Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study struct... more Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study structure-activity-relationships of the gilvocarcin group of antitumor antibiotics, because of a similar chemical structure and comparable bioactivity with gilvocarcin V, the principle compound of this group, and the feasibility of enzymatic modifications of its sugar moiety by auxiliary Omethyltransferases. Such enzymes were used to modify the interaction of the drug with histone H3, the biological target that interacts with the sugar moiety. Cytotoxicity assays revealed that a free 2'-OH group of the sugar moiety is essential to maintain the bioactivity of polycarcin V, apparently an important H-bond donor for the interaction with histone H3, while converting 3'-OH into an OCH 3 group improved the bioactivity. Bis-methylated polycarcin derivatives revealed weaker activity than the parent compound, indicating that at least two H-bond donors in the sugar are necessary for optimal binding.
Synthetic scheme for the preparation of a number of different derivatives of anticancer natural p... more Synthetic scheme for the preparation of a number of different derivatives of anticancer natural product Psoralidin is described. A convergent synthetic approach is followed using simple starting materials like substituted phenyl acetic esters and benzoic acids. The developed synthetic route leads us to complete the first synthesis of an analogous natural product Lespeflorin I 1 , a mild melanin synthesis inhibitor. Preliminary bioactivity studies of the synthesized compounds are carried out against two commonly used prostate cancer cell lines. Results show that the bioactivity of the compounds can be manipulated by the simple modification of the functional groups.
Journal of the American Chemical Society, Jul 19, 2012
Two enzymes of the gilvocarcin biosynthetic pathway, GilMT and GilM, with unclear functions were ... more Two enzymes of the gilvocarcin biosynthetic pathway, GilMT and GilM, with unclear functions were investigated by in vitro studies using purified, recombinant enzymes along with synthetically prepared intermediates. The studies revealed GilMT as a typical S-adenosylmethionine (SAM) dependent O-methyltransferase, but GilM was identified as a pivotal enzyme in the pathway that
Glycosylation reactions catalyzed by glycosyltransferases (GTs) are important structural modifica... more Glycosylation reactions catalyzed by glycosyltransferases (GTs) are important structural modifications that are often necessary for the biological activities of natural products. [1] Studying these is an increasingly important and rigorously pursued research field. Although GTs usually display strict substrate and regiospecificities, some examples were found showing surprising tolerance, which can be exploited in combinatorial biosynthetic approaches. [1b,c] ** This work was supported by the NIH grant CA 091901 to J.R.
The protein kinase Akt plays an important role in cell proliferation and survival in many cancers... more The protein kinase Akt plays an important role in cell proliferation and survival in many cancers, including prostate cancer. Due to its kinase activity, it serves as a molecular conduit for inhibiting apoptosis and promoting angiogenesis in most cell types. In most of the prostate tumors, Akt signaling is constitutively activated due to the deletion or mutation of the tumor suppressor PTEN, which negatively regulates phosphatidylinositol 3-kinase through lipid phosphatase activity. Recently, we identified a natural compound, psoralidin, which inhibits Akt phosphorylation, and its consequent activation in androgen-independent prostate cancer (AIPC) cells. Furthermore, ectopic expression of Akt renders AIPC cells resistant to chemotherapy; however, psoralidin overcomes Akt-mediated resistance and induces apoptosis in AIPC cells. While dissecting the molecular events, both upstream and downstream of Akt, we found that psoralidin inhibits phosphatidylinositol 3-kinase activation and transcriptionally represses the activation of nuclear factor-κB and its target genes (Bcl-2, Survivin, and Bcl-xL, etc.), which results in the inhibition of cell viability and induction of apoptosis in PC-3 and DU-145 cells. Interestingly, psoralidin selectively targets cancer cells without causing any toxicity to normal prostate epithelial cells. In vivo xenograft assays substantiate these in vitro findings and show that psoralidin inhibits prostate tumor growth in nude mice. Our findings are of therapeutic significance in the management of prostate cancer patients with advanced or metastatic disease, as they provide new directions for the development of a phytochemical-based platform for prevention and treatment strategies for AIPC. Prostate cancer is the second leading cause of cancer-related death in men. An estimated 186,320 new cases will be diagnosed in 2008 in the United States (1). Androgen ablation therapy and bilateral orchidectomy have been the main therapeutic options for locally advanced and/or metastatic androgen-independent prostate cancer (AIPC) cell (2). Although most patients initially respond to androgen ablation therapy (∼30% have androgen ablation therapy refractory disease), a majority of these patients transit from androgen-dependent prostate cancer to AIPC, for which there is no curative therapy (3). Most patients with AIPC show resistance to current chemotherapeutics, and even docetaxel-based combination therapy yield only a limited improvement in patient survival (4), suggesting that novel targeted therapies are much needed. Recent evidences suggest that Akt plays an important role in the conversion of androgen-dependent prostate cancer to AIPC. Accumulating evidence suggests that Akt is the focal point of a number of signaling pathways that regulate cell growth, survival, proliferation, immune activation, apoptosis (5), and tumor progression in many cancer types (6), including AIPC (7, 8). Akt is activated by the lipid kinase, phosphatidylinositol 3-kinase (PI3K; 9), which plays an important role in the prosurvival mechanism in a number of cell types and in a majority of cancers. PI3K is a heterodimeric protein composed of a catalytic subunit (p110α/β/γ/δ) and a regulatory subunit (p85α/β) that is involved in cell growth, transformation, and differentiation. Concurrent phosphatase and tensin homologue (PTEN) inactivation negatively regulates Akt activation; however, PTEN is frequently mutated or deleted in many cancers including prostate cancer (10). Three isoforms of Akt (Akt-1, Akt-2, and Akt-3) have been identified, and all three share a similar domain structure,
A one-pot enzymatic total synthesis of angucycline antibiotic rabelomycin was accomplished, start... more A one-pot enzymatic total synthesis of angucycline antibiotic rabelomycin was accomplished, starting from acetyl-CoA and malonyl-CoA, using a mixture of polyketide synthase (PKS) enzymes of the gilvocarcin, ravidomycin, and jadomycin biosynthetic pathways. The in vitro results were compared to in vivo catalysis using analogous sets of enzymes. The benz[a]anthraquinone antibiotics, commonly named angucyclines, 1,2 are well-known for their broad range of biological activities including antibiotic, antitumor, antiviral, antifungal, platelet aggregation, and enzyme inhibitory effects. The angucyclinone rabelomycin ( ) is a commonly found, simple member of this family. It was first isolated from the culture broth of Streptomyces olivaceus ATCC21549 in 1970 and was shown to have antibacterial activities comparable to those of tetrangomycin against Gram-positive microorganisms. It is also a proposed intermediate of the biosyntheses of various other members of the angucycline family, such as urdamycin, 4 oviedomycin, 5 and landomycin E.
Elloramycin (1), a new antibiotic produced by Streptomyces olivaceus strain Tii 2353, was detecte... more Elloramycin (1), a new antibiotic produced by Streptomyces olivaceus strain Tii 2353, was detected by chemical screening. The dark yellow compound, molecular formula C3_H36O,, is weakly active against a variety of Gram-positive bacteria, especially streptomycetes and against stem cells of L-1210 leukemia. Acidic hydrolysis of the antibiotic liberated elloramycinone (3) as aglycone and 2,3,4-tri-O-methyl-L-rhamnose, which was identified as methyl glycoside 5b. The structure of elloramycin was established by comparison of the spectra (UV, 1H NMR, 13C NMR) with those of the known tetracenomycin C (2), 3 and the fact that 2 and 3 gave the same tetramethyl ether after permethylation. Elloramycin is an anthracycline-like antibiotic, the aglycone resembles tetracenomycin C, the sugar is connected in a phenolic a-glycosidic linkage. In the course of our chemical screening2-5) we look for secondary metabolites of microorganisms, which can be detected on TLC plates by their color or by color reactions using special staining reagents. Elloramycin exhibited a striking appearance in TLC experiments by its dark yellow color, and by fluorescence at 366 nm in UV light. In the following study we describe the fermentation, isolation, physico-chemical characterization, structure elucidation and the biological properties of the new compound. Subject of another publication will be the special chemistry of elloramycin and its de-rivatives6). To produce elloramycin in larger amounts, 20-liter, 25-liter and 200-liter fermentations were performed. The time course of the fermentation of Streptomyces olivaceus in a 25-liter fermentor is shown in Fig. . The production of the antibiotic starts early during the beginning of the logarithmical growing phase, and reaches its maximum after about 112 hours, yielding an antibiotic concentration of about 200 mg/liter in the culture medium. The crude product, which was obtained by extraction from mycelium (methanol) and the culture filtrate (ethyl acetate) of strain Tu 2353, was chromatographically purified on silica gel and Sephadex 1292 THE JOURNAL
Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resista... more Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resistance to platinum-based chemotherapy bodes a particularly abysmal prognosis, underscoring the need for novel therapeutic agents and strategies. The use of mithramycin, an antineoplastic antibiotic, has been previously limited by its narrow therapeutic window. Recent advances in semisynthetic methods have led to mithramycin analogs with improved pharmacological profiles. Mithramycin inhibits the activity of the transcription factor Sp1, which is closely linked with ovarian tumorigenesis and platinum-resistance. This article summarizes recent clinical developments related to mithramycin and postulates a role for the use of mithramycin, or its analog, in the treatment of platinum-resistant ovarian cancer.
The citrus black spot (CBS), caused by Phyllosticta citricarpa, is one of the most important citr... more The citrus black spot (CBS), caused by Phyllosticta citricarpa, is one of the most important citrus diseases in subtropical regions of Africa, Asia, Oceania, and the Americas, and fruits with CBS lesions are still subject to quarantine regulations in the European Union. Despite the high application of fungicides, the disease remains present in the citrus crops of Central and South America. In order to find alternatives to help control CBS and reduce the use of fungicides, we explored the antifungal potential of endophytic actinomycetes isolated from the Brazilian medicinal plant Vochysia divergens found in the Pantanal biome. Two different culture media and temperatures were selected to identify the most efficient conditions for the production of active secondary metabolites. The metabolites produced by strain Microbacterium sp. LGMB471 cultured in SG medium at 36 °C considerably inhibited the development of P. citricarpa. Three isoflavones and five diketopiperazines were identified, and the compounds 7-O-β-D-glucosylgenistein and 7-O-β-D-glucosyl-daidzein showed high activity against P. citricarpa, with the MIC of 33 μg/mL and inhibited the production of asexual spores of P. citricarpa on leaves and citrus fruits. Compounds that inhibit conidia formation may be a promising alternative to reduce the use of fungicides in the control of CBS lesions, especially in regions where sexual reproduction does not occur, as in the USA. Our data suggest the use of Microbacterium sp. LGMB471 or its metabolites as an ecological alternative to be used in association with the fungicides for the control of CBS disease.
The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancerrelated... more The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancerrelated hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me 2+ )-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM = MTM SDK > MTM SA-Trp > MTM SA-Ala. An X(G/C) (G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.
Early acting cyclases play critical roles in programming the polyketide biosynthesis towards cert... more Early acting cyclases play critical roles in programming the polyketide biosynthesis towards 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.
A base catalyzed condensation of phenyl acetate with acid chloride followed by intramolecular cyc... more A base catalyzed condensation of phenyl acetate with acid chloride followed by intramolecular cyclization and microwave assisted cross metathesis reaction lead to the first total synthesis of psoralidin, a natural product with a broad range of biological activities, in a highly convergent and regio-selective manner.
The chemical structure of the new angucycline antibiotic landomycin A has been elucidated via che... more The chemical structure of the new angucycline antibiotic landomycin A has been elucidated via chemical and spectroscopic methods, in particular by 2D NMRcorrelation spectroscopy, e.g., 1H,1H-COSY, 13C,1H-COSY, correlation spectroscopy via long-range-couplings and heteronuclear multiple bond connectivity spectroscopy sequences. The spectroscopic investigations were carried out principally with the octaacetyl derivative of landomycin A, which is more soluble in organic solvents than landomycin A itself. The structure consists of a new, unusual angucyclinone, landomycinone A, and of six deoxy sugars, four D-olivoses and two L-rhodinoses, which are all assembled in one chain thus forming the sequence (olivose-4-* l-olivose-3 -» l-rhodinose)2. This long sugar chain is bondedas a phenolic glycoside to the aglycone moiety, a unique structural feature amongquinone glycoside antibiotics. By comparison with the main component landomycin A, the structures of three minor congeners, namely landomycins B, C and D, could be proposed.
Derivatives of the angucycline urdamycin A (1) were prepared in order to study structureactivity ... more Derivatives of the angucycline urdamycin A (1) were prepared in order to study structureactivity relationships in this group of antitumor antibiotics. Derivatives of 1 formed by methanolysis, O-acylation, hydrogenation and treatment with diazomethane were isolated and characterized by their spectroscopic data. Urdamycin G (20) was isolated from Streptomyces fradiae by shortening the fermentation time. The different glycosidation pattern of the aglycone 14 did not lead to significant differences in the biological activity. O-Acylation was shownto enhance the in vitro activity of 1 against stem cells of murine LI210 leukemia depending on the lipophilicity of the molecules. The importance of the 5,6-double bond of 1 with regard to the antitumor activities is discussed.
Plant diseases caused by phytopathogens are responsible for significant crop losses worldwide. Re... more Plant diseases caused by phytopathogens are responsible for significant crop losses worldwide. Resistance induction and biological control have been exploited in agriculture due to their enormous potential. In this study, we investigated the antimicrobial potential of endophytic fungi of leaves and petioles of medicinal plants Vochysia divergens and Stryphnodendron adstringens located in two regions of high diversity in Brazil, Pantanal, and Cerrado, respectively. We recovered 1,304 fungal isolates and based on the characteristics of the culture, were assigned to 159 phenotypes. One isolate was selected as representative of each phenotype and studied for antimicrobial activity against phytopathogens. Isolates with better biological activities were identified based on DNA sequences and phylogenetic analyzes. Among the 159 representative isolates, extracts from 12 endophytes that inhibited the mycelial growth (IG) of Colletotrichum abscissum (≥40%) were selected to expand the antimicrobial analysis. The minimum inhibitory concentrations (MIC) of the extracts were determined against citrus pathogens, C. abscissum, Phyllosticta citricarpa and Xanthomonas citri subsp. citri and the maize pathogen Fusarium graminearum. The highest activity against C. abscissum were from extracts of Pseudofusicoccum stromaticum CMRP4328 (IG: 83% and MIC: 40 µg/mL) and Diaporthe vochysiae CMRP4322 (IG: 75% and MIC: 1 µg/mL), both extracts also inhibited the development of post-bloom fruit drop symptoms in citrus flowers. The extracts were promising in inhibiting the mycelial growth of P. citricarpa and reducing the production of pycnidia in citrus leaves. Among the isolates that showed activity, the genus Diaporthe was the most common, including the new species D. cerradensis described in this study. In addition, high performance liquid chromatography, UV detection, and mass spectrometry and thin layer chromatography analyzes of extracts produced by endophytes that showed high activity, indicated D. vochysiae CMRP4322 and P. stromaticum CMRP4328 as promising strains that
The isolation and structure elucidation of one new fungal metabolite, phenguignardic acid butyl e... more The isolation and structure elucidation of one new fungal metabolite, phenguignardic acid butyl ester (1a), and four previously reported metabolites (1b, 2a, 3-4) from the citrus phytopathogen Phyllosticta citricarpa LGMF06 are described. The new dioxolanone phenguignardic acid butyl ester (1a) had low phytotoxic activity in citrus leaves and fruits (at dose of 100 μg), and its importance as virulence factor in citrus black spot disease needs to be further addressed. Beside the phytotoxic analysis, we also evaluated the antibacterial (against methicillin sensitive and resistant Staphylococcus aureus) and cytotoxic (A549 non-small cell lung cancer, PC3 prostate cancer and HEL 299 normal epithelial lung) activities of the isolated compounds, which revealed that compounds 1a, 1b and 2a were responsible for the antibacterial activity of this strain.
Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study struct... more Polycarcin V, a polyketide natural product of Streptomyces polyformus, was chosen to study structure-activity-relationships of the gilvocarcin group of antitumor antibiotics, because of a similar chemical structure and comparable bioactivity with gilvocarcin V, the principle compound of this group, and the feasibility of enzymatic modifications of its sugar moiety by auxiliary Omethyltransferases. Such enzymes were used to modify the interaction of the drug with histone H3, the biological target that interacts with the sugar moiety. Cytotoxicity assays revealed that a free 2'-OH group of the sugar moiety is essential to maintain the bioactivity of polycarcin V, apparently an important H-bond donor for the interaction with histone H3, while converting 3'-OH into an OCH 3 group improved the bioactivity. Bis-methylated polycarcin derivatives revealed weaker activity than the parent compound, indicating that at least two H-bond donors in the sugar are necessary for optimal binding.
Synthetic scheme for the preparation of a number of different derivatives of anticancer natural p... more Synthetic scheme for the preparation of a number of different derivatives of anticancer natural product Psoralidin is described. A convergent synthetic approach is followed using simple starting materials like substituted phenyl acetic esters and benzoic acids. The developed synthetic route leads us to complete the first synthesis of an analogous natural product Lespeflorin I 1 , a mild melanin synthesis inhibitor. Preliminary bioactivity studies of the synthesized compounds are carried out against two commonly used prostate cancer cell lines. Results show that the bioactivity of the compounds can be manipulated by the simple modification of the functional groups.
Journal of the American Chemical Society, Jul 19, 2012
Two enzymes of the gilvocarcin biosynthetic pathway, GilMT and GilM, with unclear functions were ... more Two enzymes of the gilvocarcin biosynthetic pathway, GilMT and GilM, with unclear functions were investigated by in vitro studies using purified, recombinant enzymes along with synthetically prepared intermediates. The studies revealed GilMT as a typical S-adenosylmethionine (SAM) dependent O-methyltransferase, but GilM was identified as a pivotal enzyme in the pathway that
Glycosylation reactions catalyzed by glycosyltransferases (GTs) are important structural modifica... more Glycosylation reactions catalyzed by glycosyltransferases (GTs) are important structural modifications that are often necessary for the biological activities of natural products. [1] Studying these is an increasingly important and rigorously pursued research field. Although GTs usually display strict substrate and regiospecificities, some examples were found showing surprising tolerance, which can be exploited in combinatorial biosynthetic approaches. [1b,c] ** This work was supported by the NIH grant CA 091901 to J.R.
The protein kinase Akt plays an important role in cell proliferation and survival in many cancers... more The protein kinase Akt plays an important role in cell proliferation and survival in many cancers, including prostate cancer. Due to its kinase activity, it serves as a molecular conduit for inhibiting apoptosis and promoting angiogenesis in most cell types. In most of the prostate tumors, Akt signaling is constitutively activated due to the deletion or mutation of the tumor suppressor PTEN, which negatively regulates phosphatidylinositol 3-kinase through lipid phosphatase activity. Recently, we identified a natural compound, psoralidin, which inhibits Akt phosphorylation, and its consequent activation in androgen-independent prostate cancer (AIPC) cells. Furthermore, ectopic expression of Akt renders AIPC cells resistant to chemotherapy; however, psoralidin overcomes Akt-mediated resistance and induces apoptosis in AIPC cells. While dissecting the molecular events, both upstream and downstream of Akt, we found that psoralidin inhibits phosphatidylinositol 3-kinase activation and transcriptionally represses the activation of nuclear factor-κB and its target genes (Bcl-2, Survivin, and Bcl-xL, etc.), which results in the inhibition of cell viability and induction of apoptosis in PC-3 and DU-145 cells. Interestingly, psoralidin selectively targets cancer cells without causing any toxicity to normal prostate epithelial cells. In vivo xenograft assays substantiate these in vitro findings and show that psoralidin inhibits prostate tumor growth in nude mice. Our findings are of therapeutic significance in the management of prostate cancer patients with advanced or metastatic disease, as they provide new directions for the development of a phytochemical-based platform for prevention and treatment strategies for AIPC. Prostate cancer is the second leading cause of cancer-related death in men. An estimated 186,320 new cases will be diagnosed in 2008 in the United States (1). Androgen ablation therapy and bilateral orchidectomy have been the main therapeutic options for locally advanced and/or metastatic androgen-independent prostate cancer (AIPC) cell (2). Although most patients initially respond to androgen ablation therapy (∼30% have androgen ablation therapy refractory disease), a majority of these patients transit from androgen-dependent prostate cancer to AIPC, for which there is no curative therapy (3). Most patients with AIPC show resistance to current chemotherapeutics, and even docetaxel-based combination therapy yield only a limited improvement in patient survival (4), suggesting that novel targeted therapies are much needed. Recent evidences suggest that Akt plays an important role in the conversion of androgen-dependent prostate cancer to AIPC. Accumulating evidence suggests that Akt is the focal point of a number of signaling pathways that regulate cell growth, survival, proliferation, immune activation, apoptosis (5), and tumor progression in many cancer types (6), including AIPC (7, 8). Akt is activated by the lipid kinase, phosphatidylinositol 3-kinase (PI3K; 9), which plays an important role in the prosurvival mechanism in a number of cell types and in a majority of cancers. PI3K is a heterodimeric protein composed of a catalytic subunit (p110α/β/γ/δ) and a regulatory subunit (p85α/β) that is involved in cell growth, transformation, and differentiation. Concurrent phosphatase and tensin homologue (PTEN) inactivation negatively regulates Akt activation; however, PTEN is frequently mutated or deleted in many cancers including prostate cancer (10). Three isoforms of Akt (Akt-1, Akt-2, and Akt-3) have been identified, and all three share a similar domain structure,
A one-pot enzymatic total synthesis of angucycline antibiotic rabelomycin was accomplished, start... more A one-pot enzymatic total synthesis of angucycline antibiotic rabelomycin was accomplished, starting from acetyl-CoA and malonyl-CoA, using a mixture of polyketide synthase (PKS) enzymes of the gilvocarcin, ravidomycin, and jadomycin biosynthetic pathways. The in vitro results were compared to in vivo catalysis using analogous sets of enzymes. The benz[a]anthraquinone antibiotics, commonly named angucyclines, 1,2 are well-known for their broad range of biological activities including antibiotic, antitumor, antiviral, antifungal, platelet aggregation, and enzyme inhibitory effects. The angucyclinone rabelomycin ( ) is a commonly found, simple member of this family. It was first isolated from the culture broth of Streptomyces olivaceus ATCC21549 in 1970 and was shown to have antibacterial activities comparable to those of tetrangomycin against Gram-positive microorganisms. It is also a proposed intermediate of the biosyntheses of various other members of the angucycline family, such as urdamycin, 4 oviedomycin, 5 and landomycin E.
Elloramycin (1), a new antibiotic produced by Streptomyces olivaceus strain Tii 2353, was detecte... more Elloramycin (1), a new antibiotic produced by Streptomyces olivaceus strain Tii 2353, was detected by chemical screening. The dark yellow compound, molecular formula C3_H36O,, is weakly active against a variety of Gram-positive bacteria, especially streptomycetes and against stem cells of L-1210 leukemia. Acidic hydrolysis of the antibiotic liberated elloramycinone (3) as aglycone and 2,3,4-tri-O-methyl-L-rhamnose, which was identified as methyl glycoside 5b. The structure of elloramycin was established by comparison of the spectra (UV, 1H NMR, 13C NMR) with those of the known tetracenomycin C (2), 3 and the fact that 2 and 3 gave the same tetramethyl ether after permethylation. Elloramycin is an anthracycline-like antibiotic, the aglycone resembles tetracenomycin C, the sugar is connected in a phenolic a-glycosidic linkage. In the course of our chemical screening2-5) we look for secondary metabolites of microorganisms, which can be detected on TLC plates by their color or by color reactions using special staining reagents. Elloramycin exhibited a striking appearance in TLC experiments by its dark yellow color, and by fluorescence at 366 nm in UV light. In the following study we describe the fermentation, isolation, physico-chemical characterization, structure elucidation and the biological properties of the new compound. Subject of another publication will be the special chemistry of elloramycin and its de-rivatives6). To produce elloramycin in larger amounts, 20-liter, 25-liter and 200-liter fermentations were performed. The time course of the fermentation of Streptomyces olivaceus in a 25-liter fermentor is shown in Fig. . The production of the antibiotic starts early during the beginning of the logarithmical growing phase, and reaches its maximum after about 112 hours, yielding an antibiotic concentration of about 200 mg/liter in the culture medium. The crude product, which was obtained by extraction from mycelium (methanol) and the culture filtrate (ethyl acetate) of strain Tu 2353, was chromatographically purified on silica gel and Sephadex 1292 THE JOURNAL
Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resista... more Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resistance to platinum-based chemotherapy bodes a particularly abysmal prognosis, underscoring the need for novel therapeutic agents and strategies. The use of mithramycin, an antineoplastic antibiotic, has been previously limited by its narrow therapeutic window. Recent advances in semisynthetic methods have led to mithramycin analogs with improved pharmacological profiles. Mithramycin inhibits the activity of the transcription factor Sp1, which is closely linked with ovarian tumorigenesis and platinum-resistance. This article summarizes recent clinical developments related to mithramycin and postulates a role for the use of mithramycin, or its analog, in the treatment of platinum-resistant ovarian cancer.
The citrus black spot (CBS), caused by Phyllosticta citricarpa, is one of the most important citr... more The citrus black spot (CBS), caused by Phyllosticta citricarpa, is one of the most important citrus diseases in subtropical regions of Africa, Asia, Oceania, and the Americas, and fruits with CBS lesions are still subject to quarantine regulations in the European Union. Despite the high application of fungicides, the disease remains present in the citrus crops of Central and South America. In order to find alternatives to help control CBS and reduce the use of fungicides, we explored the antifungal potential of endophytic actinomycetes isolated from the Brazilian medicinal plant Vochysia divergens found in the Pantanal biome. Two different culture media and temperatures were selected to identify the most efficient conditions for the production of active secondary metabolites. The metabolites produced by strain Microbacterium sp. LGMB471 cultured in SG medium at 36 °C considerably inhibited the development of P. citricarpa. Three isoflavones and five diketopiperazines were identified, and the compounds 7-O-β-D-glucosylgenistein and 7-O-β-D-glucosyl-daidzein showed high activity against P. citricarpa, with the MIC of 33 μg/mL and inhibited the production of asexual spores of P. citricarpa on leaves and citrus fruits. Compounds that inhibit conidia formation may be a promising alternative to reduce the use of fungicides in the control of CBS lesions, especially in regions where sexual reproduction does not occur, as in the USA. Our data suggest the use of Microbacterium sp. LGMB471 or its metabolites as an ecological alternative to be used in association with the fungicides for the control of CBS disease.
The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancerrelated... more The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancerrelated hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me 2+ )-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM = MTM SDK > MTM SA-Trp > MTM SA-Ala. An X(G/C) (G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.
Early acting cyclases play critical roles in programming the polyketide biosynthesis towards cert... more Early acting cyclases play critical roles in programming the polyketide biosynthesis towards 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.
A base catalyzed condensation of phenyl acetate with acid chloride followed by intramolecular cyc... more A base catalyzed condensation of phenyl acetate with acid chloride followed by intramolecular cyclization and microwave assisted cross metathesis reaction lead to the first total synthesis of psoralidin, a natural product with a broad range of biological activities, in a highly convergent and regio-selective manner.
The chemical structure of the new angucycline antibiotic landomycin A has been elucidated via che... more The chemical structure of the new angucycline antibiotic landomycin A has been elucidated via chemical and spectroscopic methods, in particular by 2D NMRcorrelation spectroscopy, e.g., 1H,1H-COSY, 13C,1H-COSY, correlation spectroscopy via long-range-couplings and heteronuclear multiple bond connectivity spectroscopy sequences. The spectroscopic investigations were carried out principally with the octaacetyl derivative of landomycin A, which is more soluble in organic solvents than landomycin A itself. The structure consists of a new, unusual angucyclinone, landomycinone A, and of six deoxy sugars, four D-olivoses and two L-rhodinoses, which are all assembled in one chain thus forming the sequence (olivose-4-* l-olivose-3 -» l-rhodinose)2. This long sugar chain is bondedas a phenolic glycoside to the aglycone moiety, a unique structural feature amongquinone glycoside antibiotics. By comparison with the main component landomycin A, the structures of three minor congeners, namely landomycins B, C and D, could be proposed.
Derivatives of the angucycline urdamycin A (1) were prepared in order to study structureactivity ... more Derivatives of the angucycline urdamycin A (1) were prepared in order to study structureactivity relationships in this group of antitumor antibiotics. Derivatives of 1 formed by methanolysis, O-acylation, hydrogenation and treatment with diazomethane were isolated and characterized by their spectroscopic data. Urdamycin G (20) was isolated from Streptomyces fradiae by shortening the fermentation time. The different glycosidation pattern of the aglycone 14 did not lead to significant differences in the biological activity. O-Acylation was shownto enhance the in vitro activity of 1 against stem cells of murine LI210 leukemia depending on the lipophilicity of the molecules. The importance of the 5,6-double bond of 1 with regard to the antitumor activities is discussed.
Plant diseases caused by phytopathogens are responsible for significant crop losses worldwide. Re... more Plant diseases caused by phytopathogens are responsible for significant crop losses worldwide. Resistance induction and biological control have been exploited in agriculture due to their enormous potential. In this study, we investigated the antimicrobial potential of endophytic fungi of leaves and petioles of medicinal plants Vochysia divergens and Stryphnodendron adstringens located in two regions of high diversity in Brazil, Pantanal, and Cerrado, respectively. We recovered 1,304 fungal isolates and based on the characteristics of the culture, were assigned to 159 phenotypes. One isolate was selected as representative of each phenotype and studied for antimicrobial activity against phytopathogens. Isolates with better biological activities were identified based on DNA sequences and phylogenetic analyzes. Among the 159 representative isolates, extracts from 12 endophytes that inhibited the mycelial growth (IG) of Colletotrichum abscissum (≥40%) were selected to expand the antimicrobial analysis. The minimum inhibitory concentrations (MIC) of the extracts were determined against citrus pathogens, C. abscissum, Phyllosticta citricarpa and Xanthomonas citri subsp. citri and the maize pathogen Fusarium graminearum. The highest activity against C. abscissum were from extracts of Pseudofusicoccum stromaticum CMRP4328 (IG: 83% and MIC: 40 µg/mL) and Diaporthe vochysiae CMRP4322 (IG: 75% and MIC: 1 µg/mL), both extracts also inhibited the development of post-bloom fruit drop symptoms in citrus flowers. The extracts were promising in inhibiting the mycelial growth of P. citricarpa and reducing the production of pycnidia in citrus leaves. Among the isolates that showed activity, the genus Diaporthe was the most common, including the new species D. cerradensis described in this study. In addition, high performance liquid chromatography, UV detection, and mass spectrometry and thin layer chromatography analyzes of extracts produced by endophytes that showed high activity, indicated D. vochysiae CMRP4322 and P. stromaticum CMRP4328 as promising strains that
The isolation and structure elucidation of one new fungal metabolite, phenguignardic acid butyl e... more The isolation and structure elucidation of one new fungal metabolite, phenguignardic acid butyl ester (1a), and four previously reported metabolites (1b, 2a, 3-4) from the citrus phytopathogen Phyllosticta citricarpa LGMF06 are described. The new dioxolanone phenguignardic acid butyl ester (1a) had low phytotoxic activity in citrus leaves and fruits (at dose of 100 μg), and its importance as virulence factor in citrus black spot disease needs to be further addressed. Beside the phytotoxic analysis, we also evaluated the antibacterial (against methicillin sensitive and resistant Staphylococcus aureus) and cytotoxic (A549 non-small cell lung cancer, PC3 prostate cancer and HEL 299 normal epithelial lung) activities of the isolated compounds, which revealed that compounds 1a, 1b and 2a were responsible for the antibacterial activity of this strain.
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Papers by Jurgen Rohr