Preclinical efficacy and safety of adeno-associated virus 5 alpha-galactosidase: A gene therapy for Fabry disease.

We developed a novel adeno-associated virus 5 gene therapy (AAV5-GLA) expressing human alpha-galactosidase A (GLA) under the control of a novel, small and strong, liver-restricted promoter. We assessed the preclinical potential of AAV5-GLA for treating Fabry disease, an X-linked hereditary metabolic disorder resulting from mutations in the gene encoding GLA that lead to accumulation of the substrates globotriaosylceramide and globotriaosylsphingosine, causing heart, kidney, and central nervous system dysfunction. Effects of intravenously administered AAV5-GLA were evaluated in (1) GLA-knockout mice aged 7-8 weeks (early in disease) and 20 weeks (nociception phenotype manifestation) and (2) cynomolgus macaques during an 8-week period.

The development of a broad-spectrum retaining β-exo-galactosidase activity-based probe.

Acid β-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-β-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease.

1,6--Cyclophellitol Cyclosulfamidate Is a Bona Fide Lysosomal α-Glucosidase Stabilizer for the Treatment of Pompe Disease.

α-Glucosidase inhibitors are potential therapeutics for the treatment of diabetes, viral infections, and Pompe disease. Herein, we report a 1,6--cyclophellitol cyclosulfamidate as a new class of reversible α-glucosidase inhibitors that displays enzyme inhibitory activity by virtue of its conformational mimicry of the substrate when bound in the Michaelis complex. The α-d-configured cyclophellitol cyclosulfamidate binds in a competitive manner the human lysosomal acid α-glucosidase (GAA), ER α-glucosidases, and, at higher concentrations, intestinal α-glucosidases, displaying an excellent selectivity over the human β-glucosidases GBA and GBA2 and glucosylceramide synthase (GCS).

Implementation of a machine learning application in preoperative risk assessment for hip repair surgery.

Background: This study aims to develop a machine learning-based application in a real-world medical domain to assist anesthesiologists in assessing the risk of complications in patients after a hip surgery.

Methods: Data from adult patients who underwent hip repair surgery at Chi-Mei Medical Center and its 2 branch hospitals from January 1, 2013, to March 31, 2020, were analyzed. Patients with incomplete data were excluded.

HEPES-buffering of bicarbonate-containing culture medium perturbs lysosomal glucocerebrosidase activity.

Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease.

Synthesis of broad-specificity activity-based probes for -β-mannosidases.

-β-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of -β-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds.

Efficacy of greater occipital nerve block for pain relief in patients with postdural puncture headache: A meta-analysis.

Background: This study aimed at assessing the therapeutic effectiveness of greater occipital nerve block (GONB) against postdural puncture headache (PDPH).

Methods: Studies investigating analgesic effects of GONB against PDPH in adults were retrieved from the MEDLINE, EMBASE, Google scholar, and Cochrane central databases from their inception dates to May, 2021. Pain score at postprocedural 24 hours was the primary endpoint, while secondary endpoints were pain score at postprocedural 1 hour and 12 hours as well as the risk of intervention failure.

Xylose-Configured Cyclophellitols as Selective Inhibitors for Glucocerebrosidase.

Glucocerebrosidase (GBA), a lysosomal retaining β-d-glucosidase, has recently been shown to hydrolyze β-d-xylosides and to transxylosylate cholesterol. Genetic defects in GBA cause the lysosomal storage disorder Gaucher disease (GD), and also constitute a risk factor for developing Parkinson's disease. GBA and other retaining glycosidases can be selectively visualized by activity-based protein profiling (ABPP) using fluorescent probes composed of a cyclophellitol scaffold having a configuration tailored to the targeted glycosidase family.

Activity-Based Protein Profiling of Retaining α-Amylases in Complex Biological Samples.

Amylases are key enzymes in the processing of starch in many kingdoms of life. They are important catalysts in industrial biotechnology where they are applied in, among others, food processing and the production of detergents. In man amylases are the first enzymes in the digestion of starch to glucose and arguably also the preferred target in therapeutic strategies aimed at the treatment of type 2 diabetes patients through down-tuning glucose assimilation.

Manno--cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcManGlcNAc to produce GlcNAcManGlcNAc, the precursor for all complex -glycans, including the branched -glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme.

Glycosphingolipids and lysosomal storage disorders as illustrated by gaucher disease.

Glycosphingolipids are important building blocks of the outer leaflet of the cell membrane. They are continuously recycled, involving fragmentation inside lysosomes by glycosidases. Inherited defects in degradation cause lysosomal glycosphingolipid storage disorders.

Role of β-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish.

β-glucosidases [GBA1 (glucocerebrosidase) and GBA2] are ubiquitous essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease, a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph). GBA1 and GBA2 also may transfer glucose from GlcCer to cholesterol, yielding glucosylated cholesterol (GlcChol).

Functionalized Cyclophellitols Are Selective Glucocerebrosidase Inhibitors and Induce a Bona Fide Neuropathic Gaucher Model in Zebrafish.

Gaucher disease is caused by inherited deficiency in glucocerebrosidase (GBA, a retaining β-glucosidase), and deficiency in GBA constitutes the largest known genetic risk factor for Parkinson's disease. In the past, animal models of Gaucher disease have been generated by treatment with the mechanism-based GBA inhibitors, conduritol B epoxide (CBE), and cyclophellitol. Both compounds, however, also target other retaining glycosidases, rendering generation and interpretation of such chemical knockout models complicated.

In vivo inactivation of glycosidases by conduritol B epoxide and cyclophellitol as revealed by activity-based protein profiling.

Glucocerebrosidase (GBA) is a lysosomal β-glucosidase-degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD).

New Irreversible α-l-Iduronidase Inhibitors and Activity-Based Probes.

Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination of l-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-l-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-l-iduronidase (rIDUA, Aldurazyme ).

Activity-Based Probes for Glycosidases: Profiling and Other Applications.

Glycosidases mediate the fragmentation of glycoconjugates in the body, including the vital recycling of endogenous molecules. Several inherited diseases in man concern deficiencies in lysosomal glycosidases degrading glycosphingolipids. Prominent is Gaucher disease caused by an impaired lysosomal β-glucosidase (glucocerebrosidase, GBA) and resulting in pathological lysosomal storage of glucosylceramide (glucocerebroside) in tissue macrophages.

Towards broad spectrum activity-based glycosidase probes: synthesis and evaluation of deoxygenated cyclophellitol aziridines.

Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of β-glucosidases and β-galactosidases.

A Fluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human Nonlysosomal Glucosylceramidase.

Human nonlysosomal glucosylceramidase (GBA2) is one of several enzymes that controls levels of glycolipids and whose activity is linked to several human disease states. There is a major need to design or discover selective GBA2 inhibitors both as chemical tools and as potential therapeutic agents. Here, we describe the development of a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) assay for the rapid identification, from a 350+ library of iminosugars, of GBA2 inhibitors.

1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor.

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors.

Correction to "Detection of Active Mammalian GH31 α-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes".

[This corrects the article DOI: 10.1021/acscentsci.6b00057.

Activity-based probes for functional interrogation of retaining β-glucuronidases.

Humans express at least two distinct β-glucuronidase enzymes that are involved in disease: exo-acting β-glucuronidase (GUSB), whose deficiency gives rise to mucopolysaccharidosis type VII, and endo-acting heparanase (HPSE), whose overexpression is implicated in inflammation and cancers. The medical importance of these enzymes necessitates reliable methods to assay their activities in tissues. Herein, we present a set of β-glucuronidase-specific activity-based probes (ABPs) that allow rapid and quantitative visualization of GUSB and HPSE in biological samples, providing a powerful tool for dissecting their activities in normal and disease states.

Detection of Active Mammalian GH31 α-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes.

The development of small molecule activity-based probes (ABPs) is an evolving and powerful area of chemistry. There is a major need for synthetically accessible and specific ABPs to advance our understanding of enzymes in health and disease. α-Glucosidases are involved in diverse physiological processes including carbohydrate assimilation in the gastrointestinal tract, glycoprotein processing in the endoplasmic reticulum (ER), and intralysosomal glycogen catabolism.

Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS.

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described.