A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS. / Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.

A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS. / Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.

A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS. / Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.

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No description available.

Im Rahmen dieser Arbeit wurden fluoreszierende Sphingomyelin-Analoga zu Studium der sauren Sphingomyelinase (ASM) synthetisiert. Ausgehend von L-Serin wurde ein Sphingosin-Derivat mit natürlicher Stereochemie dargestellt. Anschließend wurde mittels Phosphorodichloridat-Chemie eine Aminoethylphosphat-Gruppe installiert. Zweifache Fluoreszenzmarkierung ergab Sonden mit der Fähigkeit zu Förster-Resonanzenergietransfer (FRET). Diese wurden als Substrate der ASM akzeptiert und erlaubten die Verfolgung der Enzymaktivität in vitro. Durch die Analyse der photophysikalischen Eigenschaften der Fluorophore wurde das allgemeine Konzept der Phasentrennungs-gestützten Signalverstärkung (PS) abgeleitet. Dieses Konzept wurde erfolgreich bestätigt durch die Synthese einer 30-mal leistungsfähigeren zweiten Generation der FRET-Sonde. Ein homogener Assay wurde entwickelt, der die Quantifizierung der ASM-Aktivität erlaubte. Unter Verwendung von gereinigter rekombinanter humaner ASM, HeLa-Zelllysaten oder Lysaten von murinen embryonalen Fibroblasten (MEFs) als Enzymquelle wurde ausschließlich unter den von der ASM bevorzugten Bedingungen eine vollständige und spezifische Hydrolyse der Sonde beobachtet. Des Weiteren erlaubte die Sonde die Detektion relativer Unterschiede der Aktivität der ASM in kultivierten MEFs mittels Fluoreszenzmikroskopie mit Zweiphotonenanregung (2PE). / Fluorescent sphingomyelin analogues have been synthesized to probe the acid sphingomyelinase (ASM). Starting from L-serine, a sphingosine with natural stereochemistry was synthesized. Subsequently, phosphorodichloridate chemistry was used to install an aminoethyl phosphate moiety. Dual fluorescent labeling afforded probes capable of Förster resonance energy transfer (FRET). They were recognized as substrates of ASM and allowed for monitoring of the enzyme’s activity in vitro. Through analysis of the fluorophores’ photophysical properties, the general concept of partition aided amplification of a FRET probe’s signal (PS) was developed. This concept was successfully confirmed by the synthesis of a second-generation probe with 30-fold improved response. A homogenous assay was developed, which allowed for a quantitation of ASM activity. Using either purified recombinant human ASM, or lysates of HeLa cells or mouse embryonic fibroblasts (MEFs) as an enzyme source, complete and specific cleavage was observed exclusively under conditions preferred by ASM. Furthermore, the probe enabled the detection of relative levels of ASM activity in cultivated MEFs using fluorescence microscopy with two-photon excitation (2PE).

Indiana University-Purdue University Indianapolis (IUPUI) / Autophagy is a conserved cellular process that involves sequestration and degradation of cytosolic contents. The cell can engulf autophagic cargo (lipids, long-lived proteins, protein aggregates, and pathogens) through a double bound membrane called an autophagosome that fuses with a lysosome where hydrolases then degrade these contents. This process is one of the main defenses against starvation and is imperative for newborns at birth. Research on this process has increased exponentially in the last decade since its discovery almost a half a century ago. It has been found that autophagy is an important process in many diseases, continues to be at the forefront of research, and is clearly not fully understood. Our preliminary cell culture data in endothelial and epithelial cells show that a blockade of the de novo ceramide synthesis pathway, during treatment with an autophagy stimulus (cigarette smoke extract exposure), does not result in any reduction in autophagy or autophagic flux. Conversely, when acid sphingomyelinase (ASM) is pharmacologically inhibited, which prevents the generation of ceramide from sphingomyelin in an acidic environment, a profound increase in autophagy is observed. In this work, we hypothesize that (ASM) is an endogenous inhibitor of autophagy. ASM has two forms, a secreted form and a lysosomal form. N-terminal processing in the Golgi determines its cellular fate. In the lysosomal form, the phosphodiesterase is bound in the lysosomal membrane. The pharmacological inhibition mechanism is to release ASM from the membrane and allow other hydrolases to actively degrade the enzyme which, in turn, decreases the activity of ASM. This suggests that either the activity of ASM is a regulator of autophagy or that the presence of ASM, activity aside, is required for the lysosomal nutrient sensing machinery (LYNUS) to function properly. Here, we show that ASM is, in fact, an endogenous inhibitor of autophagy in vitro. The phosphorylation status of P70 S6k, a downstream effector of mammalian target of rapamycin (mTOR), which is part of the LYNUS, shows that dissociation of ASM from the membrane regulates mTOR and disturbs the LYNUS in such a manner as to signal autophagy.

Ceramid, ein zentrales Signalmolekül des Sphingolipidstoffwechsels, ist neben der de novo Synthese über die enzymatische Spaltung von Sphingomyelin und Glucosylceramid zugänglich. Genetische Mutationen, die eine Fehlfaltung der verantwortlichen Enzyme saure Sphingomyelinase (aSMase) und Glucocerebrosidase (GCase) begünstigen, könnten somit zu einer Dysregulation des gesamten Sphingolipidstoffwechsels und den damit verbundenen Signaltransduktionsprozessen führen. Niedermolekulare Inhibitoren können in Zellstudien einen Einblick in diese Prozesse geben und den Defekt eines Enzyms simulieren oder eine etwaige Überaktivität derselben Enzyme verhindern. Für derartige Studien ist die Möglichkeit einer zeitaufgelösten Inhibition von Vorteil. Für diese Methode müssten photolabile Schutzgruppen in eine bereits bekannte Inhibitorstruktur integriert werden. Im Fall der aSMase würden sich hierfür myo-Inosit-bisphosphat-Derivate anbieten, die starke, kompetitive Inhibitoren des Enzyms darstellen. Auf dieser Grundlage werden in der vorliegenden Arbeit die Synthese sowie die in vitro und in cellulo Wirkung des ersten zellpermeablen, photoaktivierbaren Inhibitors für die aSMase präsentiert. Kompetitive Inhibitoren können ebenso als sogenannte pharmakologische Chaperone fungieren, welche Proteine durch Herabsetzung der freien Energie des jeweiligen Faltungszustandes stabilisieren. Dies ist besonders bei von Mutationen betroffenen lysosomalen Enzymen von Interesse, um diese vor einem proteasomalen Abbau zu bewahren und einen geregelten Transport in die Lysosomen zu gewährleisten. So wurden in der vorliegenden Arbeit verschiedene myo-Inositderivate als potenzielle pharmakologische Chaperone für die aSMase und GCase synthetisiert. Um eine Verdrängung der Verbindungen vom aktiven Zentrum des Enzyms durch das natürliche Substrat zu beschleunigen, wurde eine Orthoesterfunktion in die Seitenkette der Inhibitorstruktur integriert, die im sauren Milieu der Lysosomen gespalten werden kann. / Ceramide, a central signaling molecule in sphingolipid metabolism, is in addition to the novo synthesis accessible via the enzymatic cleavage of sphingomyelin and glucosylceramide. Genetic mutations that promote misfolding of the responsible enzymes acid sphingomyelinase (aSMase) and glucocerebrosidase (GCase) could thus lead to a dysregulation of the entire sphingolipid metabolism and the associated signal transduction processes. Small molecule inhibitors can provide insight into these processes in cell studies and simulate the defect of an enzyme or prevent eventual overactivity of the same enzyme. For such studies, the possibility of a time-resolved inhibition would be advantageous. For this method, photolabile protecting groups would have to be integrated into the structure of a known inhibitor. In the case of aSMase, myo-inositol-diphosphate derivatives, which represent strong, competitive inhibitors of the enzyme, would be suitable for this purpose. On this basis, the synthesis as well as the in vitro and in cellulo effects of the first cell-permeable photocaged inhibitor for acid sphingomyelinase are presented in this work. Competitive inhibitors can also act as so-called pharmacological chaperones, which stabilize proteins by reducing the free energy of the respective folding state. This is of particular interest in the case of lysosomal enzymes affected by mutations, in order to protect them from proteasomal degradation and to ensure regulated transport into the lysosomes. In the present work, various myo-inositol derivatives were synthesized as potential pharmacological chaperones for aSMase and GCase. To accelerate displacement of the compounds from the enzyme's active site by the natural substrate, an orthoester function was integrated into the side chain of the inhibitor structure, which can be cleaved in the acidic environment of the lysosome.