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Streptomyces peucetius ATCC 27952 is known to produce a variety of secondary metabolites, including two important antitumor anthracyclines: daunorubicin and doxorubicin. Identification of peucemycin and 25-hydroxy peucemycin (peucemycin A), as well as their biosynthetic pathway, has expanded its biosynthetic potential. In this study, we isolated a new peucemycin derivative and identified it as 19-hydroxy peucemycin (peucemycin B). Its antibacterial activity was lower than those of peucemycin and peucemycin A. On the other hand, this newly identified peucemycin derivative had higher anticancer activity than the other two compounds for MKN45, NCI-H1650, and MDA-MB-231 cancer cell lines with IC 50 values of 76.97 µM, 99.68 µM, and 135.2 µM, respectively. Peucemycin biosynthetic gene cluster revealed the presence of a SARP regulator named PeuR whose role was unknown. The presence of the TTA codon in the peuR and the absence of global regulator BldA in S. peucetius reduced its ability to regulate the peucemycin biosynthetic gene cluster. Hence, different mutants harboring these genes were prepared. S. peucetius bldA25 harboring bldA produced 1.75 times and 1.77 times more peucemycin A (11.8 mg/L) and peucemycin B (21.2 mg/L), respectively, than the wild type. On the other hand, S. peucetius R25 harboring peuR produced 1.86 and 1.79 times more peucemycin A (12.5 mg/L) and peucemycin B (21.5 mg/L), respectively, than the wild type. Finally, strain S. peucetius bldAR25 carrying bldA and peuR produced roughly 3.52 and 2.63 times more peucemycin A (23.8 mg/L) and peucemycin B (31.5 mg/L), respectively, than the wild type. KEY POINTS: • This study identifies a new peucemycin derivative, 19-hydroxy peucemycin (peucemycin B). • The SARP regulator (PeuR) acts as a positive regulator of the peucemycin biosynthetic gene cluster. • The overexpression of peuR and heterologous expression of bldA increase the production of peucemycin derivatives., (© 2024. The Author(s).)

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152Δ matAB that could produce aklavinone, 9- epi -aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152Δ matAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Chromatinized DNA is targeted by proteins and small molecules to regulate chromatin function. For example, anthracycline cancer drugs evict nucleosomes in a mechanism that is still poorly understood. We here developed a flexible method for specific isotope labeling of nucleosomal DNA enabling NMR studies of such nucleosome interactions. We describe the synthesis of segmental one-strand 13 C-thymidine labeled 601-DNA, the assignment of the methyl signals, and demonstrate its use to observe site-specific binding to the nucleosome by aclarubicin, an anthracycline cancer drug that intercalates into the DNA minor grooves. Our results highlight intrinsic conformational heterogeneity in the 601 DNA sequence and show that aclarubicin binds an exposed AT-rich region near the DNA end. Overall, our data point to a model where the drug invades the nucleosome from the terminal ends inward, eventually resulting in histone eviction and nucleosome disruption., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Streptomyces are the primary source of bioactive specialized metabolites used in research and medicine, including many antimicrobials. These are presumed to be secreted and function as freely soluble compounds. However, increasing evidence suggests that extracellular vesicles are an alternative secretion system. We assessed environmental and lab-adapted Streptomyces (sporulating filamentous actinomycetes) and found frequent production of antimicrobial vesicles. The molecular cargo included actinomycins, anthracyclines, candicidin, and actinorhodin, reflecting both diverse chemical properties and diverse antibacterial and antifungal activity. The levels of packaged antimicrobials correlated with the level of inhibitory activity of the vesicles, and a strain knocked out for the production of anthracyclines produced vesicles that lacked antimicrobial activity. We demonstrated that antimicrobial containing vesicles achieve direct delivery of the cargo to other microbes. Notably, this delivery via membrane fusion occurred to a broad range of microbes, including pathogenic bacteria and yeast. Vesicle encapsulation offers a broad and permissive packaging and delivery system for antimicrobial specialized metabolites, with important implications for ecology and translation.IMPORTANCEExtracellular vesicle encapsulation changes our picture of how antimicrobial metabolites function in the environment and provides an alternative translational approach for the delivery of antimicrobials. We find many Streptomyces strains are capable of releasing antimicrobial vesicles, and at least four distinct classes of compounds can be packaged, suggesting this is widespread in nature. This is a striking departure from the primary paradigm of the secretion and action of specialized metabolites as soluble compounds. Importantly, the vesicles deliver antimicrobial metabolites directly to other microbes via membrane fusion, including pathogenic bacteria and yeast. This suggests future applications in which lipid-encapsulated natural product antibiotics and antifungals could be used to solve some of the most pressing problems in drug resistance., Competing Interests: The authors declare no conflict of interest.

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However, they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX), Daunorubicin (DNR), Epirubicin (EPI), and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class, or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this, we studied the effects of treatment of DOX, DNR, EPI, MTX, and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum, while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling, a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time, giving rise to four distinct gene expression response signatures, denoted as TOP2i early-acute, early-sustained, and late response genes, and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators, which mediate AC sensitivity across breast cancer patients. However, there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability, we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed, DOX response eQTLs are enriched in genes that respond to all TOP2i. Next, we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i, respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes, including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Matthews et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tumor metastasis is an intricate multistep process regulated via various proteins and enzymes modified and secreted by swollen Golgi apparatus in tumor cells. Thus, Golgi complex is considered as an important target for the remedy of metastasis. Currently, Golgi targeting technologies are mostly employed in Golgi-specific fluorescent probes for diagnosis, but their applications in therapy are rarely reported. Herein, we proposed a prodrug (INR) that can target and destroy the Golgi apparatus, which consisted of indomethacin (IMC) as the Golgi targeting moiety and retinoic acid (RA), a Golgi disrupting agent. The linker between IMC and RA was designed as a hypoxia-responsive nitroaromatic structure, which ensured the release of the prototype drugs in the hypoxic tumor microenvironment. Furthermore, INR could be assembled with pirarubicin (THP), an anthracycline, to form a carrier-free nanoparticle (NP) by emulsion-solvent evaporation method. A small amount of mPEG 2000 -DSPE was added to shield the positive charges and improve the stability of the nanoparticle to obtain PEG-modified nanoparticle (PNP). It was proved that INR released the prototype drugs in tumor cells and hypoxia promoted the release. The Golgi destructive effect of RA in INR was amplified owing to the Golgi targeting ability of IMC, and IMC also inhibited the protumor COX-2/PGE 2 signaling. Finally, PNP exhibited excellent curative efficacy on 4T1 primary tumor and its pulmonary and hepatic metastasis. The small molecular therapeutic prodrug targeting Golgi apparatus could be adapted to multifarious drug delivery systems and disease models, which expanded the application of Golgi targeting tactics in disease treatment.

Anthracycline antitumor agents, such as doxorubicin (DOX), are effective in the treatment of solid tumors and hematological malignancies, but anthracycline-induced cardiotoxicity (AIC) limits their application as chemotherapeutics. Dexrazoxane (DEX) has been adopted to prevent AIC. Using a chronic AIC mouse model, we demonstrated that DEX is insufficient to reverse DOX-induced cardiotoxicity. Although therapies targeting autophagy have been explored to prevent AIC, but whether novel autophagy inhibitors could alleviate or prevent AIC in clinically relevant models needs further investigation. Here, we show that genetic ablation of Atg7, a key regulator in the early phase of autophagy, protected mice against AIC. We further demonstrated that SAR405, a novel autophagy inhibitor, attenuated DOX-induced cytotoxicity. Intriguingly, the combination of DEX and SAR405 protected cells against DOX-induced cardiotoxicity in vivo. Using the cardiomyocyte cell lines AC16 and H9c2, we determined that autophagy was initiated during AIC. Our results suggest that inhibition of autophagy at its early phase with SAR405 combined with DEX represents an effective therapeutic strategy to prevent AIC., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Anthracycline antineoplastics are effective in the treatment of hematological malignancies and solid tumors. However, the anthracycline-induced cardiotoxicity (AIC) limits their use as chemotherapeutic agents. Autophagy-based therapies have been explored to prevent AIC. Yet, whether inhibition of autophagy during its early stage could alleviate AIC remains unclear. In this study, we firstly observed the activation of autophagy during AIC in both cardiomyocyte cell lines AC16 and H9c2. Moreover, knockdown of Atg7, a key regulatory factor in early autophagy, could ameliorate the effects of DOX-induced AIC. Importantly, the use of early autophagy inhibitor 3-MA protected cardiomyocyte cells from DOX-induced cardiotoxicity in vitro and in a chronic AIC mouse model. Our findings demonstrate that inhibiting early stage of autophagy may be an effective preventative therapeutic strategy to protect cardiac function from AIC., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Anthracycline antibiotics (ANT) are among the most widely used anticancer drugs. Unfortunately, their use is limited due to the development of drug resistance and cardiotoxicity. ANT metabolism, performed mainly by two enzymes-aldo-keto reductase 1C3 (AKR1C3) and carbonyl reductase 1 (CBR1)-is one of the proposed mechanisms generated by the described effects. In this study, we evaluated the CBR1 inhibitory properties of ASP9521, a compound already known as potent AKR1C3 inhibitor. First, we assessed the possibility of ASP9521 binding to the CBR1 catalytic site using molecular docking and molecular dynamics. The research revealed a potential binding mode of ASP9521. Moderate inhibitory activity against CBR1 was observed in studies with recombinant enzymes. Finally, we examined whether ASP9521 can improve the cytotoxic activity of daunorubicin against human lung carcinoma cell line A549 and assessed the cardioprotective properties of ASP9521 in a rat cardiomyocytes model (H9c2) against doxorubicin- and daunorubicin-induced toxicity. The addition of ASP9521 ameliorated the cytotoxic activity of daunorubicin and protected rat cardiomyocytes from the cytotoxic effect of both applied drugs. Considering the favorable bioavailability and safety profile of ASP9521, the obtained results encourage further research. Inhibition of both AKR1C3 and CBR1 may be a promising method of overcoming ANT resistance and cardiotoxicity.

Many small molecule natural products are decorated with sugar moieties that are essential for their biological activity. A considerable number of natural product glycosides and their derivatives are clinically important therapeutics. Anthracyclines like daunorubicin and doxorubicin are examples of valuable glycosylated natural products used in medicine as potent anticancer agents. The sugar moiety, l-daunosamine (a highly modified deoxyhexose), plays a key role in the bioactivity of these molecules as evidenced by semisynthetic anthracycline derivatives such as epirubicin, wherein alteration in the configuration of a single stereocenter of the sugar unit generates a chemotherapeutic drug with lower cardiotoxicity. The nucleotide activated sugar donor that provides the l-daunosamine group for attachment to the natural product scaffold in the biosynthesis of these anthracyclines is dTDP-l-daunosamine. In an in vitro system, we have reconstituted the enzymes in the daunorubicin/doxorubicin pathway involved in the biosynthesis of dTDP-l-daunosamine. Through the study of the enzymatic steps in this reconstituted pathway, we have gained several insights into the assembly of this precursor including the identification of a major bottleneck and competing reactions. We carried out kinetic analysis of the aminotransferase that catalyzes a limiting step of the pathway. Our in vitro reconstituted pathway also provided a platform to test the combinatorial enzymatic synthesis of other dTDP-activated deoxyhexoses as potential tools for "glycodiversification" of natural products. To this end, we replaced the stereospecific ketoreductase that acts in the last step of dTDP-l-daunosamine biosynthesis with an enzyme from a heterologous pathway with opposite stereospecificity and found that it is active in the in vitro pathway, demonstrating the potential for the enzymatic synthesis of nucleotide-activated sugars with regio- and stereospecific tailoring.

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-β-1,6- N -acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9- epi -aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10- seco -7-deoxy-nogalamycinone and 4- O -β-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.

Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy., (© 2022. The Author(s).)

Antibiotic-producing microorganisms usually require one or more self-resistance determinants to survive antibiotic production. The effectors of these mechanisms are proteins that inactivate the antibiotic, facilitate its transport, or modify the target to render it insensitive to the molecule. Streptomyces bacteria biosynthesize various bioactive natural products and possess resistance systems for most metabolites, which are coregulated with antibiotic biosynthesis genes. Streptomyces olindensis strain DAUFPE 5622 produces the antitumor antibiotic cosmomycin D (COSD), a member of the anthracycline family. In this study, we propose three self-resistance mechanisms, anchored or based in the COSD biosynthetic gene cluster. These include cosIJ (an ABC transporter), cosU (a UvrA class IIa protein), and a new self-resistance mechanism encoded by cosP , which shows response against peroxides by the enzyme mycothiol peroxidase (MPx). Activity-based investigations of MPx and its mutant enzyme confirmed peroxidation during the production of COSD. Overexpression of the ABC transporter, the UvrA class IIa protein, and the MPx led to an effective response against toxic anthracyclines, such as cosmomycins. Our findings help to understand how thiol peroxidases play an antioxidant role in the anthracycline producer S. olindensis DAUFPE 5622, a mechanism which has been reported for neoplastic cells that are resistant to doxorubicin (DOX). IMPORTANCE Anthracycline compounds are DNA intercalating agents widely used in cancer chemotherapeutic protocols. This work focused on the self-resistance mechanisms developed by the cosmomycin-producing bacterium Streptomyces olindensis. Our findings showed that cysteine peroxidases, such as mycothiol peroxidase, encoded by the gene cosP , protected S. olindensis against peroxidation during cosmomycin production. This observation can contribute to much better understanding of resistance both in the producers, eventually enhancing production, and in some tumoral cell lines.

Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O 2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular (NPM1c MRD, p = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model., (© 2022. The Author(s).)

Covering: January 1995 to June 2021Anthracyclines are glycosylated microbial natural products that harbour potent antiproliferative activities. Doxorubicin has been widely used as an anticancer agent in the clinic for several decades, but its use is restricted due to severe side-effects such as cardiotoxicity. Recent studies into the mode-of-action of anthracyclines have revealed that effective cardiotoxicity-free anthracyclines can be generated by focusing on histone eviction activity, instead of canonical topoisomerase II poisoning leading to double strand breaks in DNA. These developments have coincided with an increased understanding of the biosynthesis of anthracyclines, which has allowed generation of novel compound libraries by metabolic engineering and combinatorial biosynthesis. Coupled to the continued discovery of new congeners from rare Actinobacteria, a better understanding of the biology of Streptomyces and improved production methodologies, the stage is set for the development of novel anthracyclines that can finally surpass doxorubicin at the forefront of cancer chemotherapy.

Sdox is a synthetic H 2 S-releasing doxorubicin (Dox) less cardiotoxic and more effective than Dox in pre-clinical, Dox-resistant tumour models. The well-known anthracycline vascular toxicity, however, might limit Sdox clinical use. This study aimed at evaluating Sdox vascular toxicity in vitro, using Dox as reference compound. Both vascular smooth muscle A7r5 and endothelial EA.hy926 cells were more sensitive to Dox than Sdox, although both drugs equally increased intracellular free radical levels. Sdox released H 2 S in both cell lines. The H 2 S scavenger hydroxocobalamin partially reverted Sdox-induced cytotoxicity in A7r5, but not in EA.hy926 cells, suggesting a role for H 2 S in smooth muscle cell death. Markers of Sdox-induced apoptosis were significantly lower than, in A7r5 cells, and comparable to those of Dox in EA.hy926 cells. In A7r5 cells, Dox increased the activity of caspase 3, 8, and 9, Sdox affecting only that of caspase 3. Moreover, both drugs induced comparable DNA damage in A7r5 cells, while Sdox was less toxic than Dox in Ea.hy926 cells. In fresh aorta rings, only Dox weakly increased phenylephrine-induced contraction when endothelium was present. In rings cultured with both drugs for 7 days, Sdox blunted phenylephrine- and high K + -induced contractions though at a concentration 10-fold higher than that of Dox. In conclusion, Sdox may represent the prototype of an innovative anthracycline, effective against Dox-resistant tumours, displaying a more favourable vascular toxicity profile compared to the parent compound., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Anthracycline chemotherapeutics are highly effective, but their clinical usefulness is hampered by adverse side effects such as cardiotoxicity. Cytochrome P450 2J2 (CYP2J2) is a cytochrome P450 epoxygenase in human cardiomyocytes that converts arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acid (EET) regioisomers. Herein, we performed biochemical studies to understand the interaction of anthracycline derivatives (daunorubicin, doxorubicin, epirubicin, idarubicin, 5-iminodaunorubicin, zorubicin, valrubicin, and aclarubicin) with CYP2J2. We utilized fluorescence polarization (FP) to assess whether anthracyclines bind to CYP2J2. We found that aclarubicin bound the strongest to CYP2J2 despite it having large bulky groups. We determined that ebastine competitively inhibits anthracycline binding, suggesting that ebastine and anthracyclines may share the same binding site. Molecular dynamics and ensemble docking revealed electrostatic interactions between the anthracyclines and CYP2J2, contributing to binding stability. In particular, the glycosamine groups in anthracyclines are stabilized by binding to glutamate and aspartate residues in CYP2J2 forming salt bridge interactions. Furthermore, we used iterative ensemble docking schemes to gauge anthracycline influence on EET regioisomer production and anthracycline inhibition on AA metabolism. This was followed by experimental validation of CYP2J2-mediated metabolism of anthracycline derivatives using liquid chromatography tandem mass spectrometry fragmentation analysis and inhibition of CYP2J2-mediated AA metabolism by these derivatives. Taken together, we use both experimental and theoretical methodologies to unveil the interactions of anthracycline derivatives with CYP2J2. These studies will help identify alternative mechanisms of how anthracycline cardiotoxicity may be mediated through the inhibition of cardiac P450, which will aid in the design of new anthracycline derivatives with lower toxicity., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the bioremoval of anthracycline antibiotics (daunomycin-DNR, doxorubicin-DOX, and mitoxantrone-MTX) by immobilized mycelium of B. adusta CCBAS 930. The activity of oxidoreductases: versatile peroxidases (VP), superoxide dismutase (SOD), catalase (CAT), and glucose oxidase (GOX), and the levels of phenolic compounds (PhC) and free radicals (SOR) were determined during the biotransformation of anthracyclines by B. adusta strain CCBAS 930. Moreover, the phytotoxicity ( Lepidium sativum L.), biotoxicity (MARA assay), and genotoxicity of anthracyclines were evaluated after biological treatment. After 120 h, more than 90% of anthracyclines were removed by the immobilized mycelium of B. adusta CCBAS 930. The effective biotransformation of anthracyclines was correlated with detoxification and reduced genotoxicity.

Cardiotoxicity is a frequent undesirable phenomenon observed during oncological treatment that limits the therapeutic dose of antitumor drugs and thus may decrease the effectiveness of cancer eradication. Almost all antitumor drugs exhibit toxic properties towards cardiac muscle. One of the underlying causes of cardiotoxicity is the stimulation of oxidative stress by chemotherapy. This suggests that an appropriately designed diet or dietary supplements based on edible plants rich in antioxidants could decrease the toxicity of antitumor drugs and diminish the risk of cardiac failure. This comprehensive review compares the cardioprotective efficacy of edible plant extracts and foodborne phytochemicals whose beneficial activity was demonstrated in various models in vivo and in vitro. The studies selected for this review concentrated on a therapy frequently applied in cancer, anthracycline antibiotic-doxorubicin-as the oxidative stress- and cardiotoxicity-inducing agent.

Background: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines., Methods: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco 2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis., Results: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as β- and γ-rhodomycinone with CD 50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco 2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin., Conclusions: This is the first report about the production of β- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus .

Autophagy plays an important role in maintaining tumor cell progression and survival in response to metabolic stress. Thus, the regulation of autophagy can be used as a strategy for anticancer therapy. Here, we report dutomycin (DTM) as a novel autophagy enhancer that eventually induces apoptosis due to excessive autophagy. Also, human serine protease inhibitor B6 (SERPINB6) was identified as a target protein of DTM, and its novel function which is involved in autophagy was studied for the first time. We show that DTM directly binds SERPINB6 and then activates intracellular serine proteases, resulting in autophagy induction. Inhibitory effects of DTM on the function of SERPINB6 were confirmed through enzyme- and cell-based approaches, and SERPINB6 was validated as a target protein using siRNA-mediated knockdown and an overexpression test. In a zebrafish xenograft model, DTM showed a significant decrease in tumor area. Furthermore, the present findings will be expected to contribute to the expansion of novel basic knowledge about the correlation of cancer and autophagy by promoting active further research on SERPINB6, which was not previously considered the subject of cancer biology.

Although many advances have been achieved to treat aggressive tumours, cancer remains a leading cause of death and a public health problem worldwide. Among the main approaches for the discovery of new bioactive agents, the prospect of microbial secondary metabolites represents an effective source for the development of drug leads. In this study, we investigated the actinobacterial diversity associated with an endemic Antarctic species, Deschampsia antarctica, by integrated culture-dependent and culture-independent methods and acknowledged this niche as a reservoir of bioactive strains for the production of antitumour compounds. The 16S rRNA-based analysis showed the predominance of the Actinomycetales order, a well-known group of bioactive metabolite producers belonging to the Actinobacteria phylum. Cultivation techniques were applied, and 72 psychrotolerant Actinobacteria strains belonging to the genera Actinoplanes, Arthrobacter, Kribbella, Mycobacterium, Nocardia, Pilimelia, Pseudarthrobacter, Rhodococcus, Streptacidiphilus, Streptomyces and Tsukamurella were identified. The secondary metabolites were screened, and 17 isolates were identified as promising antitumour compound producers. However, the bio-guided assay showed a pronounced antiproliferative activity for the crude extracts of Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653. The TGI and LC 50 values revealed the potential of these natural products to control the proliferation of breast (MCF-7), glioblastoma (U251), lung/non-small (NCI-H460) and kidney (786-0) human cancer cell lines. Cinerubin B and actinomycin V were the predominant compounds identified in Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653, respectively. Our results suggest that the rhizosphere of D. antarctica represents a prominent reservoir of bioactive actinobacteria strains and reveals it as an important environment for potential antitumour agents.

Anthracyclines Doxorubicin, Epirubicin, Daunorubicin and Idarubicin are used to treat a variety of tumor types in the clinics, either alone or, most often, in combination therapies. While their cardiotoxicity is well known, the emergence of chemoresistance is also a major issue accounting for treatment discontinuation. Resistance to anthracyclines is associated to the acquisition of multidrug resistance conferred by overexpression of permeability glycoprotein-1 or other efflux pumps, by altered DNA repair, changes in topoisomerase II activity, cancer stemness and metabolic adaptations. This review further details the metabolic aspects of resistance to anthracyclines, emphasizing the contributions of glycolysis, the pentose phosphate pathway and nucleotide biosynthesis, glutathione, lipid metabolism and autophagy to the chemoresistant phenotype., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Here the author describes the tumor-selective delivery of a fluorescence photosensitizing agent and an antitumor agent, based on the polymer effect of an N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymer, by utilizing the enhanced permeability and retention (EPR) effect seen in solid tumors. Firstly, the tumor distribution of the photosensitizer, zinc-protoporphyrin IX (ZnPP), was significantly increased by conjugation with the HPMA polymer (P-ZnPP). The P-ZnPP suppressed tumor growth by local generation of cytotoxic singlet oxygen, and the tumor tissue was visualized by fluorescence upon light irradiation. Subsequently, a two-step mechanism for tumor selectivity was observed for the cytotoxic anthracycline, pirarubicin (THP), which conjugated the HPMA-based copolymer via a hydrazone bond (P-THP). The EPR-dependent accumulation of P-THP and the tumor-selective release of THP in the tumor tissues led to highly tumor-selective toxicity. Rapid cell uptake of THP compared to other anthracyclines, and deeper P-THP penetration of the tumor cell spheroid were attributed to the superior antitumor activity of P-THP. The molecular weight of P-THP affected its antitumor activity; oligomeric P-THP derivatives with higher molecular weights, DP-THP and SP-THP, showed even higher antitumor activity. P-THP was effective for both implanted tumor and autochthonous tumor models. These results indicate that nano-sized anticancer drugs based on polymer effect are promising clinical therapeutics.

Antibiotic-producing microorganism can develop strategies to deal with self-toxicity. Cytorhodins X and Y, cosmomycins A and B, and iremycin, are produced as final products from a marine-derived Streptomyces sp. SCSIO 1666. These C-7 reduced metabolites show reduced antimicrobial and comparable cytotoxic activities relative to their C-7 glycosylated counterparts. However, the biosynthetic mechanisms and relevant enzymes that drive C-7 reduction in cytorhodin biosynthesis have not yet been characterized. Here we report the discovery and characterization of a reductase, CytA, that mediates C-7 reduction of this anthracycline scaffold; CytA endows the producer Streptomyces sp. SCSIO 1666 with a means of protecting itself from the effects of its anthracycline products. Additionally, we identified cosmomycins C and D as two intermediates involved in cytorhodin biosynthesis and we also broadened the substrate specificity of CytA to clinically used anthracycline drugs., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

The interactions of (-)-epigallocatechin-3-Gallate (EGCG) and anthracycline drugs (doxorubicin, DOX and epirubicin, EPI) alone or in combination with human serum albumin (HSA) under physiological condition were studied by fluorescence spectroscopy, UV-vis absorption spectroscopy, circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). The cytotoxic activity of the single drug, combined drugs, and their complexes with HSA against human cervical cancer HeLa cell line was determined by MTT assay. Fluorescence quenching result and difference spectra of UV absorption revealed the formation of static complex between EGCG, DOX, or EPI and HSA. The binding of EGCG with HSA was driven by both enthalpy and entropy while the binding of DOX or EPI was mainly entropy driven. The nature of binding was expounded based on the effect of sodium chloride, tetrabutylammonium bromide, and sucrose which interfere in electrostatic, hydrophobic, and hydrogen bonding interactions, respectively. Site marker competitive experiments combined with synchronous fluorescence spectra showed that these three ligands mainly bound to subdomain IIA of HSA and were closer to tryptophan residues. In EGCG + DOX/EPI + HSA ternary system, the effect of one drug on the binding ability of another drug was discussed. The influences of the individual and combined binding of EGCG and DOX/EPI on the secondary structure and particle size of HSA were investigated by CD spectroscopy and DLS, respectively. Moreover, the synergistic cytotoxicity of EGCG and DOX/EPI as well as their complexes with HSA were discussed. Obtained results would provide beneficial information on the combination of EGCG and anthracyclines in clinic., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Late cardiotoxicity following treatment of malignancy diseases has been long established. Cancer therapeutics-related cardiac dysfunction (CTRCD), acute arrhythmias, pericardial disease, valvopathies and early atherosclerotic Cardiovascular Disease (CVD), are the clinical presentations of cardiotoxicity. Although these clinical modalities can affect adults treated for malignancies, they are more common to present in the pediatric survivors as improvement of prognosis, nowadays exists. Studies have shown that CVD can present earlier than thirty years, post treatment. If adding on this the early and late effect of cardiotoxicity on the developing in childhood cardiovascular system, we are then faced with a new Risk Factor (RF) for CVD. Anthracyclines and its derivatives have served for over fifty years as the road model of studding early, mid and late term cardiotoxicity. Today a vast number of chemical agents are used, many of them with very good results in treating the existing malignancies. Unfortunate, little or even less are known on their potential mechanism of derived cardiotoxicity when used by their own or combined with others and/or radiotherapy (RT). The 2013 existing guidelines by ACC/AHA on surveillance of the cardiovascular health of oncology survivors, are mostly addressing early cardiac adverse effects and CTRCD. Little is mentioned about the development of early CVD, its subclinical diagnosis, prevention and the need of early intervention before clinical events are present. The aim of this paper is to review the exist knowledge and practice on this condition with growing numbers of survivors facing the risk of early atherosclerotic CVD.

Naturally occurring pradimicins (PRMs) show specific recognition of d-mannose (d-Man) in aqueous media, which has never been achieved by artificial small molecules. Although the Ca 2+ -mediated dimerization of PRMs is essential for their d-Man binding, the dimeric structure has yet to be elucidated, leaving the question open as to how PRMs recognize d-Man. Thus, we herein report the structural elucidation of the dimer by a combination of X-ray crystallography and solid-state NMR spectroscopy. Coupled with our previous knowledge regarding the d-Man binding geometry of PRMs, elucidation of the dimer allowed reliable estimation of the mode of d-Man binding. Based on the binding model, we further developed an azide-functionalized PRM derivative (PRM-Azide) with d-Man binding specificity. Notably, PRM-Azide stained Candida rugosa cells having mannans on their cell surface through conjugation with the tetramethylrhodamine fluorophore. The present study provides the practical demonstration that PRMs can serve as lectin mimics for use in glycobiological studies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Microbial natural products are an important source of chemical entities for drug discovery. Recent advances in understanding the biosynthesis of secondary metabolites has revealed how this rich chemical diversity is generated through functional differentiation of biosynthetic enzymes. For instance, investigations into anthracycline anticancer agents have uncovered distinct S-adenosyl methionine (SAM)-dependent proteins: DnrK is a 4-O-methyltransferase involved in daunorubicin biosynthesis, whereas RdmB (52% sequence identity) from the rhodomycin pathway catalyzes 10-hydroxylation. Here, we have mined unknown anthracycline gene clusters and discovered a third protein subclass catalyzing 10-decarboxylation. Subsequent isolation of komodoquinone B from two Streptomyces strains verified the biological relevance of the decarboxylation activity. Phylogenetic analysis inferred two independent routes for the conversion of methyltransferases into hydroxylases, with a two-step process involving loss-of-methylation and gain-of-hydroxylation presented here. Finally, we show that simultaneously with the functional differentiation, the evolutionary process has led to alterations in substrate specificities.

Introduction: Acute myeloid leukaemia is the most common type of acute leukaemia in the world. Thus, the study of genetic alterations, such as single-nucleotide polymorphisms (SNPs), has contributed to a better understanding of the mechanisms underlying leukaemogenesis, to improve the prognosis and to increase the survival of these patients. However, there is no synthesis of evidence in the literature evaluating the quality of evidence and the risk of bias in the studies such that the results can be translated. Thus, this systematic review protocol aims to assess the impact of SNPs on genes involved in the metabolism of cytarabine and anthracyclines with respect to survival, treatment response and toxicity in patients with AML., Methods: This systematic review protocol is based on PRISMA guidelines and includes searches in six electronic databases, contact with authors, repositories of clinical trials, and cancer research. Studies published in peer-reviewed journals will be included if they meet the eligibility criteria: (a) samples composed of individuals of any age, of both sexes, with a diagnosis of AML, regardless of the time of diagnosis of disease; (b) participants who have undergone or are undergoing cytarabine- and anthracycline-associated chemotherapy or cytarabine-only chemotherapy; and (c) in vivo studies. Studies that include patients with promyelocytic leukaemia (Fab type 3) will be excluded because this disease has different treatment. The process of study selection, data extraction, and evaluation/synthesis will be performed in duplicate. Assessment of methodological quality and risk of bias will be performed using the Cochrane Risk of Bias Tool for randomized clinical studies and the Downs-Black Checklist for cohort and case-control studies. The synthesis of evidence will include the level of evidence based on the GRADE protocol. A meta-analysis of the association between SNPs and outcomes may be performed based on Cochrane guidelines., Discussion: It is expected that clinical decisions for AML patients will consider evidence-based practices to contribute to better patient management. In this way, we will be able to define how to treat patients with AML to improve their survival and quality of life., Systematic Review Registration: PROSPERO CRD42018100750.

Anthracyclines interact with DNA and topoisomerase II as well as with cell membranes, and it is these latter interactions that can cause an increase in their cytotoxic activity. In the present study a detailed computational analysis of the initial insertion, orientation and nature of the interaction occurring between Anthracyclines and two different lipid bilayers (unsaturated POPC and saturated DMPC) is explored through molecular dynamics (MD) simulations; four Anthracyclines: Doxorubicin (DOX), Epirubicin (EPI), Idarubicin (IDA) and Daunorubicin (DAU) were examined. The results indicate that the increased cytotoxicity of DOX, in comparison to the other three analogues, is correlated with its ability to diffuse at a faster rate into the bilayers. Additionally, DOX exhibited considerably different orientational behaviour once incorporated into the bilayer and exhibited a higher propensity to interact with the hydrocarbon tails in both lipids indicating a higher probability of transport to the other leaflet of the bilayer.

We have reported that cancer patients treated with anthracycline-based or nonanthracycline chemotherapy developed an early impairment of myocardial relaxation at echocardiography or persistent elevations of the cardiac hormone B-type natriuretic peptide (BNP). Post-hoc pharmacologic analyses showed that BNP elevations were induced by impaired relaxation and caused positive lusitropic effects that maintained normal relaxation. High BNP levels and impaired relaxation were therefore characterized as mutually exclusive manifestations of diastolic dysfunction, but high BNP levels resulted in positive chronotropism and inappropriate tachycardia. Some patients developed increased circulating levels of cardiac troponin I isoform (cTnI), a marker of cardiomyocyte necrosis. Here we have characterized whether cTnI elevations correlated with diastolic dysfunction that manifested as impaired relaxation or a high level of BNP. The effects of high BNP levels on cTnI elevations were also characterized. We show that impaired relaxation or high BNP levels were significantly more frequent in patients with cTnI elevations. High BNP levels diminished the plasma peak and area under the curve of cTnI, but this result was accompanied by inappropriate tachycardia. cTnI elevations occurred only in patients treated with anthracyclines; moreover, the association of impaired relaxation or high BNP levels with cTnI elevations was significantly more frequent in doxorubicin-treated patients compared with patients treated with its analog, epirubicin. These findings describe cause-and-effect relations between impaired relaxation and cardiomyocyte necrosis, illuminate the role of anthracycline analogs, denote that the beneficial effects of BNP in relieving impaired relaxation and cardiomyocyte necrosis are counterbalanced by inappropriate tachycardia. Patients showing troponin elevations and impaired relaxation or high BNP levels should be treated with lusitropic drugs that lack a positive chronotropism., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The search for new compounds effective against Mycobacterium tuberculosis is still a priority in medicine. The evaluation of microorganisms isolated from non-conventional locations offers an alternative to look for new compounds with antimicrobial activity. Endophytes have been successfully explored as source of bioactive compounds. In the present work we studied the nature and antimycobacterial activity of a compound produced by Streptomyces scabrisporus, an endophyte isolated from the medicinal plant Amphipterygium adstringens. The active compound was detected as the main secondary metabolite present in organic extracts of the streptomycete and identified by NMR spectroscopic data as steffimycin B (StefB). This anthracycline displayed a good activity against M. tuberculosis H37Rv ATCC 27294 strain, with MIC 100 and SI values of 7.8 µg/mL and 6.42, respectively. When tested against the rifampin mono resistant M. tuberculosis Mtb-209 pathogen strain, a better activity was observed (MIC 100 of 3.9 µg/mL), suggesting a different action mechanism of StefB from that of rifampin. Our results supported the endophyte Streptomyces scabrisporus as a good source of StefB for tuberculosis treatment, as this anthracycline displayed a strong bactericidal effect against M. tuberculosis, one of the oldest and more dangerous human pathogens causing human mortality.

The C7 (C9 or C10)- O-l-rhodosamine-bearing anthracycline antibiotic cytorhodins and their biosynthetic intermediates were recently isolated from Streptomyces sp. SCSIO 1666. Cosmid p17C4 from the Streptomyces lydicus genomic library, which harbors both the biosynthetic genes for l-rhodinose (or 2-deoxy-l-fucose) and its glycosyltransferase (encoded by slgG), was introduced into SCSIO 1666 to yield the recombinant strain Streptomyces sp. SCSIO 1666/17C4. Chemical investigations of this strain's secondary metabolic potential revealed the production of different anthracyclines featuring C7- O-l-rhodinose (or 2-deoxy-l-fucose) instead of the typically observed l-rhodosamine. Purification of the fermentation broth yielded 12 new anthracycline antibiotics including three new ε-rhodomycinone derivatives, 1, 4, and 8, nine new β-rhodomycinone derivatives, 2, 3, 5-7, and 9-12, and three known compounds, l-rhodinose-l-rhodinose-l-rhodinoserhodomycinone (13), ε-rhodomycinone (14), and γ-rhodomycinone (15). All compounds were characterized on the basis of detailed spectroscopic analyses and comparisons with previously reported data. These compounds exhibited cytotoxicity against a panel of human cancer cell lines. Significantly, compounds 4 and 13 displayed pronounced activity against HCT-116 as characterized by IC 50 values of 0.3 and 0.2 μM, respectively; these IC 50 values are comparable to that of the positive control epirubicin.

Aromatic polyketides are important therapeutic compounds which include front line antibiotics and anticancer drugs. Since most of the aromatic polyketides are known to be produced by soil dwelling Streptomyces, 54 Streptomyces strains were isolated from the soil samples. Five isolates, R1, B1, R3, R5 and Y8 were found to be potent aromatic polyketide producers and were identified by 16S rRNA gene sequencing as Streptomyces spectabilis, Streptomyces olivaceus, Streptomyces purpurascens, Streptomyces coeruleorubidus and Streptomyces lavendofoliae respectively. Their sequences have been deposited in the GenBank under the accession numbers KF468818, KF681280, KF395224, KF527511 and KF681281 respectively. The Streptomyces strains were cultivated in the media following critically optimised culture conditions. The resulting broth extracts were fractionated on a silica gel column and preparative TLC to obtain pure compounds. The pure compounds were tested for bioactivity and the most potent compound from each isolate was identified by UV-Vis, IR and NMR spectroscopic methods. Isolated S. spectabilis (R1), yielded one potent compound identified as dihydrodaunomycin with an MIC of 4 µg/ml against Bacillus cereus and an IC 50 value of 24 µM against HeLa. S. olivaceus (B1), yielded a comparatively less potent compound, elloramycin. S. purpurascens (R3) yielded three compounds, rhodomycin, epelmycin and obelmycin. The most potent compound was rhodomycin with an MIC of 2 µg/ml against B. cereus and IC 50 of 15 µM against HeLa. S. coeruleorubidus (R5), yielded daunomycin showing an IC 50 of 10 µM and also exhibiting antimetastatic properties against HeLa. S. lavendofoliae (Y8), yielded a novel aclacinomycin analogue with IC 50 value of 2.9 µM and potent antimetastatic properties at 1 µM concentration against HeLa. The study focuses on the characterization of aromatic polyketides from soil Streptomyces spp., which can serve as potential candidates for development of chemotherapeutic drugs in future.

Streptomyces peucetius ATCC 27952 is a filamentous soil bacterium with potential to produce anthracyclines such as doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of cancer. Here we present the complete genome sequence of S. peucetius ATCC 27952, which consists of 8,023,114 bp with a linear chromosome, 7187 protein-coding genes, 18 rRNA operons and 66 tRNAs. Bioinformatic analysis of the genome sequence revealed ∼68 putative gene clusters involved in the biosynthesis of secondary metabolites, including diverse classes of natural products. Diverse secondary metabolites of PKS (polyketide synthase) type II (doxorubicin and daunorubicin), NRPS (non-ribosomal peptide synthase) (T1-pks), terpene (hopene) etc. have already been reported for this strain. In addition, in silico analysis suggests the potential to produce diverse compound classes such as lantipeptides, lassopeptides, NRPS and polyketides. Furthermore, many catalytically-efficient enzymes involved in hydroxylation, methylation etc. have been characterized in this strain. The availability of genomic information provides valuable insight for devising rational strategies for the production and isolation of diverse bioactive compounds as well as for the industrial application of efficient enzymes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: To identify the roles of the two O-methyltransferase homologous genes pdmF and pdmT in the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2., Methods and Results: Pradimicins are pentangular polyphenol antibiotics synthesized by bacterial type II polyketide synthases (PKSs) and tailoring enzymes. Pradimicins are naturally derivatized by combinatorial O-methylation at two positions (i.e., 7-OH and 11-OH) of the benzo[α]naphthacenequinone structure. PdmF and PdmT null mutants (PFKO and PTKO) were generated. PFKO produced the 11-O-demethyl shunt metabolites 11-O-demethylpradimicinone II (1), 11-O-demethyl-7-methoxypradimicinone II (2), 11-O-demethylpradimicinone I (3) and 11-O-demethylpradimicin A (4), while PTKO generated the 7-O-demethyl derivatives pradimicinone II (5) and 7-hydroxypradimicin A (6). Pradimicinones 1, 2, 3, and 5 were fed to a heterologous host Escherichia coli harbouring expression plasmid pET-22b::pdmF or pET-28a::pdmT. PdmF catalysed 11-O-methylation of pradimicinones 1, 2, and 3 regardless of O-methylation at the C-7 position, while PdmT was unable to catalyse 7-O-methylation when the C-11 hydroxyl group was methylated (5)., Conclusions: PdmF and PdmT were involved in 11-O- and 7-O-methylations of the benzo[α]naphthacenequinone moiety of pradimicin, respectively. Methylation of the C-7 hydroxyl group precedes methylation of the C-11 hydroxyl group in pradimicin biosynthesis., Significance and Impact of the Study: This is the first reported demonstration of the functions of PdmF and PdmT for regiospecific O-methylation, which contributes to better understanding of the post-PKS modifications in pradimicin biosynthesis as well as to rational engineering of the pradimicin biosynthetic machinery., (© 2017 The Society for Applied Microbiology.)

Background: The family of DNA topoisomerases comprises a group of enzymes that catalyse the induction of topological changes to DNA. These enzymes play a role in the cell replication machinery and are, therefore, important targets for anticancer drugs - with human DNA topoisomerase IIα being one of the most prominent. Active compounds targeting this enzyme are classified into two groups with diverse mechanisms of action: DNA poisons act by stabilizing a covalent cleavage complex between DNA and the topoisomerase enzyme, transforming it into a cellular toxin, while the second diverse group of catalytic inhibitors, provides novel inhibition avenues for tackling this enzyme due to frequent occurrence of side effects observed during the DNA poison therapy., Methods: Based on a comprehensive literature search we present an overview of available bioassays and in silico methods in the identification of human DNA topoisomerase IIα inhibitors., Results and Conclusion: A comprehensive outline of the available methods and approaches that explore in detail the in vitro mechanistic and functional aspects of the topoisomerase IIα inhibition of both topo IIα inhibitor groups is presented. The utilized in vitro cell-based assays and in vivo studies to further explore the validated topo IIα inhibitors in subsequent preclinical stages of the drug discovery are discussed. The potential of in silico methods in topoisomerase IIα inhibitor discovery is outlined. A list of practical guidelines was compiled to aid new as well experienced researchers in how to optimally approach the design of targeted inhibitors and validation in the preclinical drug development stages., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Advances in genomics and metabolomics have made clear in recent years that microbial biosynthetic capacities on Earth far exceed previous expectations. This is attributable, in part, to the realization that most microbial natural product (NP) producers harbor biosynthetic machineries not readily amenable to classical laboratory fermentation conditions. Such "cryptic" or dormant biosynthetic gene clusters (BGCs) encode for a vast assortment of potentially new antibiotics and, as such, have become extremely attractive targets for activation under controlled laboratory conditions. We report here that coculturing of a Rhodococcus sp. and a Micromonospora sp. affords keyicin, a new and otherwise unattainable bis-nitroglycosylated anthracycline whose mechanism of action (MOA) appears to deviate from those of other anthracyclines. The structure of keyicin was elucidated using high resolution MS and NMR technologies, as well as detailed molecular modeling studies. Sequencing of the keyicin BGC (within the Micromonospora genome) enabled both structural and genomic comparisons to other anthracycline-producing systems informing efforts to characterize keyicin. The new NP was found to be selectively active against Gram-positive bacteria including both Rhodococcus sp. and Mycobacterium sp. E. coli-based chemical genomics studies revealed that keyicin's MOA, in contrast to many other anthracyclines, does not invoke nucleic acid damage.

The chemotherapeutic anthracycline metabolite doxorubicinol (doxOL) has been shown to interact with and disrupt the function of the cardiac ryanodine receptor Ca 2+ release channel (RyR2) in the sarcoplasmic reticulum (SR) membrane and the SR Ca 2+ binding protein calsequestrin 2 (CSQ2). Normal increases in RyR2 activity in response to increasing diastolic SR [Ca 2+ ] are influenced by CSQ2 and are disrupted in arrhythmic conditions. Therefore, we explored the action of doxOL on RyR2's response to changes in luminal [Ca 2+ ] seen during diastole. DoxOL abolished the increase in RyR2 activity when luminal Ca 2+ was increased from 0.1 to 1.5 mM. This was not due to RyR2 oxidation, but depended entirely on the presence of CSQ2 in the RyR2 complex. DoxOL binding to CSQ2 reduced both the Ca 2+ binding capacity of CSQ2 (by 48%-58%) and its aggregation, and lowered CSQ2 association with the RyR2 complex by 67%-77%. Each of these effects on CSQ2, and the lost RyR2 response to changes in luminal [Ca 2+ ], was duplicated by exposing native RyR2 channels to subphysiologic (≤1.0 µM) luminal [Ca 2+ ]. We suggest that doxOL and low luminal Ca 2+ both disrupt the CSQ2 polymer, and that the association of the monomeric protein with the RyR2 complex shifts the increase in RyR2 activity with increasing luminal [Ca 2+ ] away from the physiologic [Ca 2+ ] range. Subsequently, these changes may render the channel insensitive to changes of luminal Ca 2+ that occur through the cardiac cycle. The altered interactions between CSQ2, triadin, and/or junctin and RyR2 may produce an arrhythmogenic substrate in anthracycline-induced cardiotoxicity., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Streptomyces albus J1074 is one of the most popular and convenient hosts for heterologous expression of gene clusters directing the biosynthesis of various natural metabolic products, such as antibiotics. This fuels interest in elucidation of genetic mechanisms that may limit secondary metabolism in J1074. Here, we report the generation and initial study of J1074 mutant, deficient in gene bldA for tRNA Leu UAA , the only tRNA capable of decoding rare leucyl TTA codon in Streptomyces. The bldA deletion in J1074 resulted in a highly conditional Bld phenotype, with depleted formation of aerial hyphae on certain solid media. In addition, bldA mutant of J1074 was unable to produce endogenous antibacterial compounds and two heterologous antibiotics, moenomycin and aranciamycin, whose biosynthesis is directed by TTA-containing genes. We have employed a new TTA codon-specific β-galactosidase reporter system to provide genetic evidence that J1074 bldA mutant is impaired in translation of TTA. In addition, we have discussed the possible reasons for differences in the phenotypes of bldA mutants described here and in previous studies, providing knowledge to study bldA-based regulation of antibiotic biosynthesis.

Pradimicins are a group of antiviral and antifungal natural products from Actinomadura hibisca. Two putative O-methyltransferase genes, pdmF and pdmT, are present in the pradimicin biosynthetic gene cluster. However, there is only one methoxy group (11-OCH 3 ) in pradimicins. Through heterologous expression and in vitro reactions with various substrates, PdmF was characterized as the C-11 O-methyltransferase with a relatively broad substrate specificity. To probe the role of PdmT in pradimicin biosynthesis, the corresponding gene was disrupted through homologous recombination, leading to the production of pradimicinone II. This enzyme was then expressed in Escherichia coli with an N-terminal His 6 tag and purified by Ni-NTA chromatography. Reaction of pradimicinone II with PdmT generated 7-O-methylpradimicinone II, confirming that this enzyme is a C-7 O-methyltransferase. Characterization of PdmT suggests a novel pathway that leads to the "flip" of 7-OH to C-14 in pradimicin biosynthesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Anthracyclines, such as doxorubicin, are effective anticancer drugs composed of a tetracyclic polyketide aglycone and one or more deoxysugar moieties, which play a critical role in their biological activity. A facile one-pot combinatorial biosynthetic system was developed for the generation of a range of glycosylated derivatives of anthracyclines. Cocultivation of Streptomyces venezuelae mutants producing two anthracycline aglycones with eight different nucleotide deoxysugar-producing S. venezuelae mutants that coexpress a substrate-flexible glycosyltransferase led to the generation of 16 aklavinone or ε-rhodomycinone glycosides containing diverse deoxysugar moieties, seven of which are new. This demonstrates the potential of the one-pot combinatorial biosynthetic system based on cocultivation as a facile biological tool capable of combining diverse aglycones and deoxysugars to generate structurally diverse polyketides carrying engineered sugars for drug discovery and development.

Ranking among the most effective anticancer drugs, anthracyclines represent an important family of aromatic polyketides generated by type II polyketide synthases (PKSs). After formation of polyketide cores, the post-PKS tailoring modifications endow the scaffold with various structural diversities and biological activities. Here we demonstrate an unprecedented four-enzyme-participated hydroxyl regioisomerization process involved in the biosynthesis of kosinostatin. First, KstA15 and KstA16 function together to catalyze a cryptic hydroxylation of the 4-hydroxyl-anthraquinone core, yielding a 1,4-dihydroxyl product, which undergoes a chemically challenging asymmetric reduction-dearomatization subsequently acted by KstA11; then, KstA10 catalyzes a region-specific reduction concomitant with dehydration to afford the 1-hydroxyl anthraquinone. Remarkably, the shunt product identifications of both hydroxylation and reduction-dehydration reactions, the crystal structure of KstA11 with bound substrate and cofactor, and isotope incorporation experiments reveal mechanistic insights into the redox dearomatization and rearomatization steps. These findings provide a distinguished tailoring paradigm for type II PKS engineering.

Early acting cyclases play critical roles in programming the polyketide biosynthesis toward certain, distinguished scaffolds. Starting from acetyl-CoA and malonyl-CoA, a one-pot enzymatic total synthesis of an anthracyclinone scaffold, presteffimycinone, was achieved by mixing polyketide synthase (PKS) and early post-PKS enzymes from the biosynthetic pathways of three different types of type II-PKS driven anticancer antibiotics, namely, the mithramycin (aureolic acid-type), gilvocarcin (rearranged angucycline-type), and steffimycin (anthracycline) pathways.

The anthracycline natural product dutomycin and its precursor POK-MD1 were isolated from Streptomyces minoensis NRRL B-5482. The dutomycin biosynthetic gene cluster was identified by genome sequencing and disruption of the ketosynthase gene. Two polyketide synthase (PKS) systems are present in the gene cluster, including a type II PKS and a rare highly reducing iterative type I PKS. The type I PKS DutG repeatedly uses its active sites to create a nine-carbon triketide chain that is subsequently transferred to the α-l-axenose moiety of POK-MD1 at 4″-OH to yield dutomycin. Using a heterologous recombination approach, we disrupted a putative methyltransferase gene (dutMT1) and two glycosyltransferase genes (dutGT1 and dutGT2). Analysis of the metabolites of these mutants revealed the functions of these genes and yielded three dutomycin analogues SW140, SW91, and SW75. The major product SW91 in Streptomyces minoensis NRRL B-5482-ΔDutMT1 was identified as 12-desmethyl-dutomycin, suggesting that DutMT1 is the dedicated 12-methyltransferase. This was confirmed by the in vitro enzymatic assay. DutGT1 and DutGT2 were found to be responsible for the introduction of β-d-amicetose and α-l-axenose, respectively. Dutomycin and SW91 showed strong antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus, whereas POK-MD1 and SW75 had no obvious inhibition, which revealed the essential role of the C-4″ triketide chain in antibacterial activity. The minimal inhibitory concentration of SW91 against the two strains was 0.125 μg mL(-1), lower than that of dutomycin (0.25 μg mL(-1)), indicating that the antibacterial activity of dutomycin can be improved through biosynthetic structural modification.

Nanosecond pulsed electric field (nsPEF) is a novel method to increase cell proliferation rate. The phenomenon is based on the microporation of cellular organelles and membranes. However, we have limited information on the effects of nsPEF on cell physiology. Several studies have attempted to describe the effects of this process, however no real time measurements have been conducted to date. In this study we designed a model system which allows the measurement of cellular processes before, during and after nsPEF treatment in real time. The system employs a Vabrema Mitoplicator(TM) nsPEF field generating instrument connected to a BD Accuri C6 cytometer with a silicon tube led through a peristaltic pump. This model system was applied to observe the effects of nsPEF in mammalian C6 glioblastoma (C6 glioma) and HEK-293 cell lines. Viability (using DRAQ7 dye), intracellular calcium levels (using Fluo-4 dye) and scatter characteristics were measured in a kinetic manner. Data were analyzed using the FACSKin software. The viability and morphology of the investigated cells was not altered upon nsPEF treatment. The response of HEK-293 cells to ionomycin as positive control was significantly lower in the nsPEF treated samples compared to non-treated cells. This difference was not observed in C6 cells. FSC and SSC values were not altered significantly by the nsPEF treatment. Our results indicate that this model system is capable of reliably investigating the effects of nsPEF on cellular processes in real time. © 2016 International Society for Advancement of Cytometry., (© 2016 International Society for Advancement of Cytometry.)