asukamycin and manumycin

Molecular glues are an intriguing therapeutic modality that harness small molecules to induce interactions between proteins that typically do not interact. However, such molecules are rare and have been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention. We postulated that natural products bearing one or more electrophilic sites may be an unexplored source of new molecular glues, potentially acting through multicovalent attachment. Using chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells, and engage in molecular glue interactions with the neosubstrate tumor-suppressor TP53, leading to p53 transcriptional activation and cell death. Our results reveal an anticancer mechanism of this natural product family, and highlight the potential for combining chemoproteomics and multicovalent natural products for the discovery of new molecular glues.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cross-Linking Reagents; Drug Discovery; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Molecular Conformation; Molecular Structure; Polyenes; Polyketides; Polyunsaturated Alkamides; Static Electricity; Structure-Activity Relationship; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Many natural products contain epoxyquinone pharmacophore with unknown biosynthetic mechanisms. Recent genetic analysis of the asukamycin biosynthetic gene cluster proposed enzyme candidates related to epoxyquinone formation for manumycin-type metabolites. Our biochemical studies reveal that 3-amino-4-hydroxyl benzoic acid (3,4-AHBA) precursor is activated and loaded on aryl carrier protein (AsuC12) by ATP-dependent adenylase (AsuA2). AsuE1 and AsuE3, both single-component flavin-dependent monooxygenases, catalyze the exquisite regio- and enantiospecific postpolyketide synthase (PKS) assembly oxygenations. AsuE1 installs a hydroxyl group on the 3,4-AHB ring to form a 4-hydroxyquinone moiety, which is epoxidized by AsuE3 to yield the epoxyquinone functionality. Despite being a single-component monooxygenase, AsuE1 activity is elicited by AsuE2, a pathway-specific flavin reductase. We further demonstrate that the epoxyquinone moiety is critical for anti-MRSA activity by analyzing the bioactivity of various manumycin-type metabolites produced through mutasynthesis.

Topics: Benzoic Acid; Benzoquinones; Dinitrocresols; Mixed Function Oxygenases; Multigene Family; Oxidoreductases; Oxygen; Polyenes; Polyunsaturated Alkamides; Streptomyces; Substrate Specificity

Asukamycin, a member of the manumycin family of antibiotics, exhibits strong antibacterial, antifungal, and antineoplastic activities. However, its production in the wild-type strain of Streptomyces nodosus subsp. asukaensis ATCC 29757 is relatively low. Recently, the biosynthetic gene cluster for asukamycin was identified in the producing organism, and among the 36 genes reported in the cluster, six (asuR1-asuR6) were proposed to encode proteins that function as transcriptional regulators. In order to investigate their involvement in asukamycin biosynthesis and to engineer mutant strains of S. nodosus that are able to produce large amounts of asukamycin, we carried out in vivo gene inactivation, transcriptional analysis of the biosynthetic genes in the mutants, and gene duplication in the producing strain of S. nodosus. The results show that two of the putative regulatory genes (asuR1 and asuR5) are critical for asukamycin biosynthesis, whereas others regulate the pathway at various levels. Overexpression of a gene cassette harboring asuR1, asuR2, asuR3, and asuR4 in S. nodosus resulted in changes in morphology of the producing strain and an approximately 14-fold increase of asukamycin production. However, overexpression of the individual genes did not give a comparable cumulative level of asukamycin production, suggesting that some, if not all, of the gene products act synergistically to regulate the biosynthesis of this antibiotic.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Gene Expression Regulation, Bacterial; Genetic Engineering; Multigene Family; Polyenes; Polyunsaturated Alkamides; Streptomyces; Transcription, Genetic; Up-Regulation

Production of minor asukamycin congeners and its new derivatives by combination of targeted genetic manipulations with specific precursor feeding in the producer of asukamycin, Streptomyces nodosus ssp. asukaensis.. Structural variations of manumycins lie only in the diverse initiation of the 'upper' polyketide chain. Inactivation of the gene involved in the biosynthesis of cyclohexanecarboxylic acid (CHC) turned off the production of asukamycin in the mutant strain and allowed an increased production of other manumycins with the branched end of the upper chain. The ratio of produced metabolites was further affected by specific precursor feeding. Precursor-directed biosynthesis of a new asukamycin analogue (asukamycin I, 28%) with linear initiation of the upper chain was achieved by feeding norleucine to the mutant strain. Another asukamycin analogue with the unbranched upper chain (asukamycin H, 14%) was formed by the CHC-deficient strain expressing a heterologous gene putatively involved in the formation of the n-butyryl-CoA starter unit of manumycin A.. Combination of the described techniques proved to be an efficient tool for the biosynthesis of minor or novel manumycins.. Production of two novel asukamycin derivatives, asukamycins H and I, was achieved. Variations appeared in the upper polyketide chain, the major determinant of enzyme-inhibitory features of manumycins, affecting their cancerostatic or anti-inflammatory features.

Topics: Acyl Coenzyme A; Amino Acids; Anti-Bacterial Agents; Culture Media; Cyclohexanecarboxylic Acids; Genetic Engineering; Mutagenesis, Insertional; Mutation; Polyenes; Polyunsaturated Alkamides; Streptomyces

Asukamycin, a manumycin-type metabolite, was isolated by a rapid and easily scalable purification scheme. Thus far, studies on the biological activity of asukamycin have been limited to its role as an antibacterial and antifungal agent. By using five different tumor cell lines we demonstrate antineoplastic activity of asukamycin. It inhibited cell growth at concentrations similar to other members of the manumycin family (IC50 1-5 microM). Cytotoxicity of asukamycin was accompanied by activation of caspases 8 and 3 and was diminished by SB 202190, a specific p38 mitogen-activated protein kinase (MAPK) inhibitor. These data, in combination with earlier observations showing its low in vivo toxicity, indicate that further studies on the potential antitumor activity of asukamycin are warranted.

Topics: Antineoplastic Agents; Caspase 3; Caspase 8; Cell Death; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme Activation; Humans; Magnetic Resonance Spectroscopy; Polyenes; Polyunsaturated Alkamides; Streptomyces

Asukamycin (2), a metabolite of Streptomyces nodosus ssp. asukaensis ATCC 29757 and a member of the manumycin family of antibiotics, is assembled from three components, an "upper" polyketide chain initiated by cyclohexanecarboxylic acid, a "lower" polyketide chain initiated by the novel starter unit, 3-amino-4-hydroxybenzoic acid (3,4-AHBA), and a cyclized 5-aminolevulinic acid moiety, 2-amino-3-hydroxycyclopent-2-enone (C(5)N unit). To shed light on the order in which these components are assembled, we synthesized in labeled form various potential intermediates and evaluated their incorporation into 2. The assembly of the molecular framework of 2 from 3,4-AHBA and cyclohexanecarboxylic acid apparently does not involve free, unactivated intermediates. However, protoasukamycin (12), the total synthesis of which is reported, was efficiently converted into 2, demonstrating that the modification of the aromatic ring to the epoxyquinol structure is the terminal step in the biosynthesis. The results suggest that the two polyketide chains are synthesized separately and that the "upper" chain must be connected to the "lower" polyketide chain before the C(5)N unit.

Topics: Anti-Bacterial Agents; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Polyenes; Polyunsaturated Alkamides; Streptomyces

Five new type II manumycins, containing the hydroxyquinol mC7N unit, asukamycins A-II, B-II, C-II, D-II, E-II, were discovered in cultures of Streptomyces nodosus ssp. asukaensis. The biosynthetic origin of the type II manumycins from the type I compounds, containing an epoxyquinol mC7N unit, was deduced from the time course of production and proven by preparing [7'-13C]asukamycin A and demonstrating its incorporation into asukamycin A-II.

Topics: Anti-Bacterial Agents; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Fermentation; Magnetic Resonance Spectroscopy; Mass Spectrometry; Polyenes; Polyunsaturated Alkamides; Streptomyces