pastorianus genomes is an advantageous trait supporting opportunities for genetic alteration in otherwise sterile strains. Take‐away: •Radicicol was used to induce genetic diversity in interspecific Saccharomyces pastorianus hybrids for improved flavour profiles. This drug is known to inhibit the chaperone Hsp90. Amino acid analogues of phenylalanine were used to select strains with improved production of 2‐phenylethanol and 2‐phenylethyl acetate.•Two S. pastorianus mutants were selected based on their overproduction of higher alcohols and esters derived from the secondary metabolism of aromatic amino acids; 2‐phenylethanol and 2‐phenylethyl acetate, together with tryptophol and tyrosol.•Genomic analysis showed that Radicicol induced gross chromosomal rearrangements, chromosome gain and chromosome loss in S. pastorianus mutants. Furthermore, single‐nucleotide polymorphisms in ARO4 were found in both strains.•Transcriptome analysis shows upregulated genes of the Ehrlich pathway and unique expression patterns in mutant strains under fermentative conditions.•This study proposes radicicol as a non‐GMO mutagenesis method to induce changes in chromosomal architecture of polyploid strains and generate new strains with improved traits for different biotechnological processes. [ABSTRACT FROM AUTHOR]

The Fusarium solani species complex (FSSC) constitutes at least 77 phylogenetically distinct species including several agriculturally important and clinically relevant opportunistic pathogens. As with other Fusaria, they have been well documented to produce many secondary metabolites—compounds that are not required for the fungus to grow or develop but may be beneficial to the organism. An analysis of ten genomes from fungi within the terminal clade (clade 3) of the FSSC revealed each genome encoded 35 (F. cucurbitcola) to 48 (F. tenucristatum) secondary metabolite biosynthetic gene clusters (BGCs). A total of seventy-four different BGCs were identified from the ten FSSC genomes including seven polyketide synthases (PKS), thirteen nonribosomal peptide synthetases (NRPS), two terpene synthase BGCs, and a single dimethylallytryptophan synthase (DMATS) BGC conserved in all the genomes. Some of the clusters that were shared included those responsible for producing naphthoquinones such as fusarubins, a red pigmented compound, squalestatin, and the siderophores malonichrome, ferricrocin, and triacetylfusarinine. Eight novel NRPS and five novel PKS BGCs were identified, while BGCs predicted to produce radicicol, gibberellin, and fusaoctaxin were identified, which have not previously described in members of the FSSC. The diversity of the secondary metabolite repertoire of the FSSC may contribute to the expansive host range of these fungi and their ability to colonize broad habitats.

SLC6A14 is a plasma membrane transporter specific for neutral and basic amino acids, upregulated in many tumors. This study focused on breast cancer cell lines, showing the fully glycosylated band, known to be at the cell surface, in estrogen receptor positive lines. Inhibition of heat shock protein 90β (HSP90β) decreased the level of this band, what correlated with a decrease of SLC6A14 transport activity. A direct interaction between SLC6A14 and HSP90β was confirmed in proximity ligation assay, pointing to the role of HSP90 in folding control in endoplasmic reticulum and affecting farther transporter trafficking to the cell surface. Either inhibitor of SLC6A14 (α-methyltryptophan) or of HSP90 (radicicol) had the cytotoxic effect, when added alone, while treatment with both compounds had a synergistic effect. This points to SLC6A14 as a druggable target in breast cancer and a combination therapy being more efficient in killing cancer cells. [Display omitted] • Amino acid transporter SLC6A14 is expressed in breast cancer cells. • SLC6A14 directly interacts with heat shock protein 90β (HSP90β). • Presence of SLC6A14 active form depends on HSP90. • HSP90 inhibitor decreases SLC6A14 transport activity. • HSP90 and SLC6A14 inhibitors increase synergistically cytotoxicity in cancer cells. [ABSTRACT FROM AUTHOR]

Ilyonectria mors-panacis , previously Cylindrocarpon destructans , is the main plant pathogen responsible for the fungal disease ginseng root rot. This economically important disease, also called disappearing root rot, reduces crop yields by an average of 30% at harvest. While the disease is well studied from ecological and genomic perspectives, the role of I. mors-panacis secondary metabolites in the disease process is not well understood. Our previous metabolomics study showed Ilyonectria strains that cause ginseng root rot produce mixtures of putative resorcylic acid lactones, whereas avirulent strains did not, and collectively synthesize fewer metabolites. To confirm these metabolomics findings, we isolated and characterized the secondary metabolites from I. mors-panacis DAOMC 251601, a strain that causes ginseng root rot. From its EtOAc soluble culture filtrate extract, eight resorcylic acid lactones (1 - 8), including chlorinated and non-chlorinated congeners, were characterized by HRMS and spectroscopic approaches (NMR, OR, UV). The structure of one new metabolite, named radicicol E (1), was elucidated and additional spectroscopic data for the known compound nordinonediol (2) are reported. Further, radicicol (9) production was confirmed by comparison to a standard. The roles that resorcylic acid lactones and the siderophore N,N′,N" triacetylfusarine C have in promoting Ilyonectria ginseng root rot are also discussed. [Display omitted] • Studied the metabolites from an Ilyonectria mors-panacis strain that causes the disease ginseng root rot. • Eight resorcylic acid lactones, including one new compound, were isolated and characterized. • Resorcylic acid lactones are proposed to contribute to the disease process by supressing the plants immune system. [ABSTRACT FROM AUTHOR]

• Resorcylic acid lactones (RALs) are fungal polyketides. • RALs consist of a β-resorcylic acid residue embedded in a macrolactone ring. • RALs exhibit a broad range of biological activities, including anticancer activities. • Radicicol, a RAL, selectively inhibits Hsp90. • Several RALs inhibit protein kinases and nuclear factor kappa B (NF-κB). Resorcylic acid lactones (RALs) are fungal polyketides that consist of a β-resorcylic acid residue (2,4-dihydroxybenzoic acid) embedded in a macrolactone ring. RALs exhibit a broad range of biological activities, including anticancer activities. Following discovery of the selective Hsp90 inhibition activity of radicicol, the kinase inhibition activity of hypothemycin, monocillin II, 5Z-7-oxo-zeaenol, and L-783,277 RALs, and the nuclear factor kappa B (NF-κB) inhibition activity of the RAL zearalenone, have attracted great attention as potential therapeutics for cancer treatment. In this minireview, we focus on natural RALs that possess cytotoxic activities [IC 50 values < 10 μM (or 4–5 μg/ml)], discussing their structures, isolation, occurrence, biological activities, and anticancer molecular mechanisms. [ABSTRACT FROM AUTHOR]

ABSTRACT Leishmania donovani is a parasitic protist that causes the lethal Kala-azar fever in India and East Africa. Gene expression in Leishmania is regulated by gene copy number variation and inducible translation while RNA synthesis initiates at a small number of sites per chromosome and proceeds through polycistronic transcription units, precluding a gene-specific regulation (C. Clayton and M. Shapira, Mol Biochem Parasitol 156:93–101, 2007, https://doi.org/10.1016/j.molbiopara.2007.07.007). Here, we analyze the dynamics of chromatin structure in both life cycle stages of the parasite and find evidence for an additional, epigenetic gene regulation pathway in this early branching eukaryote. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis (J. D. Buenrostro, P. G. Giresi, L. C. Zaba, H. Y. Chang, and W. J. Greenleaf, Nat Methods 10:1213–1218, 2013, https://doi.org/10.1038/nmeth.2688) predominantly shows euchromatin at transcription start regions in fast-growing promastigotes, but mostly heterochromatin in the slowly proliferating amastigotes, the mammalian stage, reflecting a previously shown increase of histone synthesis in the latter stage. IMPORTANCE Leishmania parasites are important pathogens with a global impact and cause poverty-related illness and death. They are devoid of classic cis- and trans-acting transcription regulators but use regulated translation and gene copy number variations to adapt to hosts and environments. In this work, we show that transcription start regions present as open euchromatin in fast-growing insect stages but as less-accessible heterochromatin in the slowly proliferating amastigote stage, indicating an epigenetic control of gene accessibility in this early branching eukaryotic pathogen. This finding should stimulate renewed interest in the control of RNA synthesis in Leishmania and related parasites.

The interactions between radicicol and four proteins (catalase, trypsin, pepsin, and human serum protein) are investigated by spectroscopic techniques and molecular docking. A static quenching process is confirmed. The binding constant value between radicicol and human serum protein is the largest among the four proteins. Results reveal changes in the micro-environment of the protein by the addition of radicicol. It is found that radicicol shows an inhibitory effect on the activity of proteins (catalase, trypsin, and pepsin). Molecular docking results are consistent with the thermodynamic experimental results. This work provides clues to the elucidation of the mechanisms of the interactions between radicicol and proteins. [ABSTRACT FROM AUTHOR]

The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant–pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsis thaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant–pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, AtHSP90.1 and wheat TaHSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A. thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichia coli. In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants.

The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant–pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsis thaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant–pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, At HSP90.1 and wheat Ta HSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A. thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichia coli. In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Radicicol was isolated from a fungus occurring in the crevices of marble rocks. • Radicicol overcomes wild-type/mutant α-synuclein mediated yeast cell death. • Radicicol diminishes ROS levels, and reverses mitochondrial dysfunction. • Radicicol prevents nuclear DNA fragmentation in yeast cells. • Radicicol does not prevent yeast cell death inflicted by proapoptotic protein Bax. Abstract Aggregation/misfolding of α-synuclein and βA4 proteins cause neuronal cell death (NCD) associated with Parkinson's and Alzheimer's disease. It has been suggested that a heat shock protein-90 (Hsp90) inhibitor can prevent NCD by activating the heat shock transcription factor-1 which, in turn, upregulates molecular chaperones such as Hsp70 that targets aggregated/misfolded proteins for refolding/degradation. We have isolated radicicol, an Hsp90 inhibitor, from a fungus occurring in the crevices of marble rocks of Central India. Radicicol, which was found to be a strong antioxidant, was tested for its ability to rescue yeast cells from death induced by expression of wild-type α-synuclein, its more toxic A53T mutant, and βA4. It effectively overcomes wild-type/mutant α-synuclein mediated yeast cell death, concomitantly diminishes ROS levels, reverses mitochondrial dysfunction and prevents nuclear DNA-fragmentation, a hallmark of apoptosis. Surprisingly however, radicicol is unable to rescue yeast cells from death triggered by expression of secreted βA4. Moreover, although radicicol acts as an antioxidant it fails to prevent yeast cell death inflicted by the proapoptotic protein, Bax. Our results indicate that radicicol specifically targets aggregated/misfolded α-synuclein's toxicity and opens up the possibility of using multiple yeast assays to screen natural product libraries for compounds that would unambiguously target α-synuclein aggregation/misfolding. [ABSTRACT FROM AUTHOR]

Heat shock protein 90 (Hsp90) is a crucial component in carcinogenesis and serves as a molecular chaperone that facilitates protein maturation whilst protecting cells against temperature-induced stress. The function of Hsp90 is highly dependent on adenosine triphosphate (ATP) binding to the N-terminal domain of the protein. Thus, inhibition through displacement of ATP by means of competitive binding with a suitable organic molecule is considered an attractive topic in cancer research. Radicicol (RD) and its derivative, resorcinylic isoxazole amine NVP-AUY922 (NVP), have shown promising pharmacodynamics against Hsp90 activity. To date, the underlying binding mechanism of RD and NVP has not yet been investigated. In this study, we provide a comprehensive understanding of the binding mechanism of RD and NVP, from an atomistic perspective. Density functional theory (DFT) calculations enabled the analyses of the compounds’ electronic properties and results obtained proved to be significant in which NVP was predicted to be more favorable with solvation free energy value of −23.3 kcal/mol and highest stability energy of 75.5 kcal/mol for a major atomic delocalization. Molecular dynamic (MD) analysis revealed NVP bound to Hsp90 (NT-NVP) is more stable in comparison to RD (NT-RD). The Hsp90 protein exhibited a greater binding affinity for NT-NVP (−49.4 ± 3.9 kcal/mol) relative to NT-RD (−28.9 ± 4.5 kcal/mol). The key residues influential in this interaction are Gly 97, Asp 93 and Thr 184. These findings provide valuable insights into the Hsp90 dynamics and will serve as a guide for the design of potent novel inhibitors for cancer treatment.

ABSTRACT The 90-kDa heat shock protein (HSP90) of eukaryotes is a highly abundant and essential chaperone required for the maturation of regulatory and signal proteins. In the protozoan parasite Leishmania donovani, causative agent of the fatal visceral leishmaniasis, HSP90 activity is essential for cell proliferation and survival. Even more importantly, its inhibition causes life cycle progression from the insect stage to the pathogenic, mammalian stage. To unravel the molecular impact of HSP90 activity on the parasites’ gene expression, we performed a ribosome profiling analysis of L. donovani, comparing genome-wide protein synthesis patterns in the presence and absence of the HSP90-specific inhibitor radicicol and an ectopically expressed radicicol-resistant HSP90 variant. We find that ribosome-protected RNA faithfully maps open reading frames and represents 97% of the annotated protein-coding genes of L. donovani. Protein synthesis was found to correlate poorly with RNA steady-state levels, indicating a regulated translation as primary mechanism for HSP90-dependent gene expression. The results confirm inhibitory effects of HSP90 on the synthesis of Leishmania proteins that are associated with the pathogenic, intracellular stage of the parasite. Those include heat shock proteins, redox enzymes, virulence-enhancing surface proteins, proteolytic pathways, and a complete set of histones. Conversely, HSP90 promotes fatty acid synthesis enzymes. Complementing radicicol treatment with the radicicol-resistant HSP90rr variant revealed important off-target radicicol effects that control a large number of the above-listed proteins. Leishmania lacks gene-specific transcription regulation and relies on regulated translation instead. Our ribosome footprinting analysis demonstrates a controlling function of HSP90 in stage-specific protein synthesis but also significant, HSP90-independent effects of the inhibitor radicicol. IMPORTANCE Leishmania parasites cause severe illness in humans and animals. They exist in two developmental stages, insect form and mammalian form, which differ in shape and gene expression. By mapping and quantifying RNA fragments protected by protein synthesis complexes, we determined the rates of protein synthesis for >90% of all Leishmania proteins in response to the inhibition of a key regulatory protein, the 90-kDa heat shock protein. We find that Leishmania depends on a regulation of protein synthesis for controlling its gene expression and that heat shock protein 90 inhibition can trigger the developmental program from insect form to mammalian form of the pathogen.

The present study aimed to explore the mechanisms underlying sepsis-induced acute lung injury (ALI) and identify more effective therapeutic strategies to treat it. The gene expression data set GSE10474 was downloaded and assessed to identify differentially expressed genes (DEGs). Principal component analysis, functional enrichment analysis and differential co-expression analysis of DEGs were performed. Furthermore, potential target drugs for key DEGs were assessed. A total of 209 DEGs, including 107 upregulated and 102 downregulated genes were screened. A number of DEGs, including zinc finger and BTB domain containing 17 (ZBTB17), heat shock protein 90 kDa β, member 1 (HSP90B1) and major histocompatibility complex, class II, DR α were identified. Furthermore, gene ontology terms including antigen processing and presentation, glycerophospholipid metabolism, transcriptional misregulation in cancer, thyroid hormone synthesis and pathways associated with diseases, such as asthma were identified. In addition, a differential co-expression network containing ubiquitin-conjugating enzyme E2 D4, putative and tubulin, γ complex associated protein 3 was constructed. Furthermore, a number of gene-drug interactions, including between HSP90B1 and adenosine-5'-diphosphate and radicicol, were identified. Therefore, DEGs, including ZBTB17 and HSP90B1, may be important in the pathogenesis of sepsis-induced ALI. Furthermore, drugs including adenosine-5'-diphosphate may be novel drug candidates to treat patients with ALI. [ABSTRACT FROM AUTHOR]

CacyBP/SIP interacts with Hsp90 and is able to protect proteins from denaturation and/or aggregation induced by elevated temperature. In this work we studied the influence of different stress factors on CacyBP/SIP level in HEp-2 cells. We have found that H 2 O 2 and radicicol treatment resulted in a significant increase (up to 40%) in the CacyBP/SIP level. We have also found that HEp-2 cells overexpressing CacyBP/SIP were more resistant to stress-induced death. Further studies have revealed that the Hsf1 transcription factor binds to the CacyBP/SIP gene promoter and up-regulates CacyBP/SIP expression under stress conditions. To check whether the CacyBP/SIP protein might play a role in stress responses in vivo , we analyzed its level in selected brain structures of control and stressed mice. We have found that the level of the CacyBP/SIP protein was higher in the thalamus/hypothalamus, hippocampus and brainstem of stressed mice. Thus, the presented results clearly indicate that CacyBP/SIP is involved in cellular stress response. [ABSTRACT FROM AUTHOR]

The soil-borne ascomycete fungus Cylindrocarpon destructans causes ginseng root rot disease and produces various secondary metabolites such as brefeldin A and radicicol. The slow growth of this fungus compared with other plant pathogenic and saprophytic fungi in soil disturbs isolation of this fungus from soil and infected ginseng. In this study, we developed a selective medium for C. destructans using radicicol produced by this fungus. Supplementing 50 mg/L of radicicol to medium inhibited the mycelia growth of other fungi including Botrytis cinerea, Rhizoctonia solani and Alternaria panax, but did not affect the growth of C. destructans. In addition, conidia germination of other fungal species except for C. destructans was inhibited in submerged culture supplemented with radicicol. This medium provides a very efficient tool for isolating C. destructans and also can be used as an enrichment medium for this fungus.

Neuroblastoma (NBL) accounts for >7% of malignancies in patients younger than 15 years. Low- and intermediate-risk patients exhibit excellent or good prognosis after treatment, whereas for high-risk (HR) patients, the estimated 5-year survival rates is still <40%. The ability to stratify HR patients that will not respond to standard treatment strategies is critical for informed treatment decisions. In this study, we have generated a specific kinome gene signature, named Kinome-27, which is able to identify a subset of HR-NBL tumors, named ultra-HR NBL, with highly aggressive clinical behavior that not adequately respond to standard treatments. We have demonstrated that NBL cell lines expressing the same kinome signature of ultra-HR tumors (ultra-HR-like cell lines) may be selectively targeted by the use of two drugs [suberoylanilide hydroxamic acid (SAHA) and Radicicol], and that the synergic combination of these drugs is able to block the ultra-HR-like cells in G2/M phase of cell cycle. The use of our signature in clinical practice will allow identifying patients with negative outcome, which would benefit from new and more personalized treatments. Preclinical in vivo studies are needed to consolidate the SAHA and Radicicol treatment in ultra-HR NBL patients. [ABSTRACT FROM AUTHOR]

ABSTRACT Plasmodium falciparum topoisomerase VIB (TopoVIB)-TopoVIA (TopoVIB-VIA) complex can be potentially exploited as a drug target against malaria due to its absence from the human genome. Previous work in our laboratory has suggested that P. falciparum TopoVIB (PfTopoVIB) might be a target of radicicol since treatment of parasite cultures with this antibiotic is associated with upregulation of Plasmodium TopoVIB at the transcript level as well as at the protein level. Further studies demonstrated that radicicol treatment impaired mitochondrial replication of human malaria parasite P. falciparum. However, the technical challenge associated with the expression of the above protein complex hampered its functional characterization. Using Saccharomyces cerevisiae as a heterologous system, we expressed PfTopoVIB (Myc-tagged) and PfTopoVIA (Flag-tagged) (PfTopoVIB-VIA) proteins. Yeast two-hybrid analysis showed the formation of PfTopoVIB homodimers and PfTopoVIB/PfTopoVIA heteromers. Our study demonstrated that PfTopoVIB and PfTopoVIA together can rescue the lethal phenotype of yeast ΔtopoII mutants, whereas Plasmodium topoisomerase VIB alone cannot. Using yeast cell-free extracts harboring the PfTopoVIB-VIA protein complex, we have performed a decatenation assay and observed that PfTopoVIB-VIA can decatenate DNA in an ATP- and Mg2+-dependent manner. The specificity of this enzyme is established by abrogation of its activity in the presence of PfTopoVIB-specific antibody. Our study results show that radicicol and etoposide can specifically inhibit PfTopoVIB-VIA decatenation activity whereas the gyrase inhibitor novobiocin cannot. Such a yeast-based assay system can be employed in screening specific inhibitors against Plasmodium VIB-VIA. IMPORTANCE In this study we characterize topoisomerase VI from Plasmodium falciparum using genetic and biochemical approaches. We use various inhibitors and identify radicicol as a specific inhibitor of its decatenation activity. We establish a very simple and economical biochemical assay system that can be exploited to screen inhibitors of PfTopoVI.

The 90 kDa heat shock proteins (Hsp90) are responsible for the conformational maturation of nascent polypeptides and the rematuration of denatured proteins. Proteins dependent upon Hsp90 are associated with all six hallmarks of cancer. Upon Hsp90 inhibition, protein substrates are degraded via the ubiquitin-proteasome pathway. Consequentially, inhibition of Hsp90 offers a therapeutic opportunity for the treatment of cancer. Natural product inhibitors of Hsp90 have been identified in vitro, which have served as leads for the development of more efficacious inhibitors and analogs that have entered clinical trials. This review highlights the development of natural product analogs, as well as the development of clinically important inhibitors that arose from natural products. [ABSTRACT FROM AUTHOR]

Gap junctions (GJs) channels provide the basis for intercellular communication in the cardiovascular system for maintenance of the normal cardiac rhythm, regulation of vascular tone and endothelial function as well as metabolic interchange between the cells. They allow the transfer of small molecules and may enable slow calcium wave spreading, transfer of “death” or of “survival” signals. In the cardiomyocytes the most abundant isoform is Connexin 43 (Cx43). Alterations in Cx43 expression and distribution were observed in myocardium disease; i.e. in hypertrophic cardiomyopathy, heart failure and ischemia. Recent reports suggest the presence of Cx43 in the mitochondria as well, at least in the inner mitochondrial membrane, where it plays a central role in ischemic preconditioning. In this review, the current knowledge on the relationship between the remodeling of cardiac gap junctions and cardiac diseases are summarized. [ABSTRACT FROM AUTHOR]

Despite recent advances in precision medicine, many molecular-based antineoplastic agents do not potentiate sustainable long term remissions, warranting the investigation of novel therapeutic strategies. Heat shock protein 90 (Hsp90) is a molecular chaperone that not only has oncogenic properties, but also has distinct expression profiles in malignant and normal cells, providing a rational strategy to attain preferential damage. Prior attempts to target Hsp90 with natural product-based compounds have been hampered by their associated off target toxicities, suggesting that novel, fully synthetic inhibitors may be required to achieve the specificity necessary for therapeutic efficacy. Therefore, this review highlights the antineoplastic potential of PU-H71 (8-[(6-iodo-1,3-benzodioxol-5-yl)sulfanyl]-9-[3-(propan-2-ylamino)propyl]purin-6-amine), a novel purine based analog that has shown efficacy in many preclinical models of malignancy, and is now under clinical examination. In addition, the review suggests potential concomitant therapeutic approaches that may be particularly beneficial to molecular-based, as well as traditional cytotoxic cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Genome-wide transcript profiling to elucidate responses to HSP90 inhibition revealed strong induction of HSPA6 in MCF-7 cells treated with 17-AAG. Time- and dose dependent induction of HSPA6 (confirmed by qPCR and Western Blots) occurred also upon treatment with Radicicol, another HSP90 inhibitor. HSPA6 was not detectable in untreated cells or cells treated with toxins that do not inhibit HSP90, or upon applying oxidative stress. Thus, HSPA6 induction is not a general response to cytotoxic insults. Modulation of HSPA6 levels by siRNA-mediated inhibition or recombinant expression did not influence 17-AAG mediated cell death. HSPA6 induction as a consequence of HSP90 inhibition occurs in various (but not all) cell lines and may be a more specific marker for HSP90 inhibition than induction of other HSP70 proteins. [ABSTRACT FROM AUTHOR]

Hsp90 is an ATP-dependent chaperone of widespread interest as a drug target. Here, using an LC-MS/MS chemoproteomics platform based on a lysine-reactive ATP acyl phosphate probe, several Hsp90 inhibitors were profiled in native cell lysates. Inhibitor specificities for all four human paralogs of Hsp90 were simultaneously monitored at their endogenous relative abundances. Equipotent inhibition of probe labeling in each paralog occurred at sites both proximal to and distal from bound ATP observed in Hsp90 cocrystal structures, suggesting that the ATP probe is assaying a native conformation not predicted by available structures. Inhibitor profiling against a comprehensive panel of protein kinases and other ATP-binding proteins detected in native cell lysates identified PMS2, a member of the GHKL ATPase superfamily as an off-target of NVP-AUY922 and radicicol. Because of the endogenously high levels of Hsp90 paralogs in typical cell lysates, the measured potency of inhibitors was weaker than published IC50 values. Significant inhibition of Hsp90 required inhibitor concentrations above a threshold where off-target activity was detectable. Direct on- and off-target engagement was measured by profiling lysates derived from cells treated with Hsp90 inhibitors. These studies also assessed the downstream cellular pathway effects of Hsp90 inhibition, including the down regulation of several known Hsp90 client proteins and some previously unknown client proteins. Overall, the ATP probe-based assay methodology enabled a broad characterization of Hsp90 inhibitor activity and specificity in native cell lysates. [ABSTRACT FROM AUTHOR]

Proteostasis regulation using naturally occurring small molecules has been considered as a promising strategy for manipulating cancer sensitivity and therapy. Here, we identify a small molecule Hsp90 inhibitor radicicol that induces intracellular accumulation of cytotoxic clusterin variant. In the mechanistic basis, this variant proved to be a product disposed from the stressed ER. During this process, inhibitory effect of radicicol on protein degradation results in cytosolic accumulation of glycan-deficient clusterin variant that signals cell death. These results provide a therapeutic insight into the targeted proteostasis perturbation of clusterin as an anti-cancer strategy. [ABSTRACT FROM AUTHOR]

Studies towards the total synthesis of the natural product radicicol are described that employ a late-stage esterification and aromatization by trapping a ketene intermediate. The subsequent biomimetic aromatization of the resultant triketo ester gave highly functionalized resorcylates. Two distinct methods were examined that trap the ketene intermediate through either an intermolecular or intramolecular process. In the first approach, the synthesis of the resorcylate was followed by a ring-closing metathesis, which gave the macrolactone and protected precursors to monocillin I. In the second approach, an intramolecular ketene trapping was examined as an alternative to close the macrocycle and form the resorcylate macrolactone. These studies showcased a wide range of sensitive functional groups that tolerated the aromatization reaction conditions, which started from the corresponding dioxinone precursors. [ABSTRACT FROM AUTHOR]