Further observations on the activation of lysosomal acid alpha-glucosidase by cortisone derivatives.
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ABSTRACT:
SUBMITTER: Bourne EJ
PROVIDER: S-EPMC1177606 | biostudies-other | 1973 Mar
REPOSITORIES: biostudies-other
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Project description:Cortisone causes a marked increase in the activity of liver acid alpha-glucosidase 2h after injection into male Wistar rats. Studies on rat liver tissue slices, isolated lysosomes and cultured skin fibroblasts have demonstrated similar elevations of acid alpha-glucosidase activity after incubation with cortisone. Cortisone-treated human liver tissue, obtained by needle biopsy, also shows an increase in acid alpha-glucosidase activity. Neutral alpha-glucosidase activity was not stimulated by cortisone in vivo or in liver slices.
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Project description:1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.
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Project description:Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1-C-heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC50 value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1-C-alkyl chain. It is noteworthy that α-1-C-heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.
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Project description:Lysosomal degradation plays a crucial role in the metabolism of biological macromolecules supplied by autophagy. The regulation of the autophagy-lysosome system, which contributes to intracellular homeostasis, chemoresistance, and tumor progression, has recently been revealed as a promising therapeutic approach for pancreatic cancer (PC). However, the details of lysosomal catabolic function in PC cells have not been fully elucidated. In this study, we show evidence that suppression of acid alpha-glucosidase (GAA), one of the lysosomal enzymes, improves chemosensitivity and exerts apoptotic effects on PC cells through the disturbance of expression of the transcription factor EB. The levels of lysosomal enzyme were elevated by gemcitabine in PC cells. In particular, the levels of GAA were responsive to gemcitabine in a dose-dependent and time-dependent manner. Small interfering RNA against the GAA gene (siGAA) suppressed cell proliferation and promoted apoptosis in gemcitabine-treated PC cells. In untreated PC cells, we observed accumulation of depolarized mitochondria. Gene therapy using adenoviral vectors carrying shRNA against the GAA gene increased the number of apoptotic cells and decreased the tumor growth in xenograft model mice. These results indicate that GAA is one of the key targets to improve the efficacy of gemcitabine and develop novel therapies for PC.
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Project description:Lysosomal degradation plays a crucial role in the metabolism of biological macromolecules supplied by autophagy. Recent evidence demonstrated the regulation of the autophagy-lysosome system, which contributes to intracellular homeostasis, chemoresistance, and tumor progression, as a promising therapeutic approach for pancreatic cancer (PC). However, the details of lysosomal catabolic function in PC cells is not fully elucidated. In this study, we show evidence that suppression of acid alpha-glucosidase (GAA), one of the lysosomal enzymes, improves chemosensitivity and exerts apoptotic effects on PC cells through the disturbance of mitophagy via migration of the transcription factor EB. The levels of lysosomal enzyme were elevated by gemcitabine in PC cells. In particular, the levels of GAA were responsive to gemcitabine in a dose- and time-dependent manner. Small interfering RNA against the GAA gene (siGAA) suppressed cell proliferation and promoted apoptosis in gemcitabine-treated PC cells. In untreated PC cells, we observed accumulation of depolarized mitochondria. Gene therapy using adenoviral vector carrying short hairpin RNA against the GAA gene increased the number of apoptotic cells and decreased the tumor growth in xenograft model mice. These results indicate that GAA is one of the key targets to improve the efficacy of gemcitabine and develop novel therapies for PC.
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Project description:The gene coding for human lysosomal alpha-glucosidase was cloned and its structure was determined. The gene is approx. 20 kb long, and contains 20 exons. The first exon is non-coding. The coding sequence of the putative catalytic site domain is interrupted in the middle by an intron of 101 bp. This intron is not conserved in the highly similar region of the human and rabbit isomaltase genes. The promoter region was defined by a CAT assay and the start of the mRNA was determined by primer extension. The promoter has features characteristic of a 'housekeeping' gene. The GC content is high (80%) and distinct TATA and CCAAT motifs are lacking. Two potential binding sites for the AP-2 transcription factor are present. Four potential Sp-1 binding sites are located downstream of the 5' end of the mRNA.
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Project description:Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC50 of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC50 of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.HighlightsOleanolic acid oxime ester derivatives (3a-3t) were synthesised and screened against α-glucosidase and α-amylase.Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.
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Project description:Coumarin and chalcone, two important kinds of natural product skeletons, both exhibit α-glucosidase inhibitory activity. In this work, coumarin-chalcone derivatives 3 (a∼v) were synthesized, and their α-glucosidase inhibitory activity was screened. The results showed that all synthetic derivatives (IC50: 24.09 ± 2.36 to 125.26 ± 1.18 μM) presented better α-glucosidase inhibitory activity than the parent compounds 3-acetylcoumarin (IC50: 1.5 × 105 μM) and the positive control acarbose (IC50: 259.90 ± 1.06 μM). Among them, compound 3t displayed the highest α-glucosidase inhibitory activity (IC50: 24.09 ± 2.36 μM), which was approximately 10 times stronger than that of acarbose. The kinetic assay of 3t (KI = 18.82 μM, KIS = 59.99 μM) revealed that these compounds inhibited α-glucosidase in a mixed-type manner. Molecular docking was used to simulate the interaction between α-glucosidase and compound 3t.
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Project description:Embelin is a naturally occurring para-benzoquinone isolated from Embelia ribes (Burm. f.) of the Myrsinaceae family. It was first discovered to have potent inhibitory activity (IC50 = 4.2 μM) against α-glucosidase in this study. Then, four series of novel embelin derivatives were designed, prepared and evaluated in α-glucosidase inhibition assays. The results show that most of the embelin derivatives synthesised are effective α-glucosidase inhibitors, with IC50 values at the micromolar level, especially 10d, 12d, and 15d, the IC50 values of which are 1.8, 3.3, and 3.6 μM, respectively. Structure-activity relationship (SAR) studies suggest that hydroxyl groups in the 2/5-position of para-benzoquinone are very important, and long-chain substituents in the 3-position are highly preferred. Moreover, the inhibition mechanism and kinetics studies reveal that all of 10d, 12d, 15d, and embelin are reversible and mixed-type inhibitors. Furthermore, docking experiments were carried out to study the interactions between 10d and 15d with α-glucosidase.
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Project description:Acid alpha-glucosidase purified from pig liver showed heterogeneity in its affinity to Sephacryl S-200 gel. Acid alpha-glucosidase was separated into two fractions (S1 and S2) by Sephacryl S-200 affinity chromatography. Each fraction contained components at apparent 76 kDa and 67 kDa on SDS/PAGE. The amount of S1 fraction was about 1.3 times that of the S2 fraction. In the kidney the ratio of S1 to S2 fraction was similar to that in the liver. However, the heart contained 1.3 times as much S2 fraction as S1 fraction. The spleen acid alpha-glucosidase consisted mainly of S1 fraction, containing only a 76 kDa component. Immunohistochemically, acid alpha-glucosidase was demonstrated in the macrophages of the spleen. Thus the 76 kDa component in the spleen must come mainly from the macrophages. Lectin-binding analysis was carried out on the components present in the S1 and S2 fractions after electrophoresis and transfer to nitrocellulose sheets. Slight differences in binding observed suggest differences in the structure of the sugar side chains.
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