RESUMO
There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. Here, we establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders antifungal-resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating the protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for treating fungal infectious disease that evades resistance.
Assuntos
Antifúngicos/farmacologia , Depsipeptídeos/farmacologia , Fungos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/química , Depsipeptídeos/síntese química , Depsipeptídeos/química , Farmacorresistência Fúngica/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Fungos/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Testes de Sensibilidade Microbiana , Micoses/tratamento farmacológico , Micoses/microbiologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/química , Serina-Treonina Quinases TOR/metabolismoRESUMO
Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-ß42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.
Assuntos
Doença de Alzheimer/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilcolina/toxicidade , Serina-Treonina Quinases TOR/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Membrana Celular/efeitos dos fármacos , Ceramidas/biossíntese , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/biossíntese , Neurônios/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/biossíntese , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/biossínteseRESUMO
Targeting aberrantly expressed kinases in malignant pleural mesothelioma (MPM) is a promising therapeutic strategy. We here investigated the effect of the novel and highly selective Phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244) on MPM cells and on the immune cells in MPM microenvironment. To this aim, we analyzed the expression of PI3K-δ by immunohistochemistry in specimens from primary MPM, cell viability and death in three different MPM cell lines treated with roginolisib alone and in combination with ipatasertib (AKT inhibitor) and sapanisertib (mTOR inhibitor). In a co-culture model of patient-derived MPM cells, autologous peripheral blood mononuclear cells and fibroblasts, the tumor cell viability and changes in immune cell composition were investigated after treatment of roginolisib with nivolumab and cisplatin. PI3K-δ was detected in 66/89 (74%) MPM tumors and was associated with reduced overall survival (12 vs. 25 months, P=0.0452). Roginolisib induced apoptosis in MPM cells and enhanced the anti-tumor efficacy of AKT and mTOR kinase inhibitors by suppressing PI3K-δ/AKT/mTOR and ERK1/2 signaling. Furthermore, the combination of roginolisib with chemotherapy and immunotherapy re-balanced the immune cell composition, increasing effector T-cells and reducing immune suppressive cells. Overall, roginolisib induces apoptosis in MPM cells and increases the antitumor immune cell effector function when combined with nivolumab and cisplatin. These results provide first insights on the potential of roginolisib as a therapeutic agent in patients with MPM and its potential in combination with established immunotherapy regimen.
RESUMO
PI3K delta (PI3Kδ) inhibitors are used to treat lymphomas but safety concerns and limited target selectivity curbed their clinical usefulness. PI3Kδ inhibition in solid tumors has recently emerged as a potential novel anticancer therapy through the modulation of T-cell responses and direct antitumor activity. Here we report the exploration of IOA-244/MSC2360844, a first-in-class non-ATP-competitive PI3Kδ inhibitor, for the treatment of solid tumors. We confirm IOA-244's selectivity as tested against a large set of kinases, enzymes, and receptors. IOA-244 inhibits the in vitro growth of lymphoma cells and its activity correlates with the expression levels of PIK3CD, suggesting cancer cell-intrinsic effects of IOA-244. Importantly, IOA-244 inhibits regulatory T cell proliferation while having limited antiproliferative effects on conventional CD4+ T cells and no effect on CD8+ T cells. Instead, treatment of CD8 T cells with IOA-244 during activation, favors the differentiation of memory-like, long-lived CD8, known to have increased antitumor capacity. These data highlight immune-modulatory properties that can be exploited in solid tumors. In CT26 colorectal and Lewis lung carcinoma lung cancer models, IOA-244 sensitized the tumors to anti-PD-1 (programmed cell death protein 1) treatment, with similar activity in the Pan-02 pancreatic and A20 lymphoma syngeneic mouse models. IOA-244 reshaped the balance of tumor-infiltrating cells, favoring infiltration of CD8 and natural killer cells, while decreasing suppressive immune cells. IOA-244 presented no detectable safety concerns in animal studies and is currently in clinical phase Ib/II investigation in solid and hematologic tumors. Significance: IOA-244 is a first-in-class non-ATP-competitive, PI3Kδ inhibitor with direct antitumor in vitro activity correlated with PI3Kδ expression. The ability to modulate T cells, in vivo antitumor activity in various models with limited toxicity in animal studies provides the rationale for the ongoing trials in patients with solid tumors and hematologic cancers.
Assuntos
Linfoma , Neoplasias , Camundongos , Animais , Linfócitos T CD8-Positivos , Fosfatidilinositol 3-Quinases , Neoplasias/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Linfoma/tratamento farmacológico , Tolerância ImunológicaRESUMO
The target of rapamycin complex 2 (TORC2) plays a key role in maintaining the homeostasis of plasma membrane (PM) tension. TORC2 activation following increased PM tension involves redistribution of the Slm1 and 2 paralogues from PM invaginations known as eisosomes into membrane compartments containing TORC2. How Slm1/2 relocalization is triggered, and if/how this plays a role in TORC2 inactivation with decreased PM tension, is unknown. Using osmotic shocks and palmitoylcarnitine as orthogonal tools to manipulate PM tension, we demonstrate that decreased PM tension triggers spontaneous, energy-independent reorganization of pre-existing phosphatidylinositol-4,5-bisphosphate into discrete invaginated membrane domains, which cluster and inactivate TORC2. These results demonstrate that increased and decreased membrane tension are sensed through different mechanisms, highlighting a role for membrane lipid phase separation in mechanotransduction.
Assuntos
Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mecanotransdução Celular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas do Citoesqueleto , Ativação Enzimática , Proteínas Fúngicas/genética , Cinética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Mecanotransdução Celular/efeitos dos fármacos , Pressão Osmótica , Palmitoilcarnitina/farmacologia , Transporte Proteico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
Fundamental changes in the composition and distribution of lipids within the brain are believed to contribute to the cognitive decline associated with Alzheimer's disease (AD). The mechanisms by which these changes in lipid composition affect cellular function and ultimately cognition are not well understood. Although "candidate gene" approaches can provide insight into the effects of dysregulated lipid metabolism they require a preexisting understanding of the molecular targets of individual lipid species. In this report we combine unbiased gene expression profiling with a genome-wide chemogenomic screen to identify the mitochondria as an important downstream target of PC(O-16:0/2:0), a neurotoxic lipid species elevated in AD. Further examination revealed that PC(O-16:0/2:0) similarly promotes a global increase in ceramide accumulation in human neurons which was associated with mitochondrial-derived reactive oxygen species (ROS) and toxicity. These findings suggest that PC(O-16:0/2:0)-dependent mitochondrial dysfunction may be an underlying contributing factor to the ROS production associated with AD.