ABSTRACT
The isolation, structure elucidation, and synthesis of antalid (1), a novel secondary metabolite from Polyangium sp., is described herein. The structure elucidation of 1 was performed with the aid of mass spectrometry, high field NMR experiments, and crystal structure analysis. The absolute configuration of antalid was confirmed through the Mosher ester method and ultimately by total synthesis. In addition, the biosynthetic origin of this hybrid PKS-NRPS natural product was unraveled by the in silico analysis of its biosynthetic gene cluster.
ABSTRACT
BACKGROUND: The nuclear export of unspliced and partially spliced HIV-1 mRNA is mediated by the recognition of a leucine-rich nuclear export signal (NES) in the HIV Rev protein by the host protein CRM1/Exportin1. This makes the CRM1-Rev complex an attractive target for the development of new antiviral drugs. Here we tested the anti-HIV efficacy of ratjadone A, a CRM1 inhibitor derived from myxobacteria. RESULTS: Ratjadone A inhibits HIV infection in vitro in a dose-dependent manner with EC50 values at the nanomolar range. The inhibitory effect of ratjadone A occurs around 12 hours post-infection and is specific for the Rev/CRM1-mediated nuclear export pathway. By using a drug affinity responsive target stability (DARTS) assay we could demonstrate that ratjadone A interferes with the formation of the CRM1-Rev-NES complex by binding to CRM1 but not to Rev. CONCLUSION: Ratjadone A exhibits strong anti-HIV activity but low selectivity due to toxic effects. Although this limits its potential use as a therapeutic drug, further studies with derivatives of ratjadones might help to overcome these difficulties in the future.
Subject(s)
HIV Infections/prevention & control , HIV-1/metabolism , Karyopherins/metabolism , Myxococcales/metabolism , Pyrones/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , rev Gene Products, Human Immunodeficiency Virus/metabolism , Active Transport, Cell Nucleus/drug effects , Antiviral Agents/pharmacology , Cell Line , HIV Core Protein p24/metabolism , Humans , Karyopherins/antagonists & inhibitors , Protein Binding , Pyrones/chemistry , Pyrones/pharmacology , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , rev Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , Exportin 1 ProteinSubject(s)
Cytostatic Agents , Macrocyclic Compounds , Macrolides , Myxococcales/chemistry , Polyketides , Animals , Cell Line , Cell Proliferation/drug effects , Crystallography, X-Ray , Cytostatic Agents/chemistry , Cytostatic Agents/pharmacology , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/isolation & purification , Macrocyclic Compounds/pharmacology , Macrolides/chemistry , Macrolides/isolation & purification , Macrolides/pharmacology , Models, Molecular , Molecular Conformation , Polyketides/chemistry , Polyketides/isolation & purification , Polyketides/pharmacologyABSTRACT
Topoisomerase IIalpha (topo IIalpha) is exported from the nucleus of human myeloma cells by a CRM1-dependent mechanism at cellular densities similar to those found in patient bone marrow. When topo IIalpha is trafficked to the cytoplasm, it is not in contact with the DNA; thus, topo IIalpha inhibitors are unable to induce DNA-cleavable complexes and cell death. Using a CRM1 inhibitor or a CRM1-specific small interfering RNA (siRNA), we were able to block nuclear export of topo IIalpha as shown by immunofluorescence microscopy. Human myeloma cell lines and patient myeloma cells isolated from bone marrow were treated with a CRM1 inhibitor or CRM1-specific siRNA and exposed to doxorubicin or etoposide at high cell densities. CRM1-treated cell lines or myeloma patient cells were 4-fold more sensitive to topo II poisons as determined by an activated caspase assay. Normal cells were not significantly affected by CRM1-topo II inhibitor combination treatment. Cell death was correlated with increased DNA double-strand breaks as shown by the comet assay. Band depletion assays of CRM1 inhibitor-exposed myeloma cells showed increased topo IIalpha covalently bound to DNA. Topo IIalpha knockdown by a topo IIalpha-specific siRNA abrogated the CRM1-topo II therapy synergistic effect. These results suggest that blocking topo IIalpha nuclear export sensitizes myeloma cells to topo II inhibitors. This method of sensitizing myeloma cells suggests a new therapeutic approach to multiple myeloma.
Subject(s)
Bone Marrow Neoplasms/drug therapy , DNA-Binding Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Karyopherins/antagonists & inhibitors , Multiple Myeloma/drug therapy , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Topoisomerase II Inhibitors , Antibiotics, Antineoplastic/pharmacology , Antigens, Neoplasm/metabolism , Bone Marrow Neoplasms/enzymology , Bone Marrow Neoplasms/pathology , Cell Count , Cell Death/drug effects , Cell Line, Tumor , DNA Topoisomerases, Type II/metabolism , DNA, Neoplasm/metabolism , DNA-Binding Proteins/metabolism , Doxorubicin/pharmacology , Humans , Karyopherins/genetics , Karyopherins/metabolism , Multiple Myeloma/enzymology , Multiple Myeloma/pathology , Protein Binding , Protein Transport/drug effects , Pyrones/pharmacology , RNA, Small Interfering , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Exportin 1 ProteinABSTRACT
In addition to previously isolated ratjadone A we describe three new members of this family, ratjadones B, C, and D, from another strain of the myxobacterium Sorangium cellulosum. We have investigated the properties of these ratjadones with respect to their activity on mammalian cell lines. We found IC(50) values in the picomolar range and a significant increase in the size of nuclei. A further examination showed that they inhibit the export of the leucine-rich nuclear export signal (LR-NES) containing proteins in different cell lines. Ratjadones are able to inhibit the formation of the nuclear export complex composed of the CRM1, RanGTP, and the cargo protein, as shown by two different in vitro assays. Finally, the binding of ratjadone C to CRM1 was demonstrated. These ratjadone activities are in the same concentration range as described for the polyketide leptomycin B (LMB) from Streptomyces sp. Like LMB, it seems that the ratjadones covalently bind to CRM1, inhibit cargo protein binding via LR-NES, and thereby block nuclear export. Thus, the ratjadones represent a new class of natural compounds which inhibit proliferation in eukaryotes by blocking nuclear export.
