RESUMEN
Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid ß-oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore the presence of disease-specific markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease-specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease-specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.
Asunto(s)
Cardiomiopatías , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Errores Innatos del Metabolismo Lipídico , Lipidómica , Enfermedades Mitocondriales , Miopatías Mitocondriales , Proteína Trifuncional Mitocondrial/deficiencia , Enfermedades Musculares , Enfermedades del Sistema Nervioso , Rabdomiólisis , Humanos , Enfermedades Mitocondriales/diagnóstico , Carnitina , Cisteamina , LípidosRESUMEN
The peroxisome proliferator-activated receptors (PPARs) are a group of transcription factors belonging to the nuclear receptor superfamily. Since most target genes of PPARs are implicated in lipid and glucose metabolism, regulation by PPARs could be used as a screening tool to identify novel genes involved in lipid or glucose metabolism. Here, we identify Adtrp, a serine hydrolase enzyme that was reported to catalyze the hydrolysis of fatty acid esters of hydroxy fatty acids (FAHFAs), as a novel PPAR-regulated gene. Adtrp was significantly upregulated by PPARα activation in mouse primary hepatocytes, liver slices, and whole liver. In addition, Adtrp was upregulated by PPARγ activation in 3L3-L1 adipocytes and in white adipose tissue. ChIP-SEQ identified a strong PPAR-binding site in the immediate upstream promoter of the Adtrp gene. Adenoviral-mediated hepatic overexpression of Adtrp in diet-induced obese mice caused a modest increase in plasma nonesterified fatty acids but did not influence diet-induced obesity, liver triglyceride levels, liver lipidomic profiles, liver transcriptomic profiles, plasma cholesterol, triglyceride, glycerol, and glucose levels. Moreover, hepatic Adtrp overexpression did not lead to significant changes in FAHFA levels in plasma or liver and did not influence glucose and insulin tolerance. Finally, hepatic overexpression of Adtrp did not influence liver triglycerides and levels of plasma metabolites after a 24-h fast. Taken together, our data suggest that despite being a PPAR-regulated gene, hepatic Adtrp does not seem to play a major role in lipid and glucose metabolism and does not regulate FAHFA levels.
Asunto(s)
Esterasas/biosíntesis , Glucosa/metabolismo , Hepatocitos/enzimología , Metabolismo de los Lípidos , Lípidos/sangre , Proteínas de la Membrana/biosíntesis , Células 3T3-L1 , Adipocitos/enzimología , Animales , Modelos Animales de Enfermedad , Inducción Enzimática , Esterasas/genética , Ayuno/metabolismo , Femenino , Lipidómica , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Obesidad/enzimología , Obesidad/genética , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gamma/metabolismoRESUMEN
Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and the resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, confirmed by crystal structure analysis. The rate of covalent bond formation ( kinact) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK-mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.
Asunto(s)
Alquinos/farmacología , Catepsina K/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Alquinos/química , Catepsina K/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Humanos , Modelos Moleculares , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Poly (ADP-ribose) polymerase (PARP) inhibitors are a relatively new class of anticancer agents that have attracted attention for treatment of glioblastoma because of their ability to potentiate temozolomide chemotherapy. Previous studies have demonstrated that sufficient brain penetration is a prerequisite for efficacy of PARP inhibitors in glioma mouse models. Unfortunately, however, most of the PARP inhibitors developed to date have a limited brain penetration due to the presence of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) at the blood-brain barrier. AZD2461 is a novel PARP inhibitor that is unaffected by P-gp mediated resistance in breast cancer models and thus appears to have promising characteristics for brain penetration. We here use a comprehensive set of in vitro and in vivo models to study the brain penetration and oral bioavailability of AZD2461. We report that AZD2461 has a good membrane permeability. However, it is a substrate of P-gp and BCRP, and P-gp in particular limits its brain penetration in vivo. We show that AZD2461 has a low oral bioavailability, although it is not affected by P-gp and BCRP. Together, these findings are not in favor of further development of AZD2461 for treatment of glioblastoma.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/metabolismo , Ftalazinas/farmacocinética , Piperidinas/farmacocinética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Administración Oral , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Perros , Ensayos de Selección de Medicamentos Antitumorales , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Células de Riñón Canino Madin Darby , Ratones , Ratones Noqueados , Proteínas de Neoplasias/metabolismo , Permeabilidad , Ftalazinas/administración & dosificación , Piperidinas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificaciónRESUMEN
We describe here a user-friendly analysis protocol for semi-targeted polar metabolomics in human muscle biopsies using Zwitterionic Hydrophilic Interaction Liquid Chromatography and high-resolution full-scan mass spectrometry. Previously, this protocol has been used for Caenorhabditis elegans. Here we show that it can be successfully applied to human muscle biopsies with minor adjustments. Summarized instructions for other matrices are also provided. As peak integration in metabolomics can be challenging, we provide expected retention times and extensive peak descriptions to aid this process. For complete details on the use and execution of this protocol, please refer to Molenaars et al. (2021).