Genome Mining and Genetic Manipulation Reveal New Isofuranonaphthoquinones in Nocardia Species.

The identification of specialized metabolites isolated from microorganisms is urgently needed to determine their roles in treating cancer and controlling multidrug-resistant pathogens. Naphthoquinones act as anticancer agents in various types of cancers, but some toxicity indicators have been limited in their appropriate application. In this context, new isofuranonaphthoquinones (ifnq) that are less toxic to humans could be promising lead compounds for developing anticancer drugs. The aim of this study is to identify and characterize novel furanonaphthoquinones (fnqs) from Nocardia sp. CS682 and to evaluate their potential therapeutic applications. Analysis of the genome of Nocardia sp. CS682 revealed the presence of a furanonaphthoquinone (fnq) gene cluster, which displays a similar genetic organization and high nucleotide sequence identity to the ifnq gene cluster from Streptomyces sp. RI-77, a producer of the naphthoquinones JBIR-76 and JBIR-77. In this study, the overexpression of the Streptomyces antibiotic regulatory protein (SARP) in Nocardia sp. CS682DR (nargenicin gene-deleted mutant) explicitly produced new fnqs, namely, NOC-IBR1 and NOC-IBR2. Subsequently, the role of the SARP regulator was confirmed by gene inactivation using CRISPR-Cas9 and complementation studies. Furthermore, antioxidant, antimicrobial, and cytotoxicity assays were performed for the isolated compounds, and it was found that NOC-IBR2 exhibited superior activities to NOC-IBR1. In addition, a flexible methyltransferase substrate, ThnM3, was found to be involved in terminal methylation of NOC-IBR1, which was confirmed by in vitro enzyme assays. Thus, this study supports the importance of genome mining and genome editing approaches for exploring new specialized metabolites in a rare actinomycete called Nocardia.

A new peucemycin derivative and impacts of peuR and bldA on peucemycin biosynthesis in Streptomyces peucetius.

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 IC50 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.

Recent trends in the modification of polyphenolic compounds using hydroxylation and glycosylation.

Polyphenols are bioactive molecules that are used in therapeutics. Polyphenol hydroxylation and glycosylation have been shown to increase their bioavailability, solubility, bioactivity, and stability for use in various applications. Ortho-hydroxylation of polyphenols using tyrosinase allows high selectivity and yield without requiring a cofactor, while meta- and para-hydroxylation of polyphenols are mediated by site-specific hydroxylases and cytochrome P450s, although these processes are somewhat rare. O-glycosylation of polyphenols proceeds further after hydroxylation. The O-glycosylation reaction typically requires nucleotide diphosphate (NDP) sugar. However, amylosucrase (AS) has emerged as a promising enzyme for polyphenol glycosylation in large-scale production without requiring NDP-sugar. Overall, this review describes recent findings on the enzymatic mechanisms, enzyme engineering, and applications of enzymatic reactions.

Biosynthetic pathway of peucemycin and identification of its derivative from Streptomyces peucetius.

Streptomyces peucetius ATCC 27952 is a well-known producer of important anticancer compounds, daunorubicin and doxorubicin. In this study, we successfully identified a new macrolide, 25-hydroxy peucemycin, that exhibited an antibacterial effect on some pathogens. Based on the structure of a newly identified compound and through the inactivation of a polyketide synthase gene, we successfully identified its biosynthetic gene cluster which was considered to be the cryptic biosynthetic gene cluster. The biosynthetic gene cluster spans 51 kb with 16 open reading frames. Five type I polyketide synthase (PKS) genes encode eight modules that synthesize the polyketide chain of peucemycin before undergoing post-PKS tailoring steps. In addition to the regular starter and extender units, some modules have specificity towards ethylmalonyl-CoA and unusual butylmalonyl-CoA. A credible explanation for the specificity of the unusual extender unit has been searched for. Moreover, the enzyme responsible for the final tailoring pathway was also identified. Based on all findings, a plausible biosynthetic pathway is here proposed. KEY POINTS: • Identification of a new macrolide, 25-hydroxy peucemycin. • An FMN-dependent monooxygenase is responsible for the hydroxylation of peucemycin. • The module encoded by peuC is unique to accept the butylmalonyl-CoA as an unusual extender unit.

Increasing production of spinosad in Saccharopolyspora spinosa by metabolic engineering.

Spinosad, a combination of spinosyn A and D produced by Saccharopolyspora spinosa, is a highly efficient pesticide. There has been a considerable interest in the improvement of spinosad production because of a low yield achieved by wild-type S. spinosa. In this study, we designed and constructed a pIBR-SPN vector. pIBR-SPN is an integrative vector that can be used to introduce foreign genes into the chromosome of S. spinosa. Different combinations of genes encoding forasamine and rhamnose were synthesized and used for the construction of different recombinant plasmids. The following recombinant strains were developed: S. spinosa pIBR-SPN (only the vector), S. spinosa pIBR-SPN F (forosamine genes), S. spinosa pIBR-SPN R (rhamnose genes), S. spinosa pIBR-SPN FR (forosamine and rhamnose genes), S. spinosa pIBR-SPN FRS (forosamine, rhamnose, and SAM [S-adenosyl-L-methionine synthetase] genes), and S. spinosa MUV pIBR-SPN FR. Among these recombinant strains, S. spinosa pIBR-SPN FR produced 1394 ± 163 mg/L spinosad, which was 13-fold higher than the wild-type. S. spinosa MUV pIBR-SPN FR produced 1897 (±129) mg/L spinosad, which was seven-fold higher than S. spinosa MUV and 17-fold higher than the wild-type strain.

Microbial Biosynthesis of Chrysazin Derivatives in Recombinant Escherichia coli and Their Biological Activities.

Anthraquinone and its derivatives show remarkable biological properties such as anticancer, antibacterial, antifungal, and antiviral activities. Hence, anthraquinones derivatives have been of prime interest in drug development. This study developed a recombinant Escherichia coli strain to modify chrysazin to chrysazin-8-O-α-l-rhamnoside (CR) and chrysazin-8-O-α-l-2'-O-methylrhamnoside (CRM) using rhamnosyl transferase and sugar-O-methyltransferase. Biosynthesized CR and CRM were structurally characterized using HPLC, high-resolution mass spectrometry, and various nuclear magnetic resonance analyses. Antimicrobial effects of chrysazin, CR, and CRM against 18 superbugs, including 14 Gram-positive and 4 Gram-negative pathogens, were investigated. CR and CRM exhibited antimicrobial activities against nine pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) in a disk diffusion assay at a concentration of 40 µg per disk. There were MIC and MBC values of 7.81−31.25 µg/mL for CR and CRM against methicillin-sensitive S. aureus CCARM 0205 (MSSA) for which the parent chrysazin is more than >1000 µg/mL. Furthermore, the anti-proliferative properties of chrysazin, CR, and CRM were assayed using AGS, Huh7, HL60, and HaCaT cell lines. CR and CRM showed higher antibacterial and anticancer properties than chrysazin.

Functional Characterization of a Regiospecific Sugar-O-Methyltransferase from Nocardia.

Methyltransferases transfer a methyl group to a diverse group of natural products, thus providing structural diversity, stability, and altered pharmacological properties to the molecules. A limited number of regiospecific sugar-O-methyltransferases are functionally characterized. Thus, discovery of such an enzyme could solve the difficulties of biological production of methoxy derivatives of glycosylated molecules. In the current study, a regiospecific sugar-O-methyltransferase, ThnM1, belonging to the biosynthetic gene cluster (BGC) of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene produced by Nocardia sp. strain CS682, was analyzed and functionally characterized. ThnM1 demonstrated promiscuity to diverse chemical structures such as rhamnose-containing anthraquinones and flavonoids with regiospecific methylation at the 2'-hydroxyl group of the sugar moiety. Compared with other compounds, anthraquinone rhamnosides were found to be the preferred substrates for methylation. Thus, the enzyme was further employed for whole-cell biotransformation using engineered Escherichia coli to produce a methoxy-rhamnosyl derivative of quinizarin, an anthraquinone derivative. The structure of the newly generated derivative from Escherichia coli fermentation was elucidated by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopic analyses and identified as quinizarin-4-O-α-l-2-O-methylrhamnoside (QRM). Further, the biological impact of methylation was studied by comparing the cytotoxicity of QRM with that of quinizarin against the U87MG, SNU-1, and A375SM cancer cell lines. IMPORTANCE ThnM1 is a putative sugar-O-methyltransferase produced by the Nocardia sp. strain CS682 and is encoded by a gene belonging to the biosynthetic gene cluster (BGC) of 1-(α-l-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene. We demonstrated that ThnM1 is a promiscuous enzyme with regiospecific activity at the 2'-OH of rhamnose. As regiospecific methylation of sugars by chemical synthesis is a challenging step, ThnM1 may fill the gap in the potential diversification of natural products by methylating the rhamnose moiety attached to them.

Aloe emodin 3-O-glucoside inhibits cell growth and migration and induces apoptosis of non-small-cell lung cancer cells via suppressing MEK/ERK and Akt signalling pathways.

AIMS: Non-small-cell lung cancer (NSCLC) is the most frequent type of lung cancer with a high mortality rate. Glycosylation of phenolic compounds may increase water-solubility and pharmacological activities and reduce the toxicity of aglycones. This study aimed to evaluate and compare the anticancer effect of aloe emodin 3-O-glucoside (AE3G) and its aglycone, aloe emodin (AE), against NSCLC. MAIN METHOD: A human adenocarcinoma cell line (A549) and other human non-small cell lung carcinoma cell lines (NCI-H460 cells and NCI-H1299 cells) and BALB/c nu/nu xenograft mice harbouring A549 cells were used as the NSCLC models. Inhibition of cell migration, disruption of mitochondrial membrane potential (MMP), DNA fragmentation, and expression levels of apoptotic proteins were measured by western blot, wound healing assay, JC-1 staining, or TUNEL staining. Histopathological changes in tumour tissues were observed by H&E and TUNEL staining. RESULTS: With no significant cytotoxicity against noncancerous cells (Vero cells), AE3G (5-50 µM) significantly and more effectively inhibited the growth, attachment, migration, Bcl-2 expression, and activation of MEK/ERK and Akt signalling proteins and induced cytochrome c release and Bax expression in A549 cells than AE. AE3G also significantly decreased the growth of other NSCLC cells, NCI-H460 cells and NCI-H1299 cells. AE3G suppressed the mRNA expression of matrix metalloproteinases, MMP2 and MMP9, and augmented the collapse of the mitochondrial MMP, cleavage of caspases (caspase 9, 8, and 3) and PARP, and DNA fragmentation. Intraperitoneal injection of AE3G (13 and 26 mg/kg/day) reduced the tumour volume and weight and induced apoptotic cell death in tumour tissues of xenograft NSCLC mice. SIGNIFICANCE: The present study demonstrated that AE3G significantly and more effectively diminished human NSCLC cell growth and migration by triggering mitochondria-dependent intrinsic apoptosis than AE, providing AE3G as a new potent candidate to prevent or treat human NSCLC.

UPLC-PDA coupled HR-TOF ESI/MS2 -based identification of derivatives produced by whole-cell biotransformation of epothilone A using Nocardia sp. CS692 and a cytochrome P450 overexpressing strain.

Epothilone A, a microtubule-stabilizing agent used as therapeutics for the treatment of cancers, was biotransformed into three metabolites using Nocardia sp. CS692 and recombinant Nocardia overexpressing a cytochrome P450 from Streptomyces venezuelae (PikC). Among three metabolites produced in the biotransformation reaction mixtures, ESI/MS2 analysis predicted two metabolites (M1 and M2) as novel hydroxylated derivatives (M1 is hydroxylated at the C-8 position and M2 is hydroxylated at C-10 position), each with an opened-epoxide ring in their structure. Interestingly, metabolite M3 lacks an epoxide ring and is known as deoxyepothilone A, which is also called epothilone C. Metabolite M1 was produced only in PikC overexpressing strain. The endogenous enzymes of Nocardia sp. catalyzed hydroxylation of epothilone A to produce metabolite M2 and removed epoxide ring to produce metabolite M3. All the metabolites were identified based on UV-vis analysis and rigorous ESI/MS2 fragmentation based on epothilone A standard. The newly produced metabolites are anticipated to display novel cytotoxic effects and could be subjects of further pharmacological studies.

Identification of Cyclophilin A as a Potential Anticancer Target of Novel Nargenicin A1 Analog in AGS Gastric Cancer Cells.

We recently discovered a novel nargenicin A1 analog, 23-demethyl 8,13-deoxynargenicin (compound 9), with potential anti-cancer and anti-angiogenic activities against human gastric adenocarcinoma (AGS) cells. To identify the key molecular targets of compound 9, that are responsible for its biological activities, the changes in proteome expression in AGS cells following compound 9 treatment were analyzed using two-dimensional gel electrophoresis (2-DE), followed by MALDI/TOF/MS. Analyses using chemical proteomics and western blotting revealed that compound 9 treatment significantly suppressed the expression of cyclophilin A (CypA), a member of the immunophilin family. Furthermore, compound 9 downregulated CD147-mediated mitogen-activated protein kinase (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) by inhibiting the expression of CD147, the cellular receptor of CypA. Notably, the responses of AGS cells to CypA knockdown were significantly correlated with the anticancer and antiangiogenic effects of compound 9. CypA siRNAs reduced the expression of CD147 and phosphorylation of JNK and ERK1/2. In addition, the suppressive effects of CypA siRNAs on proliferation, migration, invasion, and angiogenesis induction of AGS cells were associated with G2/M cell cycle arrest, caspase-mediated apoptosis, inhibition of MMP-9 and MMP-2 expression, inactivation of PI3K/AKT/mTOR pathway, and inhibition of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression. The specific interaction between compound 9 and CypA was also confirmed using the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) approaches. Moreover, in silico docking analysis revealed that the structure of compound 9 was a good fit for the cyclosporin A binding cavity of CypA. Collectively, these findings provide a novel molecular basis for compound 9-mediated suppression of gastric cancer progression through the targeting of CypA.

Trilobatin ameliorates bone loss via suppression of osteoclast cell differentiation and bone resorptive function in vitro and in vivo.

AIM: Due to on-going safety concerns or lack of efficacy of currently used medications for the treatment of osteoporosis (OP), identifying new therapeutic agents is an important part of research. In the present study, potential anti-osteoporotic activity of a natural flavonoid glycoside, trilobatin (phloretin 4-O-glucoside, Tri) was evaluated. MATERIAL AND METHODS: Osteoclastic cells were established by treating the RAW264.7 macrophage cells with RANKL and ovariectomized (OVX) C57BL/6 female mice were used as an animal model of postmenopausal OP. Actin ring formation, expression levels of osteoclastogenic marker genes and bone resorptive proteins were measured by RT-PCR, western blot, or fluorometric assays. Bone mineral density (BMD) was determined by pDEXA densitometric measurement and serum osteoprotegerin (OPG) and RANKL were measured by ELISA. KEY FINDING: Tri (5-20 µM) significantly inhibited osteoclast formation and actin ring formation in RANKL-induced osteoclasts. Tri attenuated expression of osteoclastogenic genes (MMP-9 and cathepsin K), bone resorptive proteins (CA II and integrin ß3), and osteoclastogenic signalling proteins (TRAF6, p-Pyk2, c-Cbl, and c-Src). Oral administration of Tri to OVX mice augmented BMD and serum OPG/RANKL ratio. Interestingly, while Tri and phloretin aglycone (Phl) showed similar levels of in vitro anti-osteoclastogenic activity, Tri more potently ameliorated bone loss than Phl in OVX mice. SIGNIFICANCE: This study demonstrated that Tri inhibits osteoclastic cell differentiation and bone resorption by down-regulating the expression of osteoclastogenic marker genes and signalling proteins, bone resorptive proteins, and by augmenting serum OPG/RANKL ratio, suggesting that Tri can be a novel anti-osteoporotic compound for treating senile and postmenopausal OP.

Identification and enhancing production of a novel macrolide compound in engineered Streptomyces peucetius.

Streptomyces peucetius produces doxorubicin and daunorubicin, which are important anticancer drugs. In this study, we activate peucemycin, a new antibacterial compound, using an OSMAC strategy. In general, bioactive compounds are produced in a higher amount at room temperature; however, in this study, we have demonstrated that a bioactive novel compound was successfully activated at a low temperature (18 °C) in S. peucetius DM07. Through LC-MS/MS, IR spectroscopy, and NMR analysis, we identified the structure of this compound as a γ-pyrone macrolide. This compound was found to be novel, thus named peucemycin. It is an unusual 14-membered macrocyclic γ-pyrone ring with cyclization. Also, peucemycin exhibits potential antibacterial activity and a suppressive effect on the viability of various cancer cell lines.

Biosynthesis of bioactive tamarixetin in recombinant Escherichia coli.

Tamarixetin, a monomethylated derivative of quercetin, has been reported to possess many important biological activities. In the present study, a whole cell biotransformation system was used for regiospecific methylation of quercetin to produce 4'-O-methylated quercetin (tamarixetin) using methyltransferase from Streptomyces sp. KCTC 0041BP in Escherichia coli Bl21 (DE3). Its production was enhanced by adding a plasmid containing S-adenosine-l-methionine (SAM) synthase from E. coli K12 (MetK) with subsequent feeding of l-methionine and glycerol in the culture. The best condition produced ∼279 µM (88.2 mg/L) of tamarixetin. The biological activity of tamarixetin was tested and compared with quercetin, 7-O-methylated quercetin, and 3-O-methylated quercetin. Results showed that the growth of all tested cancer cell lines (AGS, B16F10, C6, and HeLa) were inhibited by tamarixetin more effectively than other methylated derivatives of quercetin or quercetin. Tamarixetin also exhibited the best antimelanogenic activity among all compounds tested.

Emodin 8-O-glucoside primes macrophages more strongly than emodin aglycone via activation of phagocytic activity and TLR-2/MAPK/NF-κB signalling pathway.

Emodin (Emo) is a natural plant anthraquinone derivative with a wide spectrum of pharmacological properties, including anticancer, antioxidant, and hepatoprotective activities. Glycosylation of natural anthraquinones with various sugar moieties can affect their physical, chemical, and biological functions. In this study, the potential immunomodulatory activities of Emo and its glycosylated derivative, emodin 8-O-glucoside (E8G), were evaluated and compared using murine macrophage RAW264.7 cells and human monocytic THP-1 cells. The results showed that E8G (20 µM) induced the secretion of TNF-α and IL-6 from RAW264.7 cells more effectively than unglycosylated Emo aglycone, by 4.9- and 1.6-fold, respectively, with no significant cytotoxicity in the concentration range tested (up to 20 µM). E8G (2.5-20 µM) significantly and dose-dependently induced inducible nitric oxide synthase (iNOS) expression by up to 3.2-fold compared to that of untreated control following a remarkable increase in nitric oxide (NO) production. E8G also significantly increased the expression of TLR-2 mRNA and the phosphorylation of MAPKs (JNK and p38). The activation and subsequent nuclear translocation of NF-κB was substantially enhanced upon treatment with E8G (2.5-20 µM). Moreover, E8G markedly induced macrophage-mediated phagocytosis of apoptotic Jurkat T cells. These results demonstrated that E8G far more strongly stimulates the secretion of proinflammatory cytokines, such as TNF-α and IL-6, and NO production from macrophages through upregulation of the TLR-2/MAPK/NF-κB signalling pathway than its nonglycosylated form, Emo aglycone. These results suggest for the first time that E8G may represent a novel immunomodulator, enhancing the early innate immunity.

Novel Nargenicin A1 Analog Inhibits Angiogenesis by Downregulating the Endothelial VEGF/VEGFR2 Signaling and Tumoral HIF-1α/VEGF Pathway.

Targeting angiogenesis is an attractive strategy for the treatment of angiogenesis-related diseases, including cancer. We previously identified 23-demethyl 8,13-deoxynargenicin (compound 9) as a novel nargenicin A1 analog with potential anticancer activity. In this study, we investigated the antiangiogenic activity and mode of action of compound 9. This compound was found to effectively inhibit in vitro angiogenic characteristics, including the proliferation, invasion, capillary tube formation, and adhesion of human umbilical vein endothelial cells (HUVECs) stimulated by vascular endothelial growth factor (VEGF). Furthermore, compound 9 suppressed the neovascularization of the chorioallantoic membrane of growing chick embryos in vivo. Notably, the antiangiogenic properties of compound 9 were related to the downregulation of VEGF/VEGFR2-mediated downstream signaling pathways, as well as matrix metalloproteinase (MMP)-2 and MMP-9 expression in HUVECs. In addition, compound 9 was found to decrease the in vitro AGS gastric cancer cell-induced angiogenesis of HUVECs by blocking hypoxia-inducible factor-1α (HIF-1α) and VEGF expression in AGS cells. Collectively, our findings demonstrate for the first time that compound 9 is a promising antiangiogenic agent targeting both VEGF/VEGFR2 signaling in ECs and HIF-1α/VEGF pathway in tumor cells.

Regio-specific biotransformation of alizarin to alizarin methoxide with enhanced cytotoxicity against proliferative cells.

Alizarin has been reported to have an antigenotoxic activity along with an inhibitory effect on the tumor cell growth of human colon carcinoma cells. Alizarin was biotransformed into an O-methoxide derivative using O-methyltransferase from Streptomyces avermitilis MA4680 (SaOMT2) to enhance its bioefficacy. The biotransformed product was extracted, purified, and characterized using various chromatographic and spectroscopic analyses, and confirmed to be an alizarin 2-O-methoxide. The antiproliferative activity of the compound against gastric cancer cells (AGS), uterine cervical cancer (Hela), liver cancer (HepG2), and normal cell lines was investigated. Alizarin 2-O-methoxide showed an inhibitory effect on all three cancer-cell lines at very low concentrations, from 0.078 µM, with no cytotoxicity against 267B1 (human prostate epithelial) and MRC-5 (normal human fetal lung fibroblast).

Anticancer and Antiangiogenic Activities of Novel α-Mangostin Glycosides in Human Hepatocellular Carcinoma Cells via Downregulation of c-Met and HIF-1α.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a leading cause of cancer-related death worldwide. Therefore, exploring effective anticancer agents and their modes of action is essential for the prevention and treatment of HCC. Glycosylation can significantly improve the physicochemical and biological properties of small molecules, such as high solubility, stability increase, and lower toxicity. In the present study, for the first time, we evaluated the anticancer and antiangiogenic activities of α-mangostin-3-O-ß-D-2-deoxyglucopyranoside (Man-3DG) and α-mangostin 6-O-ß-D-2-deoxyglucopyranoside (Man-6DG), glycosides of α-mangostin, against human HCC cells. Our results demonstrated that Man-3DG and Man-6DG significantly suppressed the growth of three different HCC cells (Hep3B, Huh7, and HepG2) as well as the migration of Hep3B cells. Furthermore, they induced cell cycle arrest in the G0/G1 phases and apoptotic cell death by regulating apoptosis-related proteins of mitochondria in Hep3B cells. Noticeably, Man-3DG and Man-6DG also caused autophagy, while co-treatment of the α-mangostin glycosides with an autophagy inhibitor 3-MA enhanced the inhibitory effect on Hep3B cell growth in comparison to single agent treatment. Moreover, Man-3DG and Man-6DG inhibited the c-Met signaling pathway that plays a critical role in the pathogenesis of HCC. Furthermore, the α-mangostin glycosides decreased Hep3B cell-induced angiogenesis in vitro through the downregulation of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Notably, Man-6DG more effectively inhibited the growth, tumorsphere formation, and expression of cancer stemness regulators compared to α-mangostin and Man-3DG in 3D spheroid-cultured Hep3B cells. These findings suggest that the α-mangostin glycosides might be promising anticancer agents for HCC treatment with superior pharmacological properties than the parent molecule α-mangostin.

Recent Advances in Strategies for Activation and Discovery/Characterization of Cryptic Biosynthetic Gene Clusters in Streptomyces.

Streptomyces spp. are prolific sources of valuable natural products (NPs) that are of great interest in pharmaceutical industries such as antibiotics, anticancer chemotherapeutics, immunosuppressants, etc. Approximately two-thirds of all known antibiotics are produced by actinomycetes, most predominantly by Streptomyces. Nevertheless, in recent years, the chances of the discovery of novel and bioactive compounds from Streptomyces have significantly declined. The major hindrance for obtaining such bioactive compounds from Streptomyces is that most of the compounds are not produced in significant titers, or the biosynthetic gene clusters (BGCs) are cryptic. The rapid development of genome sequencing has provided access to a tremendous number of NP-BGCs embedded in the microbial genomes. In addition, the studies of metabolomics provide a portfolio of entire metabolites produced from the strain of interest. Therefore, through the integrated approaches of different-omics techniques, the connection between gene expression and metabolism can be established. Hence, in this review we summarized recent advancements in strategies for activating cryptic BGCs in Streptomyces by utilizing diverse state-of-the-art techniques.

Characterization of Tailoring Steps of Nargenicin A1 Biosynthesis Reveals a Novel Analogue with Anticancer Activities.

Nargenicin A1(1) is an antibacterial macrolide with effective activity against various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Due to the promising properties of this compound in inhibiting cell proliferation, immunomodulation, and the cell protective effect, there has been significant interest in this molecule. Recently, the biosynthetic gene cluster (BGC) of 1 was reported from Nocardia argentinesis and Nocardia arthritidis. In addition, two crucial enzymes involved in the formation of the core decalin moiety and postmodification of the decalin moiety by an ether bridge were characterized. This study reports on the BGC of 1 from Nocardia sp. CS682. In addition, the direct capture and heterologous expression of nar BGC from Nocardia sp. CS682 in Streptomyces venezuelae led to the production of 1. Further metabolic profiling of wild type, Nocardia sp. CS682 in optimized media (DD media) resulted in the isolation of two acetylated derivatives, 18-O-acetyl-nodusmicin and 18-O-acetyl-nargenicin. The post-PKS modification pathway in biosynthesis of 1 was also deciphered by identifying intermediates and/or in vitro enzymatic reactions of NgnP1, NgnM, and NgnO3. Different novel analogues of 1, such as compound 6, compound 7, 23-demethyl 8,13-deoxy-nodusmicin (8), 23-demethyl 8,13-deoxynargenicin (9), 8,13-deoxynodusmicin (10), and 8,13-deoxynargenicin (11), were also characterized, which extended our understanding of key post-PKS modification steps during the biosynthesis of 1. In addition, the antimicrobial and anticancer activities of selected analogues were also evaluated, whereas compound 9 was shown to exhibit potent antitumor activity by induction of G2/M cell cycle arrest, apoptosis, and autophagy.

Streptomyces sp. VN1, a producer of diverse metabolites including non-natural furan-type anticancer compound.

Streptomyces sp. VN1 was isolated from the coastal region of Phu Yen Province (central Viet Nam). Morphological, physiological, and whole genome phylogenetic analyses suggested that strain Streptomyces sp. VN1 belonged to genus Streptomyces. Whole genome sequencing analysis showed its genome was 8,341,703 base pairs in length with GC content of 72.5%. Diverse metabolites, including cinnamamide, spirotetronate antibiotic lobophorin A, diketopiperazines cyclo-L-proline-L-tyrosine, and a unique furan-type compound were isolated from Streptomyces sp. VN1. Structures of these compounds were studied by HR-Q-TOF ESI/MS/MS and 2D NMR analyses. Bioassay-guided purification yielded a furan-type compound which exhibited in vitro anticancer activity against AGS, HCT116, A375M, U87MG, and A549 cell lines with IC50 values of 40.5, 123.7, 84.67, 50, and 58.64 µM, respectively. In silico genome analysis of the isolated Streptomyces sp. VN1 contained 34 gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including different types of terpene, T1PKS, T2PKS, T3PKS, NRPS, and hybrid PKS-NRPS. Genome mining with HR-Q-TOF ESI/MS/MS analysis of the crude extract confirmed the biosynthesis of lobophorin analogs. This study indicates that Streptomyces sp. VN1 is a promising strain for biosynthesis of novel natural products.