ABSTRACT
Chromatinized DNA is targeted by proteins and small molecules to regulate chromatin function. For example, anthracycline cancer drugs evict nucleosomes in a mechanism that is still poorly understood. We here developed a flexible method for specific isotope labeling of nucleosomal DNA enabling NMR studies of such nucleosome interactions. We describe the synthesis of segmental one-strand 13C-thymidine labeled 601-DNA, the assignment of the methyl signals, and demonstrate its use to observe site-specific binding to the nucleosome by aclarubicin, an anthracycline cancer drug that intercalates into the DNA minor grooves. Our results highlight intrinsic conformational heterogeneity in the 601 DNA sequence and show that aclarubicin binds an exposed AT-rich region near the DNA end. Overall, our data point to a model where the drug invades the nucleosome from the terminal ends inward, eventually resulting in histone eviction and nucleosome disruption.
Subject(s)
DNA , Isotope Labeling , Nucleosomes , Nucleosomes/metabolism , Nucleosomes/chemistry , DNA/chemistry , DNA/metabolism , Anthracyclines/chemistry , Anthracyclines/metabolism , Anthracyclines/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Aclarubicin/chemistry , Aclarubicin/pharmacology , Aclarubicin/metabolism , Nuclear Magnetic Resonance, BiomolecularABSTRACT
Carbonyl reduction biotransformation pathway of anthracyclines (doxorubicin, daunorubicin) is a significant process, associated with drug metabolism and elimination. However, it also plays a pivotal role in anthracyclines-induced cardiotoxicity and cancer resistance. Herein, carbonyl reduction of eight anthracyclines, at in vivo relevant concentrations (20 µM), was studied in human liver cytosol, to describe the relationship between their structure and metabolism. Significant differences of intrinsic clearance between anthracyclines, ranging from 0,62-74,9 µL/min/mg were found and associated with data from in silico analyses, considering their binding in active sites of the main anthracyclines-reducing enzymes: carbonyl reductase 1 (CBR1) and aldo-keto reductase 1C3 (AKR1C3). Partial atomic charges of carbonyl oxygen atom were also determined and considered as a factor associated with reaction rate. Structural features, including presence or absence of side-chain hydroxy group, a configuration of sugar chain hydroxy group, and tetracyclic rings substitution, affecting anthracyclines susceptibility for carbonyl reduction were identified.
Subject(s)
Aclarubicin/metabolism , Cytosol/metabolism , Doxorubicin/analogs & derivatives , Hepatocytes/metabolism , Oxidoreductases/metabolism , Aclarubicin/chemistry , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Aldo-Keto Reductase Family 1 Member C3/genetics , Aldo-Keto Reductase Family 1 Member C3/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Binding Sites , Biotransformation , Doxorubicin/chemistry , Doxorubicin/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Humans , Models, Molecular , Molecular Docking Simulation , Molecular Structure , Protein ConformationABSTRACT
Anthracycline anticancer drugs doxorubicin and aclarubicin have been used in the clinic for several decades to treat various cancers. Although closely related structures, their molecular mode of action diverges, which is reflected in their biological activity profile. For a better understanding of the structure-function relationship of these drugs, we synthesized ten doxorubicin/aclarubicin hybrids varying in three distinct features: aglycon, glycan, and amine substitution pattern. We continued to evaluate their capacity to induce DNA breaks, histone eviction, and relocated topoisomerase IIα in living cells. Furthermore, we assessed their cytotoxicity in various human tumor cell lines. Our findings underscore that histone eviction alone, rather than DNA breaks, contributes strongly to the overall cytotoxicity of anthracyclines, and structures containing N,N-dimethylamine at the reducing sugar prove that are more cytotoxic than their nonmethylated counterparts. This structural information will support further development of novel anthracycline variants with improved anticancer activity.
Subject(s)
Aclarubicin/chemistry , Antineoplastic Agents/chemistry , DNA Topoisomerases, Type II/metabolism , Doxorubicin/chemistry , Polysaccharides/chemistry , Anthracyclines/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , DNA Breaks, Double-Stranded/drug effects , Drug Screening Assays, Antitumor , Histones/metabolism , Humans , Structure-Activity RelationshipABSTRACT
In the rapidly evolving coronavirus disease (COVID-19) pandemic, repurposing existing drugs and evaluating commercially available inhibitors against druggable targets of the virus could be an effective strategy to accelerate the drug discovery process. The 3C-Like proteinase (3CLpro) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as an important drug target due to its role in viral replication. The lack of a potent 3CLpro inhibitor and the availability of the X-ray crystal structure of 3CLpro (PDB-ID 6LU7) motivated us to perform computational studies to identify commercially available potential inhibitors. A combination of modeling studies was performed to identify potential 3CLpro inhibitors from the protease inhibitor database MEROPS ( https://www.ebi.ac.uk/merops/index.shtml ). Binding energy evaluation identified key residues for inhibitor design. We found 15 potential 3CLpro inhibitors with higher binding affinity than that of an α-ketoamide inhibitor determined via X-ray structure. Among them, saquinavir and three other investigational drugs aclarubicin, TMC-310911, and faldaprevir could be suggested as potential 3CLpro inhibitors. We recommend further experimental investigation of these compounds.
Subject(s)
Betacoronavirus/enzymology , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Aclarubicin/chemistry , Aclarubicin/metabolism , Aminoisobutyric Acids , Betacoronavirus/isolation & purification , Binding Sites , COVID-19 , Coronavirus 3C Proteases , Coronavirus Infections/pathology , Coronavirus Infections/virology , Cysteine Endopeptidases/metabolism , Databases, Factual , Humans , Hydrogen Bonding , Leucine/analogs & derivatives , Oligopeptides/chemistry , Oligopeptides/metabolism , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Proline/analogs & derivatives , Protease Inhibitors/metabolism , Quinolines , SARS-CoV-2 , Thermodynamics , Thiazoles/chemistry , Thiazoles/metabolism , Viral Nonstructural Proteins/metabolismABSTRACT
A four-enzyme catalyzed hydroxy regioisomerization of anthracycline was integrated into the biosynthetic pathway of aclacinomycin A (ALM-A), to generate a series of iso-ALMs via directed combinatorial biosynthesis combined with precursor-directed mutasynthesis. Most of the newly acquired iso-ALMs exhibit obviously (1-5-fold) improved antitumor activity. Therefore, we not only developed iso-ALMs with potential as clinical drugs but also demonstrated the utility of this tailoring tool for modification of anthracycline antibiotics in drug discovery and development.
Subject(s)
Aclarubicin/analogs & derivatives , Antibiotics, Antineoplastic/pharmacology , Polyketide Synthases/metabolism , Aclarubicin/biosynthesis , Aclarubicin/chemistry , Aclarubicin/pharmacology , Antibiotics, Antineoplastic/biosynthesis , Antibiotics, Antineoplastic/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Humans , Molecular Conformation , Streptomyces/chemistry , Streptomyces/metabolismABSTRACT
OBJECTIVE: The aim of this study was to prepare aclacinomycin A (ACM)-loaded solid lipid nanoparticles (SLNs) and to evaluate their in vitro and in vivo characteristics. METHODS: SLNs were prepared using an emulsion evaporation-solidification method, and characterized in accordance with the morphological examination, particle size distribution, entrapment efficiency, drug-loading, and in vitro release. Pharmacokinetic and biodistribution studies were employed to evaluate the in vivo of SLNs. RESULTS: The SLNs were spherical in shape, uniform in size, and appropriate for administration via intravenous injection. The drug content, encapsulation efficiency, and drug loading of prepared SLNs were 96.4% ± 4.6%, 86.7% ± 2.3%, and 4.8% ± 0.7% (n = 3), respectively, and the mean diameter was 68.2 ± 5.6 nm from three batches. The SLNs were produced with stable physical properties and demonstrated significantly sustained release. The pharmacokinetic behavior of ACM was greatly improved by lyophilized injection of SLN with sustained drug release and high bioavailability. In addition, the results obtained from tissue distribution showed that ACM-SLNs were hepatic targeting in vivo. CONCLUSIONS: The present work demonstrated the feasibility of liver-targeted delivery of ACM utilizing SLNs.
Subject(s)
Aclarubicin/chemistry , Aclarubicin/pharmacokinetics , Drug Carriers/pharmacokinetics , Lipids/pharmacokinetics , Nanoparticles/chemistry , Chemistry, Pharmaceutical , Delayed-Action Preparations , Drug Compounding , Emulsions/chemistry , Lipids/chemistryABSTRACT
In this study, long-circulating Arg-Gly-Asp (RGD)-modified aclacinomycin A (ACM) liposomes were prepared by thin film hydration method. Their morphology, particle size, encapsulation efficiency, and in vitro release were investigated. The RGD-ACM liposomes was about 160 nm in size and had the visual appearance of a yellowish suspension. The zeta potential was -22.2 mV and the encapsulation efficiency was more than 93%. The drug-release behavior of the RGD-ACM liposomes showed a biphasic pattern, with an initial burst release and followed by sustained release at a constant rate. After being dissolved in phosphate-buffered saline (pH 7.4) and kept at 4°C for one month, the liposomes did not aggregate and still had the appearance of a milky white colloidal solution. In a pharmacokinetic study, rats treated with RGD-ACM liposomes showed slightly higher plasma concentrations than those treated with ACM liposomes. Maximum plasma concentrations of RGD-ACM liposomes and ACM liposomes were 4,532 and 3,425 ng/mL, respectively. RGD-ACM liposomes had a higher AUC0-∞ (1.54-fold), mean residence time (2.09-fold), and elimination half-life (1.2-fold) when compared with ACM liposomes. In an in vivo study in mice, both types of liposomes inhibited growth of human lung adenocarcinoma (A549) cells and markedly decreased tumor size when compared with the control group. There were no obvious pathological tissue changes in any of the treatment groups. Our results indicate that RGD-modified ACM liposomes have a better antitumor effect in vivo than their unmodified counterparts.
Subject(s)
Aclarubicin/administration & dosage , Adenocarcinoma/drug therapy , Antibiotics, Antineoplastic/administration & dosage , Lipids/chemistry , Lung Neoplasms/drug therapy , Oligopeptides/metabolism , Aclarubicin/blood , Aclarubicin/chemistry , Aclarubicin/pharmacokinetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Animals , Antibiotics, Antineoplastic/blood , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacokinetics , Area Under Curve , Cell Line, Tumor , Chemistry, Pharmaceutical , Delayed-Action Preparations , Drug Stability , Half-Life , Injections, Intravenous , Liposomes , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice, Inbred BALB C , Mice, Nude , Oligopeptides/chemistry , Particle Size , Rats , Solubility , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-L-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-L-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.
Subject(s)
Anthracyclines/metabolism , Biosynthetic Pathways , Evolution, Molecular , Mixed Function Oxygenases/genetics , S-Adenosylmethionine/metabolism , Aclarubicin/chemistry , Aclarubicin/metabolism , Amino Acid Sequence , Anthracyclines/chemistry , Biocatalysis , Catalytic Domain , Chromatography, High Pressure Liquid , Genetic Engineering , Hydroxylation , Methyltransferases/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Models, Molecular , Molecular Sequence Data , Multigene Family , Mutant Proteins/metabolism , Phylogeny , Recombinant Proteins/metabolism , Sequence Alignment , Spectrometry, Mass, Electrospray Ionization , Static ElectricityABSTRACT
Many anticancer drugs induce DNA breaks to eliminate tumor cells. The anthracycline topoisomerase II inhibitors additionally cause histone eviction. Here, we performed genome-wide high-resolution mapping of chemotherapeutic effects of various topoisomerase I and II (TopoI and II) inhibitors and integrated this mapping with established maps of genomic or epigenomic features to show their activities in different genomic regions. The TopoI inhibitor topotecan and the TopoII inhibitor etoposide are similar in inducing DNA damage at transcriptionally active genomic regions. The anthracycline daunorubicin induces DNA breaks and evicts histones from active chromatin, thus quenching local DNA damage responses. Another anthracycline, aclarubicin, has a different genomic specificity and evicts histones from H3K27me3-marked heterochromatin, with consequences for diffuse large B-cell lymphoma cells with elevated levels of H3K27me3. Modifying anthracycline structures may yield compounds with selectivity for different genomic regions and activity for different tumor types.
Subject(s)
Antineoplastic Agents/pharmacology , DNA, Neoplasm/chemistry , Gene Expression Regulation, Neoplastic , Genome, Human , Neoplasms/drug therapy , Topoisomerase Inhibitors/pharmacology , Aclarubicin/chemistry , Aclarubicin/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Chromatin/chemistry , Chromatin/drug effects , Chromatin/metabolism , DNA Damage , DNA, Neoplasm/metabolism , Daunorubicin/chemistry , Daunorubicin/pharmacology , Etoposide/chemistry , Etoposide/pharmacology , Histones/antagonists & inhibitors , Histones/chemistry , Histones/genetics , Histones/metabolism , Humans , Molecular Targeted Therapy , Neoplasms/chemistry , Neoplasms/genetics , Neoplasms/pathology , Organ Specificity , Protein Transport/drug effects , Structure-Activity Relationship , Topoisomerase Inhibitors/chemistry , Topotecan/chemistry , Topotecan/pharmacologyABSTRACT
DNA topoisomerase II inhibitors are a major class of cancer chemotherapeutics, which are thought to eliminate cancer cells by inducing DNA double-strand breaks. Here we identify a novel activity for the anthracycline class of DNA topoisomerase II inhibitors: histone eviction from open chromosomal areas. We show that anthracyclines promote histone eviction irrespective of their ability to induce DNA double-strand breaks. The histone variant H2AX, which is a key component of the DNA damage response, is also evicted by anthracyclines, and H2AX eviction is associated with attenuated DNA repair. Histone eviction deregulates the transcriptome in cancer cells and organs such as the heart, and can drive apoptosis of topoisomerase-negative acute myeloid leukaemia blasts in patients. We define a novel mechanism of action of anthracycline anticancer drugs doxorubicin and daunorubicin on chromatin biology, with important consequences for DNA damage responses, epigenetics, transcription, side effects and cancer therapy.
Subject(s)
Chromatin/chemistry , Chromatin/metabolism , Doxorubicin/pharmacology , Histones/metabolism , Nucleic Acid Conformation , Aclarubicin/chemistry , Aclarubicin/pharmacology , Animals , Anthracyclines/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Blast Crisis , Cell Line, Tumor , Cell Survival/drug effects , DNA/metabolism , DNA Damage , Doxorubicin/chemistry , Etoposide/chemistry , Etoposide/pharmacology , Heart/drug effects , Humans , Intercalating Agents/pharmacology , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Nude , Nucleosomes/drug effects , Nucleosomes/metabolism , Organ Specificity/drug effects , Transcriptome/geneticsABSTRACT
To investigate the use of folate-targeted nanoemulsion-loaded aclacinomycin A (ACM) to folate receptor (FR)-positive cells, we attempted to optimize the targeting ability of nanoemulsions by modifying the chain length and amount of the folate-PEG linker. Folate-linked, nanoemulsion-loaded ACM were formulated with 0.24 mol% of folate-poly (ethylene glycol)(3400)- (folate-PEG(3400)-) and folate-PEG(5000)-distearoylphosphatidylethanolamine (DSPE), and 0.03 mol% of folate-PEG(5000)-DSPE in nanoemulsions. Selective FR-mediated uptake was achieved in a human nasopharyngeal tumor cell line, KB, which overexpresses FR, but not in a human hepatoblastoma cell line, (FR(-)) HepG2. At the same amount of folate modification, the association with KB cells was increased with increasing the PEG-chain length. The association of 0.03 and 0.24 mol% folate-PEG(5000)-linked nanoemulsions with cells was 5- and 3.3-fold higher than that of non-folate nanoemulsion, respectively, while their cytotoxicity was similar. Both 0.03 and 0.24 mol% folate-PEG(5000)-linked nanoemulsions and non-folate nanoemulsion following intravenous injection inhibited tumor growth more significantly than ACM solution on day 24 following tumor inoculation (p < 0.01). This study demonstrates that a folate-linked nanoemulsion is feasible for tumor-targeted ACM delivery, and that folate modification with a sufficiently long PEG-chain and a small amount of nanoemulsion is an effective way of targeting nanoemulsion to tumor cells.
Subject(s)
Aclarubicin/chemistry , Aclarubicin/pharmacology , Antibiotics, Antineoplastic/chemistry , Folic Acid/chemistry , Polyethylene Glycols/chemistry , Animals , Antibiotics, Antineoplastic/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Delivery Systems , Female , Folic Acid/pharmacology , Humans , Mice , Mice, Nude , Nanostructures , Nasopharynx/cytology , Neoplasms/drug therapy , Polyethylene Glycols/pharmacology , Specific Pathogen-Free Organisms , Transplantation, HeterologousABSTRACT
The tetracyclic core of anthracycline natural products with antitumor activity such as aclacinomycin A are tailored during biosynthesis by regioselective glycosylation. We report the first synthesis of TDP-L-rhodosamine and demonstrate that the glycosyltransferase AknS transfers L-rhodosamine to the aglycone to initiate construction of the side-chain trisaccharide. The partner protein AknT accelerates AknS turnover rate for L-rhodosamine transfer by 200-fold. AknT does not affect the Km but rather affects the kcat. Using these data, we propose that AknT causes a conformational change in AknS that stabilizes the transition state and ultimately enhances transfer. When the subsequent glycosyltransferase AknK and its substrate TDP-L-fucose are also added to the aglycone, the disaccharide and low levels of a fully reconstituted trisaccharide form of aclacinomycin are observed.
Subject(s)
Aclarubicin/biosynthesis , Glycosyltransferases/chemistry , Glycosyltransferases/metabolism , Hexosamines/metabolism , Macrolides/chemistry , Aclarubicin/chemistry , Aclarubicin/metabolism , Antineoplastic Agents/chemistry , Glycosylation , Kinetics , Molecular Structure , Substrate SpecificityABSTRACT
The interaction of aclacinomycin(ACR) and DNA was investigated by fluorescence spectrum, and the characteristics of the fluorescence and absorption of aclacinomycin (ACR) were studied. The results indicate that there are two situations: in the case that the concentration ratio of ACR to DNA is small, the ACR is intercalated into the stacked base pairs of DNA; in the other case that the concentration ratio of ACR to DNA is great, the interaction between ACR and DNA is complex. The binding constant of the interaction between ACR and DNA, calculated by the fluorescence titration method, is 2.7 x 10(6) mol x L(-1), and the binding site number is about 0.67 base pairs.
Subject(s)
Aclarubicin/analogs & derivatives , DNA/chemistry , Spectrometry, Fluorescence , Aclarubicin/chemistry , Animals , Cattle , Ethidium/analogs & derivatives , Ethidium/chemistry , Fluorescence , Hydrogen-Ion Concentration , Molecular StructureABSTRACT
The photodynamic response of the anthraquinone anticancer drug aclarubicin (ACL) was investigated in vitro and compared with that of mitoxantrone (MTX). Cultured immortalized rodent B14 and NIH 3T3 cells were used in the experiments as a model for cells with neoplastic phenotype. Long-term cytotoxicity and inhibition of cell proliferation assayed by the clonal growth and MTT-tetrazolium methods were estimated to compare the efficacy of aclarubicin and mitoxantrone in photosensitizing cells and their death after non-thermal exposure to monochromatic laser light. Green He-Ne (543.5 nm) or red semiconductor (670 nm) low-power laser (LPL) irradiations were applied. Different dose-responses of both cell lines to aclarubicin and mitoxantrone were found so that the cytotoxicity of MTX was considerably greater than the cytotoxicity of ACL. Phototherapy response (P < 0.0001) was observed only for B14 cells after sensitisation with aclarubicin. Under the same conditions no significant effect of red light irradiation (semiconductor 670 nm laser) on survival of both cell lines treated with mitoxantrone was found.
Subject(s)
Aclarubicin/pharmacology , Antibiotics, Antineoplastic/pharmacology , Cell Survival , Lasers , Mitoxantrone/pharmacology , Aclarubicin/chemistry , Aclarubicin/toxicity , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/toxicity , Cell Line/drug effects , Cell Line/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Mice , Mitoxantrone/chemistry , Mitoxantrone/toxicity , Molecular Structure , Neoplasms/therapy , Photochemotherapy/methods , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Photosensitizing Agents/toxicityABSTRACT
AknH is a small polyketide cyclase that catalyses the closure of the fourth carbon ring in aclacinomycin biosynthesis in Streptomyces galilaeus, converting aklanonic acid methyl ester to aklaviketone. The crystal structure analysis of this enzyme, in complex with substrate and product analogue, showed that it is closely related in fold and mechanism to the polyketide cyclase SnoaL that catalyses the corresponding reaction in the biosynthesis of nogalamycin. Similarity is also apparent at a functional level as AknH can convert nogalonic acid methyl ester, the natural substrate of SnoaL, to auraviketone in vitro and in constructs in vivo. Despite the conserved structural and mechanistic features between these enzymes, the reaction products of AknH and SnoaL are stereochemically distinct. Supplied with the same substrate, AknH yields a C9-R product, like most members of this family of polyketide cyclases, whereas the product of SnoaL has the opposite C9-S stereochemistry. A comparison of high-resolution crystal structures of the two enzymes combined with in vitro mutagenesis studies revealed two critical amino acid substitutions in the active sites, which contribute to product stereoselectivity in AknH. Replacement of residues Tyr15 and Asn51 of AknH, located in the vicinity of the main catalytic residue Asp121, by their SnoaL counter-parts phenylalanine and leucine, respectively, results in a complete loss of product stereoselectivity.
Subject(s)
Bacterial Proteins/chemistry , Isomerases/chemistry , Protein Structure, Tertiary , Streptomyces/enzymology , Aclarubicin/analogs & derivatives , Aclarubicin/biosynthesis , Aclarubicin/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Catalysis , Crystallography, X-Ray , Isomerases/genetics , Isomerases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Nogalamycin/biosynthesis , Nogalamycin/chemistry , Protein Binding , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Substrate SpecificityABSTRACT
During biosynthesis of the anthracycline antitumor agents daunomycin, adriamycin, and aclacinomycin, the polyketide-derived tetracyclic aglycone is enzymatically glycosylated at the C7-OH by dedicated glycosyltransferases (Gtfs) that transfer L-2,3,6-trideoxy-3-aminohexoses. In aclacinomycins, the first deoxyhexose is predicted to be transferred via AknS action, then subjected to further elongation to a trisaccharide by the subsequent Gtf, AknK. We report here that purified AknS has very low activity in the absence of the adjacently encoded AknT; however, at a 3:1 ratio, AknT stimulates AknS k(cat) by 40-fold up to 0.22 min(-1) for transfer of L-2-deoxyfucose (2-dF) to the aglycone aklavinone. It is likely that several other Gtfs that glycosylate polyketide aglycones also act as two-component catalytic systems. Incubations of purified AknS/AknT/AknK with two aglycones and two dTDP-2-deoxyhexoses produced previously uncharacterized anthracycline disaccharides.
Subject(s)
Anthracyclines/metabolism , Bacterial Proteins/metabolism , Glycosides/metabolism , Glycosyltransferases/metabolism , Aclarubicin/chemistry , Aclarubicin/metabolism , Anthracyclines/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Disaccharides/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors , Glycosides/chemistry , Glycosylation , Glycosyltransferases/genetics , Glycosyltransferases/isolation & purification , Naphthacenes/chemistry , Naphthacenes/metabolism , Nucleoside Diphosphate Sugars/metabolism , Protein Binding , Streptomyces/genetics , Streptomyces/metabolismABSTRACT
To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H2O2 (HRP-H2O2) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H2O2 was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H2O2. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H2O2 in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.
Subject(s)
Antibiotics, Antineoplastic/chemistry , Benzoquinones/chemistry , Creatine Kinase/antagonists & inhibitors , Doxorubicin/chemistry , Sulfhydryl Compounds/chemistry , Aclarubicin/chemistry , Daunorubicin/chemistry , Doxorubicin/analogs & derivatives , Electron Spin Resonance Spectroscopy , Epirubicin/chemistry , Free Radicals/chemistry , Glutathione/chemistry , Horseradish Peroxidase/chemistry , Hydrogen Peroxide/chemistry , Hypoxanthine/chemistry , Idarubicin/chemistry , Oxidation-Reduction , Sulfhydryl Compounds/analysis , Xanthine Oxidase/chemistryABSTRACT
Anthracyclines have been widely used as anticancer drugs against different types of human cancers. The present study evaluated the mutagenic and recombinagenic properties of two anthracycline topoisomerase II (topo II) poisons, daunorubicin (DNR) and idarubicin (IDA), as well as the related topo II catalytic inhibitor aclarubicin (ACLA), using the wing Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. The three anthracyclines were positive in this bioassay, producing mainly mitotic homologous recombination. The results for spot-size distribution and recombinagenic activity indicate that recombinational DNA damage accounts for approximately 91, 86, and 62% of DNR, IDA, and ACLA genotoxicity, respectively. Besides being a catalytic inhibitor of topo II, ACLA is also a topoisomerase I (topo I) poison. This dual topo I and II inhibitory effect, associated with its DNA-intercalating activity, could contribute to the activity of ACLA in the SMART assay.
Subject(s)
Aclarubicin/toxicity , Daunorubicin/toxicity , Idarubicin/toxicity , Mutagenesis/drug effects , Topoisomerase Inhibitors , Aclarubicin/chemistry , Animals , Biological Assay , DNA Mutational Analysis , Daunorubicin/chemistry , Dose-Response Relationship, Drug , Drosophila , Idarubicin/chemistry , Wings, Animal/anatomy & histologyABSTRACT
Aclacinomycin methylesterase (RdmC) is one of the tailoring enzymes that modify the aklavinone skeleton in the biosynthesis of anthracyclines in Streptomyces species. The crystal structures of this enzyme from Streptomyces purpurascens in complex with the product analogues 10-decarboxymethylaclacinomycin T and 10-decarboxymethylaclacinomycin A were determined to nominal resolutions of 1.45 and 1.95 A, respectively. RdmC is built up of two domains. The larger alpha/beta domain shows the common alpha/beta hydrolase fold, whereas the smaller domain is alpha-helical. The active site and substrate binding pocket are located at the interface between the two domains. Decarboxymethylaclacinomycin T and decarboxymethylaclacinomycin A bind close to the catalytic triad (Ser102-His276-Asp248) in a hydrophobic pocket, with the sugar moieties located at the surface of the enzyme. The binding of the ligands is dominated by hydrophobic interactions, and specificity appears to be controlled mainly by the shape of the binding pocket rather than through specific hydrogen bonds. Mechanistic key features consistent with the structure of complexes of RdmC with product analogues are Ser102 acting as nucleophile and transition state stabilization by an oxyanion hole formed by the backbone amides of residues Gly32 and Met103.
Subject(s)
Aclarubicin/analogs & derivatives , Aclarubicin/chemistry , Anthracyclines/chemistry , Carboxylic Ester Hydrolases/chemistry , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Catalysis , Crystallography, X-Ray , Electrons , Glycine/chemistry , Hydrogen Bonding , Methionine/chemistry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Folding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Streptomyces/enzymologyABSTRACT
The rdm genes B, C and E from Streptomyces purpurascens encode enzymes that tailor aklavinone and aclacinomycins. We report that in addition to hydroxylation of aklavinone to epsilon-rhodomycinone, RdmE (aklavinone-11-hydroxylase) hydroxylated 11-deoxy-beta-rhodomycinone to beta-rhodomycinone both in vivo and in vitro. 15-Demethoxyaklavinone and decarbomethoxyaklavinone did not serve as substrates. RdmC (aclacinomycin methyl esterase) converted aclacinomycin T (AcmT) to 15-demethoxyaclacinomycin T, which was in turn converted to 10-decarbomethoxyaclacinomycin T and then to rhodomycin B by RdmB (aclacinomycin-10-hydroxylase). RdmC and RdmB were most active on AcmT, the one-sugar derivative, with their activity decreasing by 70-90% on two- and three-sugar aclacinomycins. Aclacinomycin A competitively inhibited the AcmT modifications at C-10. The results presented here suggest that in vivo the modifications at C-10 take place principally after addition of the first sugar.