RESUMEN
Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and ß-glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Clorhidrato de Fingolimod/análogos & derivados , Clorhidrato de Fingolimod/farmacología , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Células Cultivadas , Células Endoteliales/metabolismo , Clorhidrato de Fingolimod/química , Humanos , Permeabilidad/efectos de los fármacos , Arteria Pulmonar/metabolismo , Relación Estructura-ActividadRESUMEN
Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and ß-glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.
Asunto(s)
Clorhidrato de Fingolimod/análogos & derivados , Calcio/metabolismo , Línea Celular , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Clorhidrato de Fingolimod/farmacología , Fluoruros/química , Glucurónidos/química , Humanos , Lisofosfolípidos/metabolismo , Microscopía Fluorescente , Permeabilidad/efectos de los fármacos , Fosforilación , Arteria Pulmonar/citología , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/ß-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and ß-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/ß-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteína Fosfatasa 2/metabolismo , Animales , Apoptosis , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Activadores de Enzimas/farmacología , Clorhidrato de Fingolimod , Proteínas de Fusión bcr-abl/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Humanos , Janus Quinasa 2/metabolismo , Células K562 , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/enzimología , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Esfingosina/farmacología , Vía de Señalización Wnt , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
Sphingosine kinase 2 (SK2) catalyses the conversion of sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). We report here, the stereospecific synthesis of an analogue of FTY720 called (R)-FTY720-OMe, which we show is a competitive inhibitor of SK2. (R)-FTY720-OMe failed to inhibit sphingosine kinase 1 activity, thereby demonstrating specificity for SK2. Prolonged treatment of HEK 293 cells with (R)-FTY720-OMe also induced a reduction in SK2 expression. In addition, (R)-FTY720-OMe inhibited DNA synthesis and prevented S1P-stimulated rearrangement of actin in MCF-7 breast cancer cells. These findings demonstrate that SK2 functions as a pro-survival protein and is involved in promoting actin rearrangement into membrane ruffles/lamellipodia in response to S1P in MCF-7 breast cancer cells.
Asunto(s)
Actinas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Actinas/efectos de los fármacos , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Membrana Celular/metabolismo , Pruebas de Enzimas/métodos , Femenino , Clorhidrato de Fingolimod , Células HEK293 , Humanos , Lisofosfolípidos/metabolismo , Microscopía Fluorescente , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Glicoles de Propileno/síntesis química , Seudópodos/metabolismo , Esfingosina/síntesis química , Esfingosina/metabolismo , Esfingosina/farmacologíaRESUMEN
FTY720 (Fingolimod), a synthetic analogue of sphingosine 1-phosphate (S1P), activates four of the five EDG-family S1P receptors and is in a phase-III clinical study for the treatment of multiple sclerosis. (S)-FTY720-phosphate (FTY720-P) causes S1P(1) receptor internalization and targeting to the proteasomal degradative pathway, and thus functions as an antagonist of S1P(1) by depleting the functional S1P(1) receptor from the plasma membrane. Here we describe the pharmacological characterization of two unsaturated phosphonate enantiomers of FTY720, (R)- and (S)-FTY720-vinylphosphonate. (R)-FTY720-vinylphosphonate was a full agonist of S1P(1) (EC(50) 20+/-3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P(1,3,4) (K(i) 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P(2), and S1P(5). Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with K(i) values in the low micromolar range, although with different enzyme kinetic mechanisms. When injected into mice, both enantiomers caused transient peripheral lymphopenia. (R)- and (S)-FTY720-vinylphosphonates activated ERK1/2, AKT, and exerted an antiapoptotic effect in camptothecin-treated IEC-6 intestinal epithelial cells, which primarily express S1P(2) transcripts and traces of S1P(5). (S)-FTY720-vinylphosphonate is the first pan-antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. The biological effects of the (R)- and (S)-FTY720-vinylphosphonate analogues underscore the complexity of FTY720 cellular targets.
Asunto(s)
Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Esfingosina/análogos & derivados , Compuestos de Vinilo/farmacología , Animales , Línea Celular , Humanos , Lisofosfolípidos/farmacología , Ratones , Ratones Endogámicos C57BL , Organofosfatos/farmacología , Organofosfonatos , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Ratas , Receptores de Lisoesfingolípidos/agonistas , Transducción de Señal/efectos de los fármacos , Esfingosina/química , Esfingosina/farmacología , Estereoisomerismo , Compuestos de Vinilo/químicaRESUMEN
The first enantioselective synthesis of chiral isosteric phosphonate analogues of FTY720 is described. One of these analogues, FTY720-(E)-vinylphosphonate (S)-5, but not its R enantiomer, elicited a potent antiapoptotic effect in intestinal epithelial cells, suggesting that it exerts its action via the enantioselective activation of a receptor. (S)-5 failed to activate the sphingosine 1-phosphate type 1 (S1P(1)) receptor.
Asunto(s)
Inmunosupresores/síntesis química , Organofosfonatos/síntesis química , Glicoles de Propileno/síntesis química , Esfingosina/análogos & derivados , Compuestos de Vinilo/síntesis química , Animales , Células Cultivadas , Clorhidrato de Fingolimod , Humanos , Inmunosupresores/farmacología , Mucosa Intestinal/efectos de los fármacos , Estructura Molecular , Organofosfonatos/química , Glicoles de Propileno/química , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/efectos de los fármacos , Esfingosina/síntesis química , Esfingosina/química , Esfingosina/farmacología , Estereoisomerismo , Compuestos de Vinilo/química , Compuestos de Vinilo/farmacologíaRESUMEN
Sphingosine 1-phosphate (S1P) regulates diverse cellular functions through extracellular ligation to S1P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S1P to generate intracellular S1P. We, therefore, examined the role of lipid phosphate phosphatase (LPP)-1 and sphingosine kinase1 (SphK1) in converting exogenous S1P to intracellular S1P. Exposure of (32)P-labeled HPAECs to S1P or sphingosine (Sph) increased the intracellular accumulation of [(32)P]S1P in a dose- and time-dependent manner. The S1P formed in the cells was not released into the medium. The exogenously added S1P did not stimulate the sphingomyelinase pathway; however, added [(3)H]S1P was hydrolyzed to [(3)H]Sph in HPAECs, and this was blocked by XY-14, an inhibitor of LPPs. HPAECs expressed LPP1-3, and overexpression of LPP-1 enhanced the hydrolysis of exogenous [(3)H]S1P to [(3)H]Sph and increased intracellular S1P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-1 by siRNA decreased intracellular S1P production from extracellular S1P but had no effect on the phosphorylation of Sph to S1P. Knockdown of SphK1, but not SphK2, by siRNA attenuated the intracellular generation of S1P. Overexpression of wild type SphK1, but not SphK2 wild type, increased the accumulation of intracellular S1P after exposure to extracellular S1P. These studies provide the first direct evidence for a novel pathway of intracellular S1P generation. This involves the conversion of extracellular S1P to Sph by LPP-1, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S1P by SphK1.
Asunto(s)
Endotelio Vascular/metabolismo , Lisofosfolípidos/metabolismo , Fosfatidato Fosfatasa/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Adenoviridae/genética , Biotina , Western Blotting , Células Cultivadas , Ceramidas , Cromatografía Liquida , Citoplasma/metabolismo , Endotelio Vascular/citología , Humanos , Riñón/metabolismo , Espectrometría de Masas , Fosfatidato Fosfatasa/antagonistas & inhibidores , Fosfatidato Fosfatasa/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Arteria Pulmonar/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Esfingosina/metabolismoRESUMEN
An azido group was incorporated into the immunomodulatory agent FTY720, accomplishing the first synthesis of a photoactivatable analogue of this ligand (2) in 9 steps from 2-(4-hydroxyphenyl)ethanol and in 34% overall yield. The key steps are formation of a primary amine at a quaternary center of aniline derivative 13 followed by selective diazotization of the arylamine.
Asunto(s)
Azidas/química , Inmunosupresores/química , Propanolaminas/química , Glicoles de Propileno/química , Esfingosina/análogos & derivados , Azidas/síntesis química , Clorhidrato de Fingolimod , Inmunosupresores/síntesis química , Fotoquímica , Propanolaminas/síntesis química , Esfingosina/químicaRESUMEN
D-erythro-(2S,3R,4E)-Sphingosine-1-phosphonate (1), the isosteric phosphonate analogue of naturally occurring sphingosine 1-phosphate (1a), and D-ribo-phytosphingosine 1-phosphonate (2), the isosteric phosphonate analogue of D-ribo-phytosphingosine-1-phosphate (2a), were synthesized starting with methyl 2,3-O-isopropylidene-d-glycerate (4) and D-ribo-phytosphingosine (3), respectively. Oxirane 12 was formed in eight steps from 4, and cyclic sulfamidate 22 was formed in five steps from 3. The phosphonate group was introduced via regioselective ring-opening reactions of oxirane 12 and cyclic sulfamidate 22 with lithium dialkyl methylphosphonate, affording 13 and 23, respectively. The synthesis of 1 was completed by S(N)2 displacement of chloromesylate intermediate 14b with azide ion, followed by conversion of the resulting azido group to a NHBoc group and deprotection. The synthesis of 2 was completed by cleavage of the acetal, N-benzyl, and alkyl phosphonate ester groups.