RESUMO
Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.
Assuntos
Endossomos , Oligonucleotídeos , Animais , Camundongos , Oligonucleotídeos/metabolismo , Endossomos/genética , Endocitose/fisiologia , Transporte Biológico , Oligonucleotídeos Antissenso/genética , RNA Interferente Pequeno/genéticaRESUMO
The anti-neoplastic sphingolipid analog SH-BC-893 starves cancer cells to death by down-regulating cell surface nutrient transporters and blocking lysosomal trafficking events. These effects are mediated by the activation of protein phosphatase 2A (PP2A). To identify putative PP2A substrates, we used quantitative phosphoproteomics to profile the temporal changes in protein phosphorylation in FL5.12 cells following incubation with SH-BC-893 or the specific PP2A inhibitor LB-100. These analyses enabled the profiling of more than 15,000 phosphorylation sites, of which 958 sites on 644 proteins were dynamically regulated. We identified 114 putative PP2A substrates including several nutrient transporter proteins, GTPase regulators (e.g. Agap2, Git1), and proteins associated with actin cytoskeletal remodeling (e.g. Vim, Pxn). To identify SH-BC-893-induced cell signaling events that disrupt lysosomal trafficking, we compared phosphorylation profiles in cells treated with SH-BC-893 or C2-ceramide, a non-vacuolating sphingolipid that does not impair lysosomal fusion. These analyses combined with functional assays uncovered the differential regulation of Akt and Gsk3b by SH-BC-893 (vacuolating) and C2-ceramide (non-vacuolating). Dynamic phosphoproteomics of cells treated with compounds affecting PP2A activity thus enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Fosfoproteínas/análise , Piperazinas/farmacologia , Proteína Fosfatase 2/metabolismo , Proteômica/métodos , Esfingolipídeos/farmacologia , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacologiaRESUMO
Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Nutrientes/metabolismo , Esfingolipídeos/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Cloridrato de Fingolimode/farmacologia , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Células HeLa , Humanos , Proteínas com Domínio LIM/metabolismo , Células MCF-7 , Proteínas dos Microfilamentos , Oxigenases de Função Mista , Esfingolipídeos/farmacologiaRESUMO
Targeting the inability of cancerous cells to adapt to metabolic stress is a promising alternative to conventional cancer chemotherapy. FTY720 (Gilenya), an FDA-approved drug for the treatment of multiple sclerosis, has recently been shown to inhibit cancer progression through the down-regulation of essential nutrient transport proteins, selectively starving cancer cells to death. However, the clinical use of FTY720 for cancer therapy is prohibited because of its capability of inducing immunosuppression (lymphopenia) and bradycardia when phosphorylated upon administration. A prodrug to specifically prevent phosphorylation during circulation, hence avoiding bradycardia and lymphopenia, was synthesized by capping its hydroxyl groups with polyethylene glycol (PEG) via an acid-cleavable ketal linkage. Improved aqueous solubility was also accomplished by PEGylation. The prodrug reduces to fully potent FTY720 upon cellular uptake and induces metabolic stress in cancer cells. Enhanced release of FTY720 at a mildly acidic endosomal pH and the ability to substantially down-regulate cell-surface nutrient transporter proteins in leukemia cells only by an acid-cleaved drug were confirmed. Importantly, the prodrug demonstrated nearly identical efficacy to FTY720 in an animal model of BCR-Abl-driven leukemia without inducing bradycardia or lymphopenia in vivo, highlighting its potential clinical value. The prodrug formulation of FTY720 demonstrates the utility of precisely engineering a drug to avoid undesirable effects by tackling specific molecular mechanisms as well as a financially favorable alternative to new drug development. A multitude of existing cancer therapeutics may be explored for prodrug formulation to avoid specific side effects and preserve or enhance therapeutic efficacy.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Cloridrato de Fingolimode/química , Cloridrato de Fingolimode/farmacologia , Leucemia/tratamento farmacológico , Polietilenoglicóis/química , Acetais/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Leucemia/patologia , FosforilaçãoRESUMO
Inspired by the cytotoxicity of perphenazine toward cancer cells and its ability to activate the serine/threonine protein phosphatase 2A (PP2A), we prepared series of ether-carbon linked analogs of a constrained synthetic sphingolipid analog 3, known for its cytotoxicity, nutrient transporter down-regulation and vacuolation properties, incorporating the tricyclic neuroleptics phenoxazine and phenothiazine to represent hybrid structures with possible synergistic cytotoxic activity. While the original activity of the lead compound 3 was diminished by fusion with the phenoxazine or phenothiazine tethered moieties, the corresponding 3-pyridyltetryl ether analog 10 showed cytotoxicity and nutrient transporter down-regulation similar to the lead compound 3, although it separated these PP2A-dependent phenotypes from that of vacuolation.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Oxazinas/farmacologia , Fenotiazinas/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Esfingolipídeos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Camundongos , Estrutura Molecular , Oxazinas/química , Fenotiazinas/química , Proteína Fosfatase 2/metabolismo , Esfingolipídeos/química , Relação Estrutura-AtividadeRESUMO
Constrained analogs containing a 2-hydroxymethylpyrrolidine core of the natural sphingolipids sphingosine, sphinganine, N,N-dimethylsphingosine and N-acetyl variants of sphingosine and sphinganine (C2-ceramide and dihydro-C2-ceramide) were synthesized and evaluated for their ability to down-regulate nutrient transporter proteins and trigger cytoplasmic vacuolation in mammalian cells. In cancer cells, the disruptions in intracellular trafficking produced by these sphingolipids lead to cancer cell death by starvation. Structure activity studies were conducted by varying the length of the hydrocarbon chain, the degree of unsaturation and the presence or absence of an aryl moiety on the appended chains, and stereochemistry at two stereogenic centers. In general, cytotoxicity was positively correlated with nutrient transporter down-regulation and vacuolation. This study was intended to identify structural and functional features in lead compounds that best contribute to potency, and to develop chemical biology tools that could be used to isolate the different protein targets responsible for nutrient transporter loss and cytoplasmic vacuolation. A molecule that produces maximal vacuolation and transporter loss is expected to have the maximal anti-cancer activity and would be a lead compound.
Assuntos
Morte Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Hidrocarbonetos/química , Proteínas de Membrana Transportadoras/metabolismo , Esfingolipídeos/farmacologia , Vacúolos/efeitos dos fármacos , Animais , Humanos , Esfingolipídeos/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Rapidly proliferating cancer cells increase flux through anabolic pathways to build the mass necessary to support cell division. Imported amino acids and glucose lie at the apex of the anabolic pyramid. Consistent with this, elevated expression of nutrient transporter proteins is characteristic of aggressive and highly malignant cancers. Because tumour cells are more dependent than their normal neighbours on accelerated nutrient import, these up-regulated transporters could be excellent targets for selective anti-cancer therapies. A study by Babu et al. in a recent issue of the Biochemical Journal definitively shows that SLC6A14 (where SLC is solute carrier) is one such cancer-specific amino acid transporter. Although mice completely lacking SLC6A14 are viable and exhibit normal mammary gland development, these animals are highly resistant to mammary tumour initiation and progression driven by potent oncogenes. Because SLC6A14 is essential for tumour growth yet dispensable for normal development and tissue maintenance, small molecules that block amino acid import through this transporter could be effective and selective anti-cancer agents, particularly as components of rational drug combinations.
Assuntos
Sistemas de Transporte de Aminoácidos , Proliferação de Células , Deleção de Genes , Neoplasias Mamárias Experimentais/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores , Transdução de Sinais , Animais , FemininoRESUMO
A synthetic sphingolipid related to a ring-constrained hydroxymethyl pyrrolidine analog of FTY720 that was known to starve cancer cells to death was chemically modified to include a series of alkoxy-tethered 3,6-substituted 1,2-pyridazines. These derivatives exhibited excellent antiproliferative activity against eight human cancer cell lines from four different cancer types. A 2.5- to 9-fold reduction in IC50 in these cell lines was observed relative to the lead compound, which lacked the appended heterocycle.
RESUMO
A series of compounds containing pyrrolidine and pyrrolizidine cores with appended hydrophobic substituents were prepared as constrained analogs of FTY720 and phytosphingosine. The effect of these compounds on the viability of cancer cells, on downregulation of the nutrient transport systems, and on their ability to cause vacuolation was studied. An attempt to inhibit HDACs with some phosphate esters of our analogs was thwarted by our failure to reproduce the reported inhibitory action of FTY720-phosphate.
Assuntos
Antineoplásicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Esfingosina/análogos & derivados , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Cloridrato de Fingolimode/análogos & derivados , Cloridrato de Fingolimode/química , Camundongos , Estrutura Molecular , Esfingosina/síntese química , Esfingosina/química , Esfingosina/farmacologia , Relação Estrutura-AtividadeRESUMO
FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anticancer agent in multiple animal models. Our published work indicates that the anticancer effects of FTY720 do not depend on actions at S1P receptors but instead stem from FTY720s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here, we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Cloridrato de Fingolimode/análogos & derivados , Cloridrato de Fingolimode/uso terapêutico , Neoplasias/tratamento farmacológico , Pirrolidinas/química , Pirrolidinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Cloridrato de Fingolimode/farmacologia , Humanos , Imunossupressores/química , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Pirrolidinas/farmacologia , Receptores de Lisoesfingolipídeo/metabolismoRESUMO
Oncogenic mutations drive anabolic metabolism, creating a dependency on nutrient influx through transporters, receptors, and macropinocytosis. While sphingolipids suppress tumor growth by downregulating nutrient transporters, macropinocytosis and autophagy still provide cancer cells with fuel. Therapeutics that simultaneously disrupt these parallel nutrient access pathways have potential as powerful starvation agents. Here, we describe a water-soluble, orally bioavailable synthetic sphingolipid, SH-BC-893, that triggers nutrient transporter internalization and also blocks lysosome-dependent nutrient generation pathways. SH-BC-893 activated protein phosphatase 2A (PP2A), leading to mislocalization of the lipid kinase PIKfyve. The concomitant mislocalization of the PIKfyve product PI(3,5)P2 triggered cytosolic vacuolation and blocked lysosomal fusion reactions essential for LDL, autophagosome, and macropinosome degradation. By simultaneously limiting access to both extracellular and intracellular nutrients, SH-BC-893 selectively killed cells expressing an activated form of the anabolic oncogene Ras in vitro and in vivo. However, slower-growing, autochthonous PTEN-deficient prostate tumors that did not exhibit a classic Warburg phenotype were equally sensitive. Remarkably, normal proliferative tissues were unaffected by doses of SH-BC-893 that profoundly inhibited tumor growth. These studies demonstrate that simultaneously blocking parallel nutrient access pathways with sphingolipid-based drugs is broadly effective and cancer selective, suggesting a potential strategy for overcoming the resistance conferred by tumor heterogeneity.
Assuntos
Ativadores de Enzimas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteína Fosfatase 2/antagonistas & inibidores , Esfingolipídeos/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteína Fosfatase 2/metabolismoRESUMO
Highly proliferative cells, including cancer cells, require a constant supply of molecular building blocks to support their growth. To acquire substrates such as glucose and amino acids from the extracellular space, dividing cells rely on transporter proteins in the plasma membrane. Numerous studies link transcriptional and post-translational control of nutrient transporter expression with proliferation, highlighting the importance of nutrient transporters in both physiologic and pathologic growth. Here we review recent work that spotlights the crucial role of nutrient transporters in cell growth and proliferation, discuss post-translational mechanisms for coordinating expression of different transporters, and consider the therapeutic potential of targeting these proteins in cancer and other diseases characterized by inappropriate cell division.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Aminoácidos/metabolismo , Animais , Glucose/metabolismo , Humanos , Neoplasias/metabolismoRESUMO
FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has anti-neoplastic actions. Our published work suggests that at least some of FTY720's anti-cancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720's S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective anti-tumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for anti-leukemic activity.