Progress toward the Total Synthesis of Nogalamycin Using a Benzyne Cycloaddition Strategy.

Nogalamycin (NOG) is a member of the anthracycline glycoside natural products; no total syntheses have yet been reported, and there is minimal understanding of how the aglycone substitution pattern and identities of the A- and D-ring sugars impact the anticancer activity and toxicity. This paper reports progress toward a modular approach to NOG that could enable systematic structure-activity relationship studies. Key steps include a regioselective benzyne cycloaddition and reductive ring-opening to assemble a versatile AB core for analogue synthesis.

Inhibitory effects of anthracyclines on partially purified 5'-3' DNA helicase of Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum has been becoming resistant to the currently used anti-malarial drugs. Searching for new drug targets is urgently needed for anti-malarial development. DNA helicases separating double-stranded DNA into single-stranded DNA intermediates are essential in nearly all DNA metabolic transactions, thus they may act as a candidate for new drug targets against malarial parasites. METHODS: In this study, a P. falciparum 5' to 3' DNA helicase (PfDH-B) was partially purified from the crude extract of chloroquine- and pyrimethamine-resistant P. falciparum strain K1, by ammonium sulfate precipitation and three chromatographic procedures. DNA helicase activity of partially purified PfDH-B was examined by measuring its ability to unwind 32P-labelled partial duplex DNA. The directionality of PfDH-B was determined, and substrate preference was tested by using various substrates. Inhibitory effects of DNA intercalators such as anthracycline antibiotics on PfDH-B unwinding activity and parasite growth were investigated. RESULTS: The native PfDH-B was partially purified with a specific activity of 4150 units/mg. The PfDH-B could unwind M13-17-mer, M13-31-mer with hanging tail at 3' or 5' end and a linear substrate with 3' end hanging tail but not blunt-ended duplex DNA, and did not need a fork-like substrate. Anthracyclines including aclarubicin, daunorubicin, doxorubicin, and nogalamycin inhibited the unwinding activity of PfDH-B with an IC50 value of 4.0, 7.5, 3.6, and 3.1 µM, respectively. Nogalamycin was the most effective inhibitor on PfDH-B unwinding activity and parasite growth (IC50 = 0.1 ± 0.002 µM). CONCLUSION: Partial purification and characterization of 5'-3' DNA helicase of P. falciparum was successfully performed. The partially purified PfDH-B does not need a fork-like substrate structure found in P. falciparum 3' to 5' DNA helicase (PfDH-A). Interestingly, nogalamycin was the most potent anthracycline inhibitor for PfDH-B helicase activity and parasite growth in culture. Further studies are needed to search for more potent but less cytotoxic inhibitors targeting P. falciparum DNA helicase in the future.

The Rieske Oxygenase SnoT Catalyzes 2''-Hydroxylation of l-Rhodosamine in Nogalamycin Biosynthesis.

Nogalamycin is an anthracycline anti-cancer agent that intercalates into the DNA double helix. The binding is facilitated by two carbohydrate units, l-nogalose and l-nogalamine, that interact with the minor and major grooves of DNA, respectively. However, recent investigations have shown that nogalamycin biosynthesis proceeds through the attachment of l-rhodosamine (2''-deoxy-4''-epi-l-nogalamine) to the aglycone. Herein, we demonstrate that the Rieske enzyme SnoT catalyzes 2''-hydroxylation of l-rhodosamine as an initial post-glycosylation step. Furthermore, we establish that the reaction order continues with 2-5'' carbocyclization and 4'' epimerization by the non-heme iron and 2-oxoglutarate-dependent enzymes SnoK and SnoN, respectively. These late-stage tailoring steps are important for the bioactivity of nogalamycin due to involvement of the 2''- and 4''-hydroxy groups of l-nogalamine in hydrogen bonding interactions with DNA.

Evolution-guided engineering of non-heme iron enzymes involved in nogalamycin biosynthesis.

Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. The key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison with their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to nonheme iron and 2-oxoglutarate-dependent mono-oxygenases. In spite of structural similarity, the two proteins catalyze distinct chemical reactions; SnoK is a C2-C5″ carbocyclase, whereas SnoN catalyzes stereoinversion at the adjacent C4″ position. Here, we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyze stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C-terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for the formation of the C2-C5″ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents. ENZYMES: EC number 1.14.11.

How a cofactor-free protein environment lowers the barrier to O2 reactivity.

Molecular oxygen (O2)-utilizing enzymes are among the most important in biology. The abundance of O2, its thermodynamic power, and the benign nature of its end products have raised interest in oxidases and oxygenases for biotechnological applications. Although most O2-dependent enzymes have an absolute requirement for an O2-activating cofactor, several classes of oxidases and oxygenases accelerate direct reactions between substrate and O2 using only the protein environment. Nogalamycin monooxygenase (NMO) from Streptomyces nogalater is a cofactor-independent enzyme that catalyzes rate-limiting electron transfer between its substrate and O2 Here, using enzyme-kinetic, cyclic voltammetry, and mutagenesis methods, we demonstrate that NMO initially activates the substrate, lowering its pKa by 1.0 unit (ΔG* = 1.4 kcal mol-1). We found that the one-electron reduction potential, measured for the deprotonated substrate both inside and outside the protein environment, increases by 85 mV inside NMO, corresponding to a ΔΔG0' of 2.0 kcal mol-1 (0.087 eV) and that the activation barrier, ΔG‡, is lowered by 4.8 kcal mol-1 (0.21 eV). Applying the Marcus model, we observed that this suggests a sizable decrease of 28 kcal mol-1 (1.4 eV) in the reorganization energy (λ), which constitutes the major portion of the protein environment's effect in lowering the reaction barrier. A similar role for the protein has been proposed in several cofactor-dependent systems and may reflect a broader trend in O2-utilizing proteins. In summary, NMO's protein environment facilitates direct electron transfer, and NMO accelerates rate-limiting electron transfer by strongly lowering the reorganization energy.

In silico identification and in vitro validation of nogalamycin N-oxide (NSC116555) as a potent anticancer compound against non-small-cell lung cancer cells.

The epidermal growth factor receptor (EGFR) was found to be overexpressed in several cancers, especially in lung cancers. Finding new effective drug against EGFR is the key to cancer treatment. In this study, the GOLD docking algorithm was used to virtually screen for novel human EGFR inhibitors from the NCI database. Thirty-four hit compounds were tested for EGFR-tyrosine kinase (TK) inhibition. Two potent compounds, 1-amino-4-(4-[4-amino-2-sulfophenyl]anilino)-9,10-dioxoanthracene-2-sulfonic acid (NSC125910), and nogalamycin N-oxide (NSC116555) were identified with IC50 values against EGFR-TK comparable to gefitinib; 16.14 and 37.71 nM, respectively. However, only NSC116555 demonstrated cytotoxic effects against non-small-cell lung cancer, A549, shown in the cell cytotoxicity assay with an IC50 of 0.19 + 0.01 µM, which was more potent than gefitinib. Furthermore, NSC116555 showed cytotoxicity against A549 via apoptosis in a dose-dependent manner.

Induction of HRR genes and inhibition of DNMT1 is associated with anthracycline anti-tumor antibiotic-tolerant breast carcinoma cells.

The aim of the study was to understand the role of homologous recombination repair (HRR) pathway genes in development of chemotolerance in breast cancer (BC). For this purpose, chemotolerant BC cells were developed in MCF-7 and MDA MB 231 cell lines after treatment with two anthracycline anti-tumor antibiotics doxorubicin and nogalamycin at different concentrations for 48 h with differential cell viability. The drugs were more effective in MCF-7 (IC50: 0.214-0.242 µM) than in MDA MB 231 (IC50: 0.346-0.37 µM) as shown by cell viability assay. The drugs could reduce the protein expression of PCNA in the cell lines. Increased mRNA/protein expression of the HRR (BRCA1, BRCA2, FANCC, FANCD2, and BRIT1) genes was seen in the cell lines in the presence of the drugs at different concentrations (lower IC50, IC50, and higher IC50) irrespective of the cell viability (68-41%). Quantitative methylation assay showed an increased percentage of hypomethylation of the promoters of these genes after drug treatment in the cell lines. Similarly, chemotolerant neoadjuvant chemotherapy (NACT) treated primary BC samples showed significantly higher frequency of hypomethylation of the genes than the pretherapeutic BC samples. The drugs in different concentrations could reduce m-RNA and protein expression of DNMT1 (DNA methyltransferase 1) in the cell lines. Similar phenomenon was also evident in the NACT samples than in the pretherapeutic BC samples. Thus, our data indicate that reduced DNMT1 expression along with promoter hypomethylation and increased expression of the HRR genes might have importance in chemotolerance in BC.

Enzymatic Synthesis of the C-Glycosidic Moiety of Nogalamycin R.

Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units. The former of these is attached through a canonical O-glycosidic linkage, but the latter is connected via an unusual dual linkage composed of C-C and O-glycosidic bonds. In this work, we have utilized enzyme immobilization techniques and synthesized l-rhodosamine-thymidine diphosphate (TDP) from α-d-glucose-1-TDP using seven enzymes. In a second step, we assembled the dual linkage system by attaching the aminosugar to an anthracycline aglycone acceptor using the glycosyl transferase SnogD and the α-ketoglutarate dependent oxygenase SnoK. Furthermore, our work indicates that the auxiliary P450-type protein SnogN facilitating glycosylation is surprisingly associated with attachment of the neutral sugar l-nogalose rather than the aminosugar l-nogalamine in nogalamycin biosynthesis.

ATPase activity of Plasmodium falciparum MLH is inhibited by DNA-interacting ligands and dsRNAs of MLH along with UvrD curtail malaria parasite growth.

Malaria caused by Plasmodium falciparum is the major disease burden all over the world. Recently, the situation has deteriorated because the malarial parasites are becoming progressively more resistant to numerous commonly used antimalarial drugs. Thus, there is a critical requirement to find other means to restrict and eliminate malaria. The mismatch repair (MMR) machinery of parasite is quite unique in several ways, and it can be exploited for finding new drug targets. MutL homolog (MLH) is one of the major components of MMR machinery, and along with UvrD, it helps in unwinding the DNA. We have screened several DNA-interacting ligands for their effect on intrinsic ATPase activity of PfMLH protein. This screening suggested that several ligands such as daunorubicin, etoposide, ethidium bromide, netropsin, and nogalamycin are inhibitors of the ATPase activity of PfMLH, and their apparent IC50 values range from 2.1 to 9.35 µM. In the presence of nogalamycin and netropsin, the effect was significant because in their presence, the V max value dropped from 1.024 µM of hydrolyzed ATP/min to 0.596 and 0.643 µM of hydrolyzed ATP/min, respectively. The effect of double-stranded RNAs of PfMLH and PfUvrD on growth of P. falciparum 3D7 strain was studied. The parasite growth was significantly inhibited suggesting that these components belonging to MMR pathway are crucial for the survival of the parasite.

[THE ROLE OF (p)ppGpp MOLECULES IN FORMATION OF "STRICT RESPONSE" IN BACTERIA AND BIOSYNTHESIS OF ANTIBIOTICS AND MORPHOLOGICAL DIFFERENTIATION IN ACTINOMYCETES].

Strict response is a pleiotropic physiological response of cells caused by lack of aminoacetylated tRNAs. Experimentally, this response occurs due to the lack of amino acids in the environment and the limitation of tRNA aminoacylation even in the presence of the corresponding amino acids in the cell. Many features of this response indicate its dependence on the accumulation of ppGpp molecules. There is a correlation between the growth rate of actinomycetes and biosynthesis of their secondary metabolites. Introduction of additional relA gene copies of ppGpp synthetase can affect the production of antibiotics in streptomycetes. The article presents the authors' own experimental data, dedicated to the influence of heterologous relA gene expression in Streptomyces nogalater cells.

Investigating the impacts of DNA binding mode and sequence on thermodynamic quantities and water exchange values for two small molecule drugs.

Doxorubicin and nogalamycin are antitumor antibiotics that interact with DNA via intercalation and threading mechanisms, respectively. Because the importance of water, particularly its impact on entropy changes, has been established in other biological processes, we investigated the role of water in these two drug-DNA binding events. We used the osmotic stress method to calculate the number of water molecules exchanged (Δnwater), and isothermal titration calorimetry to measure Kbinding, ΔH, and ΔS for two synthetic DNAs, poly(dA·dT) and poly(dG·dC), and calf thymus DNA (CT DNA). For nogalamycin, Δnwater<0 for CT DNA and poly(dG·dC). For doxorubicin, Δnwater>0 for CT DNA and Δnwater<0 for poly(dG·dC). For poly(dA·dT), Δnwater~0 with both drugs. Net enthalpy changes were always negative, but net entropy changes depended on the drug. The effect of water exchange on the overall sign of entropy change appears to be smaller than other contributions.

Discriminating Intercalative Effects of Threading Intercalator Nogalamycin, from Classical Intercalator Daunomycin, Using Single Molecule Atomic Force Spectroscopy.

DNA threading intercalators are a unique class of intercalating agents, albeit little biophysical information is available on their intercalative actions. Herein, the intercalative effects of nogalamycin, which is a naturally-occurring DNA threading intercalator, have been investigated by high-resolution atomic force microscopy (AFM) and spectroscopy (AFS). The results have been compared with those of the well-known chemotherapeutic drug daunomycin, which is a non-threading classical intercalator bearing structural similarity to nogalamycin. A comparative AFM assessment revealed a greater increase in DNA contour length over the entire incubation period of 48 h for nogalamycin treatment, whereas the contour length increase manifested faster in case of daunomycin. The elastic response of single DNA molecules to an externally applied force was investigated by the single molecule AFS approach. Characteristic mechanical fingerprints in the overstretching behaviour clearly distinguished the nogalamycin/daunomycin-treated dsDNA from untreated dsDNA-the former appearing less elastic than the latter, and the nogalamycin-treated DNA distinguished from the daunomycin-treated DNA-the classically intercalated dsDNA appearing the least elastic. A single molecule AFS-based discrimination of threading intercalation from the classical type is being reported for the first time.

Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway.

Nogalamycin, an aromatic polyketide displaying high cytotoxicity, has a unique structure, with one of the carbohydrate units covalently attached to the aglycone via an additional carbon-carbon bond. The underlying chemistry, which implies a particularly challenging reaction requiring activation of an aliphatic carbon atom, has remained enigmatic. Here, we show that the unusual C5''-C2 carbocyclization is catalyzed by the non-heme iron α-ketoglutarate (α-KG)-dependent SnoK in the biosynthesis of the anthracycline nogalamycin. The data are consistent with a mechanistic proposal whereby the Fe(IV) = O center abstracts the H5'' atom from the amino sugar of the substrate, with subsequent attack of the aromatic C2 carbon on the radical center. We further show that, in the same metabolic pathway, the homologous SnoN (38% sequence identity) catalyzes an epimerization step at the adjacent C4'' carbon, most likely via a radical mechanism involving the Fe(IV) = O center. SnoK and SnoN have surprisingly similar active site architectures considering the markedly different chemistries catalyzed by the enzymes. Structural studies reveal that the differences are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species. Our findings significantly expand the repertoire of reactions reported for this important protein family and provide an illustrative example of enzyme evolution.

[THE ROLE OF STREPTOMYCES NOGALATER Lv65 snoaM, snoaL and snoaE GENES IN NOGALAMYCIN BIOSYNTHESIS].

The results of phylogenetic analysis indicate high similarity of SnoaM, SnoaL SnoaE to the cyclases involved in the biosynthesis of various antibiotics. Genes snoaM, snoaL and snoaE disruption in S. nogalater chromosome was carried on and S. nogalater MI, LI and EI strains were generated. The gene replacement events in M1, L1 and E1 were verified by Southern hybridization. Recombinant strains were characterised by lack of nogalamycin biosynthesis. Originally, M1, L1 and E1 were complemented with plasmids expressing putative cyclase genes from S. nogalater leading to restoration of nogalamycine production. The complementation results clearly indicate that obtained strains are cyclase deficient mutants. Furthermore, complementation of M1, L1 and E1 with a cyclase genes from wild-type strain is consistent with the suggested function of these enzymes.

Inactivation and identification of three genes encoding glycosyltransferase required for biosynthesis of nogalamycin.

Nogalamycin is an anthracycline antitumor antibiotic, consisting of the aromatic aglycone attached with a nogalose and a nogalamine. At present, the biosynthesis pathway of nogalamycin, especially the glycosylation mechanism of the two deoxysugar moieties, had still not been extensively investigated in vivo. In this study, we inactivated the three glycotransferase genes in the nogalamycin-produced strain, and investigated the function of these genes by analyzing the metabolites profiles in the fermentation broth. The in-frame deletion of snogD and disruption of snogE abolished the production of nogalamycin completely, indicating that the gene products of snogD and snogE are essential to the biosynthesis of nogalamycin. On the other hand, in-frame deletion of snogZ does not abolish the production of nogalamycin, but production yield was reduced to 28% of the wild type, implying that snogZ gene may involved in the activation of other glycotransferases in nogalamycin biosynthesis. This study laid the foundation of modification of nogalamycin biosynthesis/production by genetic engineering methods.

Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth.

BACKGROUND: Human malaria parasite infection and its control is a global challenge which is responsible for ~0.65 million deaths every year globally. The emergence of drug resistant malaria parasite is another challenge to fight with malaria. Enormous efforts are being made to identify suitable drug targets in order to develop newer classes of drug. Helicases play crucial roles in DNA metabolism and have been proposed as therapeutic targets for cancer therapy as well as viral and parasitic infections. Genome wide analysis revealed that Plasmodium falciparum possesses UvrD helicase, which is absent in the human host. RESULTS: Recently the biochemical characterization of P. falciparum UvrD helicase revealed that N-terminal UvrD (PfUDN) hydrolyses ATP, translocates in 3' to 5' direction and interacts with MLH to modulate each other's activity. In this follow up study, further characterization of P. falciparum UvrD helicase is presented. Here, we screened the effect of various DNA interacting compounds on the ATPase and helicase activity of PfUDN. This study resulted into the identification of daunorubicin (daunomycin), netropsin, nogalamycin, and ethidium bromide as the potential inhibitor molecules for the biochemical activities of PfUDN with IC50 values ranging from ~3.0 to ~5.0 µM. Interestingly etoposide did not inhibit the ATPase activity but considerable inhibition of unwinding activity was observed at 20 µM. Further study for analyzing the importance of PfUvrD enzyme in parasite growth revealed that PfUvrD is crucial/important for its growth ex-vivo. CONCLUSIONS: As PfUvrD is absent in human hence on the basis of this study we propose PfUvrD as suitable drug target to control malaria. Some of the PfUvrD inhibitors identified in the present study can be utilized to further design novel and specific inhibitor molecules.

Personalized chemotherapy profiling using cancer cell lines from selectable mice.

PURPOSE: High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells. EXPERIMENTAL DESIGN: We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. RESULTS: Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)-approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. CONCLUSION: Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice.

Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater.

The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3',4'-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5'-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.

Discovery of a two-component monooxygenase SnoaW/SnoaL2 involved in nogalamycin biosynthesis.

Nogalamycin is an anthracycline polyketide antibiotic that contains two deoxysugars, at positions C-1 and C-7. Previous biosynthetic studies conducted in vivo affiliated snoaL2 with an unusual C-1 hydroxylation reaction, but in vitro activity was not established. Here, we demonstrate that inactivation of either snoaL2 or snoaW resulted in accumulation of two nonhydroxylated metabolites, nogalamycinone and a novel anthracycline 3',4'-demethoxy-nogalose-nogalamycinone. The C-1 hydroxylation activity was successfully reconstructed in vitro in the presence of the two enzymes, NAD(P)H and the substrates. Based on relative reaction efficiencies, 3',4'-demethoxy-nogalose-nogalamycinone was identified as the likely natural substrate. A biosynthetic model was established where the atypical short-chain alcohol dehydrogenase SnoaW reduces the anthraquinone to a dihydroquinone using NADPH, which enables activation of oxygen and formation of a hydroperoxy intermediate. Finally, protonation of the intermediate by SnoaL2 yields the 1-hydroxylated product.