RESUMO
Aphidicolin, a potent DNA polymerase α inhibitor, has been explored in clinical trials for the treatment of cancer. So far, about 300 modified aphidicolins have been discovered. However, none have shown a stronger effect. Herein, we report 71 new (aphidicolins A1-A71, 1-71) and eight known (72-79) aphidicolin congeners from Botryotinia fuckeliana MCCC 3A00494, a fungus isolated from the western Pacific Ocean (-5572 m). The structures of 1-71 were determined through extensive spectroscopic analysis, X-ray crystallography, chemical derivatization, modified Mosher's method, and the ECD exciton chirality method. Compounds 54-57 and 58-64 are novel 6/6/5/6/5 pentacyclic aphidicolins featuring tetrahydrofuran and dihydrofuran rings, respectively, while compounds 65-71 are rare noraphidicolins. Aphidicolin A8 (8) significantly induced apoptosis in T24 (IC50 = 2.5 µM) and HL-60 (IC50 = 6.1 µM) cancer cells by causing DNA damage. By docking its structure to the human DNA polymerase α binding pocket, 8 was found to form tight intermolecular contacts, elaborating aphidicolin A8 as a potently cytotoxic lead compound.
Assuntos
Afidicolina/química , Botrytis/química , Biologia Marinha , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Cristalografia por Raios X , Estrutura Molecular , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
G-quadruplex DNA has been viewed as a prospective anti-cancer target owing to its potential biological relevance. Real-time monitoring of DNA G-quadruplex structures in living cells can provide valuable insights into the relationship between G-quadruplex formation and its cellular consequences. However, the probes capable of detecting DNA G-quadruplexes in living cells are still very limited. Herein, we reported a new fluorescent probe, IMT, for real-time visualization of DNA G-quadruplex structures in living cells. Using IMT as a fluorescent indicator, the quantity changes of DNA G-quadruplex at different points in time during continuous cellular progression responding to Aphidicolin and Hydroxyurea treatment have been directly visualized. Our data demonstrate that IMT will be a valuable tool for exploring DNA G-quadruplexes in live cells. Further application of IMT in fluorescence imaging may reveal more information on the roles of DNA G-quadruplexes in biological systems.
Assuntos
DNA/química , Corantes Fluorescentes/química , Quadruplex G/efeitos dos fármacos , Afidicolina/química , Linhagem Celular Tumoral , Células HeLa , Humanos , Hidroxiureia/química , Microscopia de Fluorescência , Espectrometria de FluorescênciaRESUMO
The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.
Assuntos
Afidicolina/química , Aspergillus oryzae/genética , Retículo Endoplasmático/química , Farnesiltranstransferase/química , Citoplasma/química , Citoplasma/enzimologia , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Farnesiltranstransferase/genética , Fosfatos de Poli-Isoprenil/químicaRESUMO
Inflatin G (1), a new aphidicolin analogue, together with seven known compounds inflatin A (2), inflatin B (3), aphidicolin (4), aphidicolin-17-monoacetate (5), gulypyrone A (6), pyridoxatin rotamers A (7) and B (8), were isolated from the ascomycete fungus Tolypocladium inflatum. Their structures were determined through NMR analyses and the circular dichroism data of the in situ formed [Rh2(OCOCF3)4] complexes. Compounds 1, 4, 5, 7, and 8 showed modest cytotoxicity against four human cancer cell lines A549, CNE1-MP1, A375, and MCF-7.
Assuntos
Antineoplásicos/isolamento & purificação , Afidicolina/análogos & derivados , Afidicolina/isolamento & purificação , Hypocreales/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Afidicolina/química , Afidicolina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , HumanosRESUMO
Chagas disease continues to be a difficult disease to eradicate, largely because of the widespread populations it affects as well as the highly toxic effects of current therapies. Thus, the exploration of innovative scaffolds, ideally with distinct mechanisms of action, is urgently needed. The natural product aphidicolin and its effects on cell cycle division have been widely studied; it is a potent inhibitor of parasitic cells. In the present study, we report for the first time the semisynthesis of a series of aphidicolin derivatives, their unique structural features, and demonstration of their activity against Trypanosoma cruzi cells. Two demonstrated high potency and selectivity against parasitic amastigote cells, and thus show promise as new leads for Chagas disease treatment.
Assuntos
Afidicolina/química , Afidicolina/farmacologia , Tripanossomicidas/síntese química , Trypanosoma cruzi/efeitos dos fármacos , Afidicolina/uso terapêutico , Doença de Chagas/tratamento farmacológico , Humanos , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêuticoRESUMO
Cell synchronization techniques have been used for the studies of mechanisms involved in cell cycle regulation. Synchronization involves the enrichment of subpopulations of cells in specific stages of the cell cycle. These subpopulations are then used to study regulatory mechanisms of the cell cycle such as DNA synthesis, gene expression, protein synthesis, protein phosphorylation, protein degradation, and development of new drugs (e.g., CDK inhibitors). Here, we describe several protocols for synchronization of cells from different phases of the cell cycle. We also describe protocols for determining cell viability and mitotic index and for validating the synchrony of the cells by flow cytometry.
Assuntos
Técnicas de Cultura de Células/métodos , Proteínas Inibidoras de Quinase Dependente de Ciclina/química , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Animais , Afidicolina/química , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , DNA/química , Replicação do DNA , Citometria de Fluxo , Células HeLa , Humanos , Camundongos , Mitose , Índice Mitótico , Células NIH 3T3 , Nocodazol/química , Timidina/química , Fatores de Tempo , Azul Tripano/químicaRESUMO
Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.
Assuntos
Afidicolina/química , DNA Polimerase I/química , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores da Síntese de Ácido Nucleico/química , Afidicolina/análogos & derivados , Domínio Catalítico , Guanina/química , Humanos , Modelos Moleculares , RNA/químicaRESUMO
Six new secondary metabolites including two aphidicolin analogues, inflatins A (1) and B (2), and four chlamydosporol derivatives, inflatins C-F (3-6), have been isolated from the crude extract of Tolypocladium inflatum. The structures of 1-6 were determined mainly by NMR experiments, and 4 and 5 were further confirmed by X-ray crystallography. The absolute configurations of C-16 in 1 and C-5 in 3 were deduced via the circular dichroism data of the in situ formed [Rh2(OCOCF3)4] complexes, whereas that of 4 was assigned by X-ray crystallography using Cu Kα radiation. Compounds 1 and 2 showed modest cytotoxicity against a panel of eight human tumor cell lines.
Assuntos
Afidicolina , Ascomicetos/química , Pironas/isolamento & purificação , Afidicolina/análogos & derivados , Afidicolina/química , Afidicolina/isolamento & purificação , Afidicolina/farmacologia , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Pironas/química , Pironas/farmacologiaRESUMO
A new metabolite, 3,16-diketoaphidicolan (1), was isolated together with four known compounds: aphidicolin (2), 17-acetyl-aphidicolin (3), (+)-eupenoxide (4), and phomoxanthone A (5) from the endophytic fungus Phoma sp. The structure of the new compound 1 was determined by spectroscopic methods (mainly extensive 1D and 2D NMR experiments and by mass spectral measurements) and confirmed by X-ray crystallography. Its absolute configuration was assigned by means of the solid-state CD/TDDFT approach comparing the solid-state CD spectrum with the TDDFT-calculated one on the X-ray geometry.
Assuntos
Afidicolina/análogos & derivados , Ascomicetos/metabolismo , Plantas/microbiologia , Afidicolina/química , Dicroísmo Circular , Cristalografia por Raios X , Espectroscopia de Ressonância MagnéticaRESUMO
Recently, the three-dimensional structure of the active site of human DNA polymerase alpha (pol alpha) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol alpha in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , DNA Polimerase I/antagonistas & inibidores , Ceratose Actínica/tratamento farmacológico , Modelos Químicos , Modelos Moleculares , Inibidores da Síntese de Ácido Nucleico , Neoplasias Cutâneas/tratamento farmacológico , Afidicolina/química , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/enzimologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA Polimerase I/química , DNA Polimerase I/metabolismo , Humanos , Queratinócitos , Ceratose Actínica/enzimologia , Necrose , Inibidores da Síntese de Ácido Nucleico/síntese química , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas de Transporte de Nucleotídeos/genética , Proteínas de Transporte de Nucleotídeos/metabolismo , Ligação Proteica , Purinas/química , Neoplasias Cutâneas/enzimologia , Timidina/químicaRESUMO
Synthesis of a tricyclic enone (B/C/D ring system), a common key precursor for the aphidicolane- and stemodane-type diterpene, is described. The key reaction for the construction of the quaternary carbon center is allylation of epoxide at the more substituted carbon with an organotitanium reagent. Asymmetric reduction with DIP-Cl followed by stereoselective cyclization of spirocyclic ketone and the functional group modification gave the desired tricyclic enone in good yield.
Assuntos
Acetais/química , Afidicolina/química , Diterpenos/química , Pironas/química , Triterpenos/química , Estrutura MolecularRESUMO
We have recently described the selection of rapidly dividing Arabidopsis cell suspension cultures MM1 and MM2d that provide a powerful platform for plant cell-cycle research. Here we provide detailed protocols and procedures to achieve high levels of synchronization, either by starving the cell cultures of sucrose or by applying the toxin aphidicolin. Cell-cycle activity during cell-cycle reentry (starvation-induced synchrony) or further cell-cycle progression (aphidicolin-induced synchrony) can be conveniently followed by using various validation procedures, such as determination of labeling index and metaphase/anaphase index or flow cytometry. We also describe a procedure that allows clonal transformed cell-suspension lines to be produced using Agrobacterium-mediated transformation, and an optimized and straightforward method for the cryopreservation and recovery of both parental and transformed lines which is applicable both to Arabidopsis and the tobacco BY2 cell lines. Cell-cycle synchronization capacity of the parental lines is maintained after both transformation and recovery from cryopreservation. The techniques described here require no specialized equipment and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures. The ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for future high-throughput cell-cycle analysis.
Assuntos
Arabidopsis/citologia , Botânica/métodos , Técnicas de Cultura de Células/métodos , Antimetabólitos/farmacologia , Afidicolina/química , Bromodesoxiuridina/farmacologia , Ciclo Celular , Células Cultivadas , Criopreservação , Plantas Geneticamente Modificadas , Rhizobium/metabolismo , Sacarose/metabolismo , Fatores de Tempo , TransgenesRESUMO
In nature there are several terpenoids with a characteristic gamma-dioxygenated system on the A ring, and many of them show interesting pharmacological properties. We have developed a novel strategy for the synthesis of these terpenoids involving three stages: (a) the selective epoxidation of commercial polyenes, (b) titanium(III)-catalyzed cyclization of the epoxypolyprenes thus obtained, and (c) Pd-mediated remote functionalization of the equatorial methyl group attached at C-4 on ring A of the cyclic terpenoid thus formed. This strategy has proved to be useful for the synthesis of the natural labdane rostratone (1) and related terpenoids, as well as for advanced synthetic approaches toward the pharmacologically active products aphidicolin (2) and pyripyropene A (3).
Assuntos
Afidicolina/síntese química , Diterpenos/síntese química , Oxigênio/química , Paládio/química , Piridinas/síntese química , Sesquiterpenos/síntese química , Terpenos/síntese química , Titânio/química , Antivirais/síntese química , Antivirais/química , Afidicolina/química , Diterpenos/química , Estrutura Molecular , Piridinas/química , Sesquiterpenos/químicaRESUMO
Aphidicolin, a tetracyclic diterpene antibiotic produced by Cephalosporium aphidicola, is under investigation as anti-cancer drug. Because of its poor solubility in water, it cannot be administered directly in vivo. Systemic application of aphidicolin glycinate or aphidicolin gamma-cyclodextrin complexes resulted in tumour growth inhibition but not in cures. To improve the pharmacokinetics, a liposomal preparation of aphidicolin was developed and tested in neuroblastoma-bearing (UKF-NB-3) mice. The loading capacity of these liposomes was limited. Therefore, 4.5 mg aphidicolin/kg body weight was the maximum aphidicolin dose that could be applied as liposomal preparation in this approach. Comparison of effects on tumour growth exhibited by aphidicolin liposomes (4.5 mg aphidicolin/kg) given for 15 consecutive days to those of gamma-cyclodextrin inclusion complexes (15 mg aphidicolin/kg) revealed comparable tumour growth inhibition, although aphidicolin concentrations were approximately 3-fold lower. This shows that liposomal encapsulation is a promising strategy for the improvement of systemic anti-cancer activity of aphidicolin.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Afidicolina/administração & dosagem , Neuroblastoma/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/uso terapêutico , Afidicolina/química , Afidicolina/uso terapêutico , Portadores de Fármacos , Feminino , Humanos , Lipossomos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
A total synthesis of (+/-)-stemodinone, a tetracyclic stemodane diterpene, from the known tricyclic methyl olefin 11 is described. The key steps involve an efficient ring-exchange reaction and palladium(0)-catalyzed lactone migration. The ring-exchange strategy for controlling the stereochemistry was based on an initial Diels-Alder reaction to form a new ring followed by cleavage of the original ring. Cleavage of the original ring of the Diels-Alder adduct 9 was achieved by an initial regio- and chemoselective Baeyer-Villiger oxidation followed by the Pd(0)-catalyzed lactone-migration reaction reported by us.
Assuntos
Antivirais/síntese química , Diterpenos/síntese química , Plantas Medicinais/química , Antivirais/química , Afidicolina/química , Diterpenos/química , EstereoisomerismoRESUMO
[reaction: see text] Aphidicolin unnatural derivative (2) was synthesized using a new tandem transannular Diels-Alder/aldol methodology. The 8-epi-aphidicolane skeleton is constructed in a highly diastereoselective manner and converted into (11R)-(-)-8-epi-11-hydroxyaphidicolin (2). An efficient method for the difficult C16 funtionalization is presented.
Assuntos
Afidicolina/análogos & derivados , Afidicolina/química , Acremonium/química , Antineoplásicos/química , Antivirais/química , Diterpenos/química , Inibidores Enzimáticos/química , EstereoisomerismoRESUMO
A series of labdans and their derivatives have been identified as novel potential antileishmanial drugs using an in vitro test system against extracellular promastigotes and intracellular amastigotes of Leishmania donovani in murine macrophages (Kayser, O., Kiderlen, A.F., 1998. In vitro activity of leishmanicidal labdanes and related compounds. Proceedings of the Ninth International Congress of Parasitology, Monduzi Editore, Bologna, 925-929). Of these compounds, aphidicolin, a tetradecanhydro-3,9-dihydroxy-4,11b-dimethyl-8, 11a-methano-11aH-cyclo-hepta[a]naphthalin-4,9-dimethanol+ ++ (Fig. 1), was shown to be highly active at concentrations in the microgram range (EC(50) = 0.16 microg/ml). To improve drug targeting effects aphidicolin was formulated as nanosuspension and retested for its enhanced activity (EC(50) = 0.003 microg/ml).
Assuntos
Afidicolina/farmacologia , Leishmania/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Suspensões/farmacologia , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Afidicolina/química , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/parasitologia , Dimetil Sulfóxido/química , Dimetil Sulfóxido/farmacologia , Sistemas de Liberação de Medicamentos , Concentração Inibidora 50 , Leishmania/crescimento & desenvolvimento , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Polissorbatos/química , Polissorbatos/farmacologia , Suspensões/químicaRESUMO
A variety of isosteres of the DNA polymerase inhibitor aphidicolin were synthesized as potential antiherpes agents. Modeling studies indicated that the bicyclooctane C, D rings of aphidicolin could be replaced by an aromatic moiety while maintaining the spatial arrangement of the hydroxyl group equivalent to the essential C18 hydroxyl group of aphidicolin. Of the racemic isosteres synthesized only 13, the compound with the greatest structural similarity to aphidicolin, showed any significant antiviral activity in primary assays. An enantioselective synthesis of the compound was carried out and the 4aS isomer 36 was shown to account for the observed antiviral activity noted against herpes simplex virus 1 and human cytomegalovirus.