RESUMEN
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.
Asunto(s)
Afidicolina/química , Botrytis/química , Biología Marina , Espectroscopía de Resonancia Magnética con Carbono-13 , Cristalografía por Rayos X , Estructura Molecular , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
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.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , G-Cuádruplex/efectos de los fármacos , Afidicolina/química , Línea Celular Tumoral , Células HeLa , Humanos , Hidroxiurea/química , Microscopía Fluorescente , Espectrometría de FluorescenciaRESUMEN
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.
Asunto(s)
Afidicolina/química , Aspergillus oryzae/genética , Retículo Endoplásmico/química , Farnesiltransferasa/química , Citoplasma/química , Citoplasma/enzimología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Farnesiltransferasa/genética , Fosfatos de Poliisoprenilo/químicaRESUMEN
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.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Afidicolina/análogos & derivados , Afidicolina/aislamiento & purificación , Hypocreales/química , Antineoplásicos/química , Antineoplásicos/farmacología , Afidicolina/química , Afidicolina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , HumanosRESUMEN
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.
Asunto(s)
Afidicolina/química , Afidicolina/farmacología , Tripanocidas/síntesis química , Trypanosoma cruzi/efectos de los fármacos , Afidicolina/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Humanos , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Tripanocidas/farmacología , Tripanocidas/uso terapéuticoRESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Animales , Afidicolina/química , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , ADN/química , Replicación del ADN , Citometría de Flujo , Células HeLa , Humanos , Ratones , Mitosis , Índice Mitótico , Células 3T3 NIH , Nocodazol/química , Timidina/química , Factores de Tiempo , Azul de Tripano/químicaRESUMEN
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.
Asunto(s)
Afidicolina/química , ADN Polimerasa I/química , Replicación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores de la Síntesis del Ácido Nucleico/química , Afidicolina/análogos & derivados , Dominio Catalítico , Guanina/química , Humanos , Modelos Moleculares , ARN/químicaRESUMEN
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.
Asunto(s)
Afidicolina , Ascomicetos/química , Pironas/aislamiento & purificación , Afidicolina/análogos & derivados , Afidicolina/química , Afidicolina/aislamiento & purificación , Afidicolina/farmacología , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Pironas/química , Pironas/farmacologíaRESUMEN
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.
Asunto(s)
Afidicolina/análogos & derivados , Ascomicetos/metabolismo , Plantas/microbiología , Afidicolina/química , Dicroismo Circular , Cristalografía por Rayos X , Espectroscopía de Resonancia MagnéticaRESUMEN
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.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , ADN Polimerasa I/antagonistas & inhibidores , Queratosis Actínica/tratamiento farmacológico , Modelos Químicos , Modelos Moleculares , Inhibidores de la Síntesis del Ácido Nucleico , Neoplasias Cutáneas/tratamiento farmacológico , Afidicolina/química , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , Humanos , Queratinocitos , Queratosis Actínica/enzimología , Necrosis , Inhibidores de la Síntesis del Ácido Nucleico/síntesis química , Inhibidores de la Síntesis del Ácido Nucleico/química , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteínas de Transporte de Nucleótidos/genética , Proteínas de Transporte de Nucleótidos/metabolismo , Unión Proteica , Purinas/química , Neoplasias Cutáneas/enzimología , Timidina/químicaRESUMEN
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.
Asunto(s)
Acetales/química , Afidicolina/química , Diterpenos/química , Pironas/química , Triterpenos/química , Estructura MolecularRESUMEN
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.
Asunto(s)
Arabidopsis/citología , Botánica/métodos , Técnicas de Cultivo de Célula/métodos , Antimetabolitos/farmacología , Afidicolina/química , Bromodesoxiuridina/farmacología , Ciclo Celular , Células Cultivadas , Criopreservación , Plantas Modificadas Genéticamente , Rhizobium/metabolismo , Sacarosa/metabolismo , Factores de Tiempo , TransgenesRESUMEN
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).
Asunto(s)
Afidicolina/síntesis química , Diterpenos/síntesis química , Oxígeno/química , Paladio/química , Piridinas/síntesis química , Sesquiterpenos/síntesis química , Terpenos/síntesis química , Titanio/química , Antivirales/síntesis química , Antivirales/química , Afidicolina/química , Diterpenos/química , Estructura Molecular , Piridinas/química , Sesquiterpenos/químicaRESUMEN
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.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Afidicolina/administración & dosificación , Neuroblastoma/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/uso terapéutico , Afidicolina/química , Afidicolina/uso terapéutico , Portadores de Fármacos , Femenino , Humanos , Liposomas , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Antivirales/síntesis química , Diterpenos/síntesis química , Plantas Medicinales/química , Antivirales/química , Afidicolina/química , Diterpenos/química , EstereoisomerismoRESUMEN
[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.
Asunto(s)
Afidicolina/análogos & derivados , Afidicolina/química , Acremonium/química , Antineoplásicos/química , Antivirales/química , Diterpenos/química , Inhibidores Enzimáticos/química , EstereoisomerismoRESUMEN
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).
Asunto(s)
Afidicolina/farmacología , Leishmania/efectos de los fármacos , Macrófagos/efectos de los fármacos , Suspensiones/farmacología , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacología , Afidicolina/química , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/parasitología , Dimetilsulfóxido/química , Dimetilsulfóxido/farmacología , Sistemas de Liberación de Medicamentos , Concentración 50 Inhibidora , Leishmania/crecimiento & desarrollo , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Polisorbatos/química , Polisorbatos/farmacología , Suspensiones/químicaRESUMEN
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.