RESUMEN
The antimalarial agents artemisinins inhibit cytomegalovirus (CMV) in vitro and in vivo, but their target(s) has been elusive. Using a biotin-labeled artemisinin, we identified the intermediate filament protein vimentin as an artemisinin target, validated by detailed biochemical and biological assays. We provide insights into the dynamic and unique modulation of vimentin, depending on the stage of human CMV (HCMV) replication. In vitro, HCMV entry and viral progeny are reduced in vimentin-deficient fibroblasts, compared with control cells. Similarly, mouse CMV (MCMV) replication in vimentin knockout mice is significantly reduced compared with controls in vivo, confirming the requirement of vimentin for establishment of infection. Early after HCMV infection of human foreskin fibroblasts vimentin level is stable, but as infection proceeds, vimentin is destabilized, concurrent with its phosphorylation and virus-induced calpain activity. Intriguingly, in vimentin-overexpressing cells, HCMV infection is reduced compared with control cells. Binding of artesunate, an artemisinin monomer, to vimentin prevents virus-induced vimentin degradation, decreasing vimentin phosphorylation at Ser-55 and Ser-83 and resisting calpain digestion. In vimentin-deficient fibroblasts, the anti-HCMV activity of artesunate is reduced compared with controls. In summary, an intact and stable vimentin network is important for the initiation of HCMV replication but hinders its completion. Artesunate binding to vimentin early during infection stabilizes it and antagonizes subsequent HCMV-mediated vimentin destabilization, thus suppressing HCMV replication. Our target discovery should enable the identification of vimentin-binding sites and compound moieties for binding.
Asunto(s)
Antivirales/farmacología , Artemisininas/farmacología , Citomegalovirus/efectos de los fármacos , Vimentina/antagonistas & inhibidores , Antivirales/química , Antivirales/metabolismo , Artemisininas/química , Artemisininas/metabolismo , Artesunato/farmacología , Sitios de Unión , Calpaína/metabolismo , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/metabolismo , Reposicionamiento de Medicamentos , Humanos , Espectrometría de Masas , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Vimentina/genética , Vimentina/metabolismo , Replicación Viral/efectos de los fármacos , Witanólidos/farmacologíaRESUMEN
Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition.
Asunto(s)
Antimaláricos/farmacología , Antivirales/farmacología , Artemisininas/farmacología , Ciclo Celular/efectos de los fármacos , Citomegalovirus/efectos de los fármacos , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/biosíntesis , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/biosíntesis , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/biosíntesis , Farmacorresistencia Viral , Inhibidores Enzimáticos/farmacología , Fibroblastos/virología , Fase G1/efectos de los fármacos , Ganciclovir/farmacología , Humanos , Fosforilación , Purinas/farmacología , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Roscovitina , Replicación Viral/efectos de los fármacosRESUMEN
Eight new artemisinin-derived trioxane dimer esters 5 have been prepared and tested for antimalarial efficacy in malaria-infected mice. At a single oral dose of only 6mg/kg combined with 18mg/kg of mefloquine, each of the dimer esters 5 outperformed the antimalarial drug artemether (2). The most efficacious dimer, dichlorobenzoate ester 5h, prolonged mouse survival past day 30 of infection with three of the four mice in this group having no detectable parasitemia and appearing and acting healthy on day 30.
Asunto(s)
Antimaláricos/administración & dosificación , Antimaláricos/química , Artemisininas/administración & dosificación , Artemisininas/química , Malaria/tratamiento farmacológico , Plasmodium berghei/efectos de los fármacos , Administración Oral , Animales , Dimerización , RatonesRESUMEN
Conventional therapy for human cytomegalovirus (CMV) relies on inhibition of the viral DNA polymerase. Ganciclovir (GCV) is the first-line therapy, but when GCV-resistant strains emerge, alternative therapies are extremely limited and are associated with significant toxicities. Combination of anti-CMV agents that act on different targets or stages of virus replication has not been well studied, mostly because of the limited number of anti-CMV agents. We report our investigation of combinations of agents that inhibit CMV by targeting the viral DNA polymerase, cellular kinases, or other cell/virus mechanisms yet to be discovered. The selected compounds differed by the slopes of their dose-response curve: compounds with a slope of 1 (GCV) representing one target or noncooperativity and compounds with high slopes indicating positive cooperativity. Analysis of anti-CMV drug combinations using the Bliss model (which accounts for the slope parameter) distinguished between combinations with synergistic, antagonistic, and additive activities. The combination of GCV and foscarnet was slightly synergistic; strong synergism was found when GCV was used with artemisinin-derived monomers or dimers or the MEK inhibitor U0126. The combination of GCV and cardiac glycosides (digoxin, digitoxin, and ouabain) was additive. The monomeric artemisinin artesunate was synergistic when combined with U0126 or the multikinase inhibitor sunitinib. However, the combination of artemisinin-derived dimers (molecular weights, 606 and 838) and U0126 or sunitinib was antagonistic. These results demonstrate that members of a specific drug class show similar patterns of combination with GCV and that the slope parameter plays an important role in the evaluation of drug combinations. Lastly, antagonism between different classes of CMV inhibitors may assist in target identification and improve the understanding of CMV inhibition by novel compounds.
Asunto(s)
Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Artemisininas/farmacología , Butadienos/farmacología , Línea Celular , Citomegalovirus/crecimiento & desarrollo , Digitoxina/farmacología , Digoxina/farmacología , Combinación de Medicamentos , Interacciones Farmacológicas , Fibroblastos/virología , Foscarnet/farmacología , Ganciclovir/farmacología , Humanos , Indoles/farmacología , Nitrilos/farmacología , Ouabaína/farmacología , Pirroles/farmacología , SunitinibRESUMEN
Several 2-carbon-linked trioxane dimer secondary alcohol carbonates 14 and thiocarbonates 15, combined with mefloquine and administered in a low single oral dose, prolonged the survival times of malaria-infected mice much more effectively than the popular monomeric antimalarial drug artemether plus mefloquine. Three dimer carbonates 14 and one dimer thiocarbonate 15 partially cured malaria-infected mice.
Asunto(s)
Antimaláricos/uso terapéutico , Artemisininas/química , Carbonatos/uso terapéutico , Malaria/tratamiento farmacológico , Plasmodium berghei/efectos de los fármacos , Compuestos de Sulfhidrilo/uso terapéutico , Animales , Antimaláricos/administración & dosificación , Antimaláricos/química , Carbonatos/administración & dosificación , Carbonatos/química , Dimerización , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/administración & dosificación , Compuestos de Sulfhidrilo/químicaRESUMEN
Sixteen new artemisinin-derived 2-carbon-linked trioxane dimers were prepared to study chemical structure/antimalarial activity relationships (SAR). Administering a very low single oral dose of only 5mg/kg of dimer secondary alcohol 6a or 6b plus 15 mg/kg of mefloquine hydrochloride prolonged the lives of Plasmodium berghei-infected mice to an average of 25 days after infection. This ACT chemotherapy result is of high medicinal significance because the antimalarial efficacy of the popular trioxane drug artemether (2) plus mefloquine under the same conditions was significantly lower (only 20 day average survival). NH-aryl carbamate derivatives 7e, 7i, and 7j of 2-carbon-linked dimer alcohol 6b also significantly outperformed artemether (2) in prolonging the survival times (25-27 days) of malaria-infected mice.
Asunto(s)
Antimaláricos/química , Artemisininas/química , Carbamatos/química , Animales , Antimaláricos/síntesis química , Antimaláricos/farmacología , Arteméter , Artemisininas/farmacología , Artemisininas/uso terapéutico , Carbamatos/farmacología , Carbamatos/uso terapéutico , Carbono/química , Cristalografía por Rayos X , Dimerización , Quimioterapia Combinada , Malaria/tratamiento farmacológico , Malaria/mortalidad , Mefloquina/farmacología , Mefloquina/uso terapéutico , Ratones , Conformación Molecular , Plasmodium berghei/efectos de los fármacos , Relación Estructura-Actividad , Análisis de SupervivenciaRESUMEN
We report that the artemisinin-derived dimer diphenyl phosphate (DPP; dimer 838) is the most selective inhibitor of human cytomegalovirus (CMV) replication among a series of artemisinin-derived monomers and dimers. Dimer 838 was also unique in being an irreversible CMV inhibitor. The peroxide unit within artemisinins' chemical structures is critical to their activities, and its absence results in loss of anti-CMV activities. Surprisingly, the deoxy dimer of 838 retained modest anti-CMV activity, suggesting that the DPP moiety of dimer 838 contributes to its anti-CMV activities. DPP alone did not inhibit CMV replication, but triphenyl phosphate (TPP) had modest CMV inhibition, although its selectivity index was low. Artemisinin DPP derivatives dimer 838 and monomer diphenyl phosphate (compound 558) showed stronger CMV inhibition and a higher selectivity index than their analogs lacking the DPP unit. An add-on and removal assay revealed that removing DPP derivatives (compounds 558 and 838) but not the non-DPP backbones (artesunate and compound 606) at 24 h postinfection (hpi) already resulted in dominant CMV inhibition. CMV inhibition was fully irreversible with 838 and partially irreversible with 558, while non-DPP artemisinins were reversible inhibitors. While all artemisinin derivatives and TPP reduced the expression of the CMV immediate early 2 (IE2), UL44, and pp65 proteins at or after 48 hpi, only TPP inhibited the expression of both IE1 and IE2. Combination of a non-DPP dimer (compound 606) with TPP was synergistic in CMV inhibition, while ganciclovir and TPP were additive. Although TPP shared structural similarity with monomer DPP (compound 558) and dimer DPP (compound 838), its pattern of CMV inhibition was significantly different from the patterns of the artemisinins. These findings demonstrate that the DPP group contributes to the unique activities of compound 838.
Asunto(s)
Antivirales/farmacología , Artemisininas/farmacología , Citomegalovirus/efectos de los fármacos , ADN Viral/antagonistas & inhibidores , Organofosfatos/química , Replicación Viral/efectos de los fármacos , Antivirales/química , Artemisininas/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citomegalovirus/crecimiento & desarrollo , ADN Viral/metabolismo , Dimerización , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Genes Reporteros , Células HeLa , Humanos , Concentración 50 Inhibidora , Luciferasas/genética , Luciferasas/metabolismo , Relación Estructura-Actividad CuantitativaRESUMEN
We recently reported the anti-cancer and anti-cytomegalovirus (CMV) activity of artemisinin-derived trioxane diphenylphosphate dimer 838. To probe the relationship between chemical structure and anti-CMV and anti-cancer activities, we now report synthesis and evaluation of a series of eight new dimer phosphate ester analogs of 838. This series of novel molecules was screened against human foreskin fibroblasts (HFFs) infected with CMV and against the human Jurkat T cell acute lymphoblastic leukemia cell line. This SAR study confirms the very high anti-CMV and anti-cancer potencies of dimer diphenyl phosphate ester 838 without its being toxic to normal cells.
Asunto(s)
Antineoplásicos/química , Antivirales/química , Artemisininas/química , Infecciones por Citomegalovirus/tratamiento farmacológico , Citomegalovirus/efectos de los fármacos , Leucemia/tratamiento farmacológico , Antineoplásicos/farmacología , Antivirales/farmacología , Artemisininas/farmacología , Dimerización , Ésteres , Humanos , Células Jurkat , Fosfatos/química , Fosfatos/farmacologíaRESUMEN
We previously reported that among a series of artemisinin-derived monomers and dimers, dimer diphenyl phosphate (838) was the most potent inhibitor of human cytomegalovirus (CMV) replication. Our continued investigation of a prototypic artemisinin monomer (artesunate [AS]) and dimer (838) now reveals that both compounds have specific activity against CMV but do not inhibit lytic replication of human herpesvirus 1 or 2 or Epstein-Barr virus. AS and 838 inhibited CMV replication during the first 24 h of the virus replication cycle, earlier than the time of ganciclovir (GCV) activities and prior to DNA synthesis. Neither compound inhibited virus entry. Quantification of DNA replication and virus yield revealed a similar level of inhibition by GCV, but AS and 838 had a 10-fold-higher inhibition of virus yield than of DNA replication, suggesting that artemisinins could inhibit CMV through multiple steps: a predominant early inhibition and possibly an additional step following DNA replication. During the strong early CMV inhibition, the transcription of immediate-early genes was not significantly downregulated, and viral protein expression was reduced only after 48 h. AS and GCV were reversible CMV inhibitors, but the inhibition of CMV replication by 838 was irreversible. Combinations of GCV and 838 as well as GCV and AS were highly synergistic. Finally, treatment with 838, but not AS, prior to CMV infection demonstrated strong anti-CMV activity. These findings illustrate the unique activities of dimer 838, including early and irreversible CMV inhibition, possibly by tight binding to its target.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Artemisininas/química , Citomegalovirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Western Blotting , Línea Celular , Chlorocebus aethiops , Citomegalovirus/genética , Replicación del ADN/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Humanos , Células VeroRESUMEN
We have previously demonstrated that the active vitamin D hormone, 1α,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) and a cis-locked non-genomic analogue, protect skin cells from ultraviolet radiation (UV)-induced skin cell loss, DNA damage, immunosuppression and skin carcinogenesis. Herein, we used a low-calcaemic analogue, 1α-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bis-homovitamin D3 (QW), which has some transactivating capacity and is approximately 80-100 times less calcaemic than 1,25(OH)(2)D(3). QW (0.1-10 nM) significantly (p < 0.05-0.01) reduced UV-induced DNA lesions (CPD) in skin fibroblasts and keratinocytes and reduced cell death after UV exposure. Moreover, both 1,25(OH)(2)D(3) and QW (1 nM) were equally effective in significantly (p < 0.01) increasing levels of tumour suppressive p53 in cultured human keratinocytes at 3 and 6 h after UV exposure. In a hairless mouse model, both 1,25(OH)(2)D(3) and QW (22.8 ρmol cm(-2)) reduced UV-immunosuppression from 13.7 ± 1.3% to 0.1 ± 1.1% (p < 0.01) and 5.4 ± 1.5% (p < 0.01) respectively. When tested alongside 1,25(OH)(2)D(3) in a murine model of skin carcinogenesis. QW (22.8 ρmol cm(-2)) was not as effective as 1α,25(OH)(2)D(3) or a cis-locked analogue in reducing tumour formation or inhibiting tumour progression. It is possible that the dose required for QW to be effective as an anti-photocarcinogenesis agent in vivo is higher than for protection against the acute effects of UV exposure, but the dissociation between clear acute photo-protective effects and limited long term photoprotection is as yet unexplained.
Asunto(s)
Calcitriol/análogos & derivados , Calcitriol/farmacología , Queratinocitos/efectos de los fármacos , Protectores contra Radiación/farmacología , Piel/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/efectos de la radiación , Células Cultivadas , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Humanos , Terapia de Inmunosupresión , Queratinocitos/citología , Queratinocitos/efectos de la radiación , Ratones , Ratones Pelados , Piel/efectos de la radiación , Proteína p53 Supresora de Tumor/metabolismo , Rayos UltravioletaRESUMEN
Recombinant Towne CMV expressing luciferase under the control of CMV-DNA polymerase (POL) or the late pp28 (UL99) promoters were evaluated for potential application in high-throughput screening of anti-viral compounds. POL-and pp28-luciferase displayed maximal expression 48 and 72 hours post infection, respectively. The pp28-luciferase virus achieved a wider dynamic range of luciferase expression (6-7 logs) and was selected for testing of inhibition by five anti-viral compounds. Luciferase expression highly correlated with plaque reduction and real-time PCR. The pp28-luciferase reporter system is rapid, reproducible, and highly sensitive. It may be applied to screening of novel anti-CMV compounds.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/virología , Citomegalovirus/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Luciferasas/genética , Línea Celular , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/tratamiento farmacológico , Evaluación Preclínica de Medicamentos/instrumentación , Expresión Génica/efectos de los fármacos , Humanos , Luciferasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
5-(Z)-alkylidene-2-thioxo-1,3-thiazolidin-4-ones (rhodanine derivatives) were prepared by reaction of in situ generated dithiocarbamates with recently reported racemic α-chloro-ß,γ-alkenoate esters. This multicomponent sequential transformation performed in one reaction flask represents a general route to this medicinally valuable class of sulfur/nitrogen heterocycles. Using this convergent procedure, we prepared an analogue of the drug epalrestat, an aldose reductase inhibitory rhodanine.
Asunto(s)
Técnicas de Química Sintética/métodos , Inhibidores Enzimáticos/síntesis química , Rodanina/síntesis química , Aldehído Reductasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Rodanina/químicaRESUMEN
New chlorodiols (±)-3 and (±)-5 are densely functionalized and versatile synthons. They are converted in one step on a gram scale into 2-chlorolactones (±)-6 and (±)-7 and into 4-hydroxy glycidate esters (±)-9 and (±)-10. The 4-hydroxy glycidate esters (±)-9 and (±)-10 are converted stereospecifically and regiospecifically into oxazolines (±)-13 and (±)-14 and into cyclic carbamates (±)-18-(±)-20. The 4-hydroxy glycidate ester (±)-10 undergoes stereocontrolled and regiocontrolled epoxide opening by sodium azide to form the 2-azido-3,4-dihydroxy alkanoate (±)-21. Finally, chlorodiol (±)-5 reacts stereospecifically with silver triflate to form the 2,3-dihydroxyfuranone (±)-26.
RESUMEN
Four 5-carbon-linked trioxane dimer orthoesters (6a-6d) have been prepared in 4 or 5 chemical steps from the natural trioxane artemisinin (1). When administered orally to malaria-infected mice using a single dose of only 6 mg/kg body weight along with 18 mg/kg of mefloquine hydrochloride, trioxane dimer orthoester sulfone 6d completely and safely cured the mice; after 30 days, the cured mice showed no detectable parasitemia, gained at least as much weight as the control mice (no infection), and behaved normally.
Asunto(s)
Antimaláricos/uso terapéutico , Ésteres/uso terapéutico , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Compuestos Heterocíclicos/uso terapéutico , Mefloquina/uso terapéutico , Plasmodium berghei/efectos de los fármacos , Administración Oral , Animales , Antimaláricos/administración & dosificación , Artemisininas/uso terapéutico , Cristalografía por Rayos X , Quimioterapia Combinada , Ésteres/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Malaria/tratamiento farmacológico , Ratones , Estructura MolecularRESUMEN
A central paradox of vitamin D biology is that 1alpha,25-(OH)(2) D(3) exposure inversely relates to colorectal cancer (CRC) risk despite a capacity for activation of both pro- and anti-oncogenic mediators including osteopontin (OPN)/CD44 and E-cadherin, respectively. Most sporadic CRCs arise from adenomatous polyposis coli (APC) gene mutation but understanding of its effects on vitamin D growth control is limited. Here we investigate effects of the Apc(Min/+) genotype on 1alpha,25-(OH)(2) D(3) regulation of OPN/CD44/E-cadherin signalling and intestinal tumourigenesis, in vivo. In untreated Apc(Min/+) versus Apc(+/+) intestines, expression levels of OPN and its CD44 receptor were increased, whereas E-cadherin tumour suppressor signalling was attenuated. Treatment by 1alpha,25-(OH)(2) D(3) or rationally designed analogues (QW or BTW) enhanced OPN but inhibited expression of CD44, the OPN receptor implicated in cell growth. These treatments also enhanced E-cadherin tumour suppressor activity, characterized by inhibition of beta-catenin nuclear localization, T-cell factor 1 and c-myelocytomatosis protein expression in Apc(Min/+) intestine. All secosteroids suppressed Apc(Min/+)-driven tumourigenesis although QW and BTW had lower calcium-related toxicity. Taken together, these data indicate that the Apc(Min/+) genotype modulates vitamin D secosteroid actions to promote functional predominance of E-cadherin tumour suppressor activity within antagonistic molecular networks. APC heterozygosity may promote favourable tissue- or tumour-specific conditions for growth control by vitamin D secosteroid treatment.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Vitamina D/análogos & derivados , Vitamina D/farmacología , Proteína de la Poliposis Adenomatosa del Colon/fisiología , Animales , Cadherinas/genética , Cadherinas/fisiología , Colon/patología , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/prevención & control , Cartilla de ADN , Femenino , Genotipo , Humanos , Pólipos Intestinales/genética , Pólipos Intestinales/patología , Ratones , Ratones Endogámicos C57BL , Osteopontina/fisiología , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Receptores de Calcitriol/genética , Factores de Riesgo , Vitamina D/uso terapéutico , Aumento de Peso/genéticaRESUMEN
Vitamin D receptor (VDR) can modulate functionally antagonistic growth regulatory pathways, involving beta-catenin/E-cadherin on one hand and osteopontin (OPN) on the other. This study investigates effects of VDR ligand treatment on the balance of these discordant signals and on associated cell behavior. Treatment of Rama 37 or SW480 cells by 1alpha,25-(OH)(2) D(3) or analogs suppressed beta-catenin/Lef-1/Tcf signaling and upregulated E-cadherin, consistent with a cancer-inhibitory action. Conversely, treatment also increased transcription of OPN that may be implicated in tumor progression. Molecular crosstalk was observed between the antagonistic VDR-dependent signals, in that beta-catenin/Lef-1/Tcf molecules modulated VDR activation of OPN. Treatment effects on cell growth were related to a constitutive balance of OPN and E-cadherin expression. No growth effects were observed in Rama 37 cells that have low OPN and high E-cadherin expression. Conversely, treatment of Rama 37 stably transfected subclones that had high OPN and/or low level E-cadherin induced small but significant increases of cell attachment to fibronectin, anchorage-independent growth or invasion. This study shows that relative expression levels of key VDR downstream genes may influence growth regulation by 1alpha,25-(OH)(2) D(3) or analogs. These findings may be relevant to the cell- or tissue-specificity of vitamin D growth regulation.
Asunto(s)
Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/patología , Osteopontina/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Elemento de Respuesta a la Vitamina D/genética , Animales , Western Blotting , Cadherinas/metabolismo , Adhesión Celular , Neoplasias Colorrectales/metabolismo , Femenino , Fibronectinas/metabolismo , Humanos , Luciferasas/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Mutagénesis Sitio-Dirigida , Osteopontina/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Ratas , Transducción de Señal , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Vitamina D/análogos & derivados , Vitamina D/farmacología , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
OBJECTIVES: We sought to improve upon the usefulness of artemisinins as anti-Toxoplasma agents by synthesizing new unsaturated, carba derivatives and then testing them for in vitro efficacy against three steps of the lytic cycle of Toxoplasma gondii tachyzoites. METHODS: Novel derivatives of ART were synthesized and then tested for in vitro antiparasitic activity using T. gondii tachyzoites constitutively expressing beta-galactosidase and human fibroblast host cells. Compounds were evaluated for parasite growth inhibition and cytotoxicity, inhibition of replication and inhibition of parasite invasion of host cells. RESULTS: Five of the seven new derivatives, 3a-c, 3e and 3f, effectively inhibited T. gondii growth (IC50=1.0-4.4 microM); however, only three of these proved to be relatively non-cytotoxic (TD50>or=200 microM). The same five derivatives also inhibited tachyzoite replication, and attachment to and invasion of host cells as effectively as or better than the parent compound ART. In addition, one of the derivatives incapable of inhibiting growth, deoxy-3a, was found to inhibit parasite invasion. CONCLUSIONS: These new artemisinin derivatives have the ability to inhibit multiple steps of T. gondii's lytic cycle. Synthetic unsaturated, carba derivatives of ART have potential as therapeutic agents for the prevention and treatment of toxoplasmosis in humans.
Asunto(s)
Antiprotozoarios/farmacología , Antiprotozoarios/toxicidad , Artemisininas/farmacología , Artemisininas/toxicidad , Toxoplasma/efectos de los fármacos , Animales , Antiprotozoarios/síntesis química , Artemisininas/síntesis química , Células Cultivadas , Fibroblastos/parasitología , Humanos , Concentración 50 Inhibidora , Toxoplasma/crecimiento & desarrolloRESUMEN
The replacement of a t-butyl group with a trifluoromethyl group has profound effects on the biological profile of 1alpha,25-dihydroxyvitamin D(3) sulfone analogs. Investigation of whether the improved biological activities are due to steric and electronic factors of the trifluoromethyl group led to the design, synthesis and biological evaluation of two analogous alkyl sulfone molecules, methyl sulfone (AU-16-ene-25-SO(2)-CH(3)) and isopropyl sulfone (AU-16-ene-25-SO(2)-i-Pr). These alkyl sulfones are sterically comparable to, but electronically very different from a trifluoromethyl group. The syntheses, antiproliferative activities and calcemic activities of these new alkyl sulfones are presented herein. In comparing the in vitro antiproliferative profiles of the new alkyl sulfone 1alpha,25-dihydroxyvitamin D(3) analogs with the trifluoromethylsulfone and an analogous t-butyl sulfone, the activities increase in the following order: CH(3) < t-Bu approximately = i-Pr < CF(3). In contrast to the calcemic t-butyl sulfone, the novel alkyl sulfones and trifluoromethyl sulfone display desirable low calcemic levels.
Asunto(s)
Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Sulfonas/química , Sulfonas/farmacología , Vitamina D/análogos & derivados , Animales , Calcitriol/farmacología , Células Cultivadas , Queratinocitos/citología , Ratas , Relación Estructura-Actividad , Sulfonas/síntesis química , Vitamina D/síntesis química , Vitamina D/química , Vitamina D/farmacología , Vitaminas/farmacologíaRESUMEN
Three new 5-carbon-linked trioxane dimer carboxylate esters have been prepared from the natural trioxane, artemisinin in only 3-steps and 40-50% overall yields. Each one of these new chemical entities is at least as efficacious as the clinically used trioxane antimalarial drug artemether when combined with mefloquine hydrochloride in a low single oral dose cure.
RESUMEN
In four or five chemical steps from the 1,2,4-trioxane artemisinin, a new series of 23 trioxane dimers has been prepared. Eleven of these new trioxane dimers cure malaria-infected mice via oral dosing at 3 x 30 mg/kg. The clinically used trioxane drug sodium artesunate prolonged mouse average survival to 7.2 days with this oral dose regimen. In comparison, animals receiving no drug die typically on day 6-7 postinfection. At only 3 x 10 mg/kg oral dosing, seven dimers prolong the lifetime of malaria-infected mice to days 14-17, more than double the chemotherapeutic effect of sodium artesunate. Ten new trioxane dimers at only a single oral dose of 30 mg/kg prolong mouse average survival to days 8.7-13.7, and this effect is comparable to that of the fully synthetic trioxolane drug development candidate OZ277, which is in phase II clinical trials.