Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synthetase independent siderophore (NIS) synthetase, including SbnE in staphyloferrin B and AsbA in petrobactin. In this study, we developed a biochemical assay specific for NIS synthetases to screen for inhibitors of SbnE and AsbA against a library of marine microbial-derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces tempisquensis led to the isolation of the novel antibiotics baulamycins A (BmcA, 6) and B (BmcB, 7). BmcA and BmcB displayed in vitro activity with IC50 values of 4.8 µM and 19 µM against SbnE and 180 µM and 200 µM against AsbA, respectively. Kinetic analysis showed that the compounds function as reversible competitive enzyme inhibitors. Liquid culture studies with S. aureus, B. anthracis, E. coli and several other bacterial pathogens demonstrated the capacity of these natural products to penetrate bacterial barriers and inhibit growth of both Gram-positive and Gram-negative species. These studies provide proof-of-concept that natural product inhibitors targeting siderophore virulence factors can provide access to novel broad-spectrum antibiotics, which may serve as important leads for the development of potent anti-infective agents
Pathogenic microorganisms often have the ability to attach to a surface, building a complex matrix where they colonize to form a biofilm. This cellular superstructure can display increased resistance to antibiotics and cause serious, persistent health problems in humans. Here we describe a high-throughput in vitro screen to identify inhibitors of Acinetobacter baumannii biofilms using a library of natural product extracts derived from marine microbes. Analysis of extracts derived from Streptomyces gandocaensis results in the discovery of three peptidic metabolites (cahuitamycins A–C), with cahuitamycin C being the most effective inhibitor (IC50=14.5 μM). Biosynthesis of cahuitamycin C proceeds via a convergent biosynthetic pathway, with one of the steps apparently being catalysed by an unlinked gene encoding a 6-methylsalicylate synthase. Efforts to assess starter unit diversification through selective mutasynthesis lead to production of unnatural analogues cahuitamycins D and E of increased potency (IC50=8.4 and 10.5 μM).
C. elegans development and lifespan are controlled by the nuclear hormone receptor DAF-12, an important model for vertebrate vitamin D and liver-X receptors. Similar to its mammalian homologs, DAF-12 function is regulated by bile acid-like steroidal ligands, the dafachronic acids; however, tools for investigating their biosynthesis and function in vivo are lacking. We report a flexible synthesis for DAF-12 ligands and masked ligand derivatives that enable precise temporal control of DAF-12 function. For ligand masking, we introduce photocleavable amides of 5-methoxy-N-methyl-2-nitroaniline (MMNA). MMNA-masked ligands are bioavailable and after incorporation into the worm can be used to trigger expression of DAF-12 target genes and initiate development from dauer larvae to adults by brief, innocuous UV-irradiation. In-vivo release of DAF-12 ligands and other small-molecule signals using MMNA-based probes will enable functional studies with precise spatial and temporal resolution.
Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P 2) is critical for synaptic vesicle docking and fusion and generation of the second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. PI-4,5-P 2 can be generated by two families of kinases: type 1 phosphatidylinositol-4-phosphate 5-kinases, encoded by PIP5K1A, PIP5K1B and PIP5K1C, and type 2 phosphatidylinositol-5-phosphate 4-kinases, encoded by PIP4K2A, PIP4K2B, and PIP4K2C. While the roles of the type 1 enzymes in brain function have been extensively studied, the roles of the type 2 enzymes are poorly understood. Using selective antibodies validated by genetic deletion of pip4k2a or pip4k2b in mouse brain, we characterized the location of the enzymes, PI5P4Kα and PI5P4Kβ, encoded by these genes. In mice, we demonstrate that PI5P4Kα is expressed in adulthood, whereas PI5P4Kβ is expressed early in development. PI5P4Kα localizes to white matter tracts, especially the corpus callosum, and at a low level in neurons, while PI5P4Kβ is expressed in neuronal populations, especially hippocampus and cortex. Dual labeling studies demonstrate that PI5P4Kα co-localizes with the oligodendrocyte marker, Olig2, whereas PI5P4Kβ co-localizes with the neuronal marker, NeuN. Ultrastructural analysis demonstrates that both kinases are contained in axon terminals and dendritic spines adjacent to the synaptic membrane, which support a potential role in synaptic transmission. Immunoperoxidase analysis of macaque and human brain tissue demonstrate a conserved pattern for PI5P4Kα and PI5P4Kβ. These results highlight the diverse cell-autonomous expression of PI5P4Kα and PI5P4Kβ and support further exploration into their role in synaptic function in the brain.
Background Insulin feedback is a critical mechanism responsible for poor clinical efficacy of PI3K inhibition in cancer, and hyperglycemia is an independent factor associated with poor prognosis in glioblastoma. We investigated combination anti-hyperglycemic therapy in a mouse model of glioblastoma and evaluated the association of glycemic control in clinical trial data from patients with glioblastoma. Methods The effect of the anti-hyperglycemic regimens metformin and the ketogenic diet were evaluated in combination with PI3K inhibition in patient-derived glioblastoma cells and an orthotopic glioblastoma mouse model. Insulin feedback and the immune microenvironment were retrospectively evaluated in blood and tumor tissue from a Phase 2 clinical trial of buparlisib in patients with recurrent glioblastoma. Results We found that PI3K inhibition induces hyperglycemia and hyperinsulinemia in mice and that combining metformin with PI3K inhibition improves treatment efficacy in an orthotopic glioblastoma xenograft model. Through examination of clinical trial data, we found that hyperglycemia was an independent factor associated with poor progression-free survival in patients with glioblastoma. We also found that PI3K inhibition increased insulin receptor activation and T cell and microglia abundance in tumor tissue from these patients. Conclusion Reducing insulin feedback improves the efficacy of PI3K inhibition in glioblastoma in mice, and hyperglycemia worsens progression-free survival in patients with glioblastoma treated with PI3K inhibition. These findings indicate that hyperglycemia is a critical resistance mechanism associated with PI3K inhibition in glioblastoma and that anti-hyperglycemic therapy may enhance PI3K inhibitor efficacy in glioblastoma patients.
Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P 2 ) is critical for synaptic vesicle docking and fusion and generation of the second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. PI-4,5-P 2 can be generated by two families of kinases: type 1 phosphatidylinositol-4-phosphate 5kinases, encoded by PIP5K1A, PIP5K1B and PIP5K1C, and type 2 phosphatidylinositol-5phosphate 4-kinases, encoded by PIP4K2A, PIP4K2B, and PIP4K2C. While the roles of the type 1 enzymes in brain function have been extensively studied, the roles of the type 2 enzymes are poorly understood. Using selective antibodies validated by genetic deletion of pip4k2a or pip4k2b in mouse brain, we characterized the location of the enzymes, PI5P4Kα and PI5P4Kß, encoded by these genes. In mice, we demonstrate that PI5P4Kα is expressed in adulthood, whereas PI5P4Kß is expressed early in development. PI5P4Kα localizes to white matter tracts, especially the corpus callosum, and at a low level in neurons, while PI5P4Kß is expressed in neuronal populations, especially hippocampus and cortex. Dual labeling studies demonstrate that PI5P4Kα co-localizes with the oligodendrocyte marker, Olig2, whereas PI5P4Kß co-localizes with the neuronal marker, NeuN. Immunohistochemical subcellular distribution studies demonstrate that PI5P4Kα and PI5P4Kß are expressed in the early endosome system. Ultrastructural analysis demonstrates that both kinases are contained in axon terminals and dendritic spines adjacent to the synaptic membrane, which support a potential role in synaptic transmission. Immunohistochemical analysis of macaque and human brain tissue demonstrate a conserved pattern for PI5P4Kα and PI5P4Kß. These results highlight the diverse cell-autonomous expression of PI5P4Kα and PI5P4Kß and support further exploration into their role in synaptic function in the brain.
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