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

Acid ß-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-ß-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. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various ß-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.

1,6-epi-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-epi-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-glc-configured cyclophellitol cyclosulfamidate 4 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 ß-glucosidases GBA and GBA2 and glucosylceramide synthase (GCS). Cyclosulfamidate 4 stabilizes recombinant human GAA (rhGAA, alglucosidase alfa, Myozyme) in cell medium and plasma and facilitates enzyme trafficking to lysosomes. It stabilizes rhGAA more effectively than existing small-molecule chaperones and does so in vitro, in cellulo, and in vivo in zebrafish, thus representing a promising therapeutic alternative to Miglustat for Pompe disease.

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. A total of 22 features were included in the algorithms, including demographics, comorbidities, and major preoperative laboratory data from the database. The primary outcome was a composite of adverse events (in-hospital mortality, acute myocardial infarction, stroke, respiratory, hepatic and renal failure, and sepsis). Secondary outcomes were intensive care unit (ICU) admission and prolonged length of stay (PLOS). The data obtained were imported into 7 machine learning algorithms to predict the risk of adverse outcomes. Seventy percent of the data were randomly selected for training, leaving 30% for testing. The performances of the models were evaluated by the area under the receiver operating characteristic curve (AUROC). The optimal algorithm with the highest AUROC was used to build a web-based application, then integrated into the hospital information system (HIS) for clinical use. RESULTS: Data from 4,448 patients were analyzed; 102 (2.3%), 160 (3.6%), and 401 (9.0%) patients had primary composite adverse outcomes, ICU admission, and PLOS, respectively. Our optimal model had a superior performance (AUROC by DeLong test) than that of ASA-PS in predicting the primary composite outcomes (0.810 vs. 0.629, p < 0.01), ICU admission (0.835 vs. 0.692, p < 0.01), and PLOS (0.832 vs. 0.618, p < 0.01). CONCLUSIONS: The hospital-specific machine learning model outperformed the ASA-PS in risk assessment. This web-based application gained high satisfaction from anesthesiologists after online use.

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. GCase undergoes extensive modification of its four N-glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent activity-based probes (ABPs) that covalently label GCase in reaction-based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage-like RAW264.7 cells. Specific attention was paid to the impact of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate-buffered medium. Here, we report how HEPES-buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES-containing medium was also found to reduce maturation of other lysosomal enzymes (α-glucosidase and ß-glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD-patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD.

Synthesis of broad-specificity activity-based probes for exo-ß-mannosidases.

Exo-ß-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 exo-ß-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. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-ß-mannosidases, we present a concise synthesis of ß-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-ß-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-ß-mannosidases in biological and biomedical research.

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. RESULTS: Of the 7 studies (randomized controlled trials [RCTs], n = 4; non-RCTs, n = 3) that recruited 275 patients, 2 investigated female patients undergoing cesarean section and the other 5 were conducted in both obstetric and nonobstetric settings. Pooled results showed a lower mean pain score at 24 hours (i.e., primary outcome) (mean difference [MD] = -2.66, 95%: CI: -3.98 to -1.33, P < .001; I2 = 97%, 6 studies), 1 hour (MD = -4.23, 95% confidence interval [CI]: -5.08 to -3.37, P < .00001; I2 = 86%, 5 studies), and 6 hours (MD = -2.78, 95% CI: -4.99 to -0.57, P = .01; I2 = 98%, 4 studies) in patients with GONB compared to those without. Trial sequential analysis supported the robustness of evidence at postprocedural 24 hours. The use of GONB also decreased the risk of intervention failure (relative ratio [RR] = 0.4, 95% CI: 0.19 to 0.82, P = .01; I2 = 96%, 6 studies, 277 patients). CONCLUSION: Our results suggested a therapeutic effect of greater occipital nerve block against postdural puncture headache up to postprocedural 24 hours. Further large-scale studies are warranted to evaluate its therapeutic benefit beyond the acute stage.

Xylose-Configured Cyclophellitols as Selective Inhibitors for Glucocerebrosidase.

Glucocerebrosidase (GBA), a lysosomal retaining ß-d-glucosidase, has recently been shown to hydrolyze ß-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. GBA processes ß-d-xylosides in addition to ß-d-glucosides, this in contrast to the other two mammalian cellular retaining ß-d-glucosidases, GBA2 and GBA3. Here we show that the xylopyranose preference also holds up for covalent inhibitors: xylose-configured cyclophellitol and cyclophellitol aziridines selectively react with GBA over GBA2 and GBA3 in vitro and in vivo, and that the xylose-configured cyclophellitol is more potent and more selective for GBA than the classical GBA inhibitor, conduritol B-epoxide (CBE). Both xylose-configured cyclophellitol and cyclophellitol aziridine cause accumulation of glucosylsphingosine in zebrafish embryo, a characteristic hallmark of GD, and we conclude that these compounds are well suited for creating such chemically induced GD models.

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. Efficient and sensitive assays that report selectively on retaining amylase activities irrespective of the nature and complexity of the biomaterial studied are of great value both in finding new and effective human amylase inhibitors and in the discovery of new microbial amylases with potentially advantageous features for biotechnological application. Activity-based protein profiling (ABPP) of retaining glycosidases is inherently suited for the development of such an assay format. We here report on the design and synthesis of 1,6-epi-cyclophellitol-based pseudodisaccharides equipped with a suite of reporter entities and their use in ABPP of retaining amylases from human saliva, murine tissue as well as secretomes from fungi grown on starch. The activity and efficiency of the inhibitors and probes are substantiated by extensive biochemical analysis, and the selectivity for amylases over related retaining endoglycosidases is validated by structural studies.

Manno-epi-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 GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-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. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.

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. The relatively common glycosphingolipidosis Gaucher disease is highlighted here to discuss new insights in the molecular basis and pathophysiology of glycosphingolipidoses reached by fundamental research increasingly using chemical biology tools. We discuss improvements in the detection of glycosphingolipid metabolites by mass spectrometry and review new developments in laboratory diagnosis and disease monitoring as well as therapeutic interventions.

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

ß-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). Here, we aimed to clarify the role of zebrafish Gba2 in glycosphingolipid metabolism during Gba1 deficiency in zebrafish (Danio rerio), which are able to survive total Gba1 deficiency. We developed Gba1 (gba1-/-), Gba2 (gba2-/-), and double (gba1-/-:gba2-/-) zebrafish knockouts using CRISPR/Cas9 and explored the effects of both genetic and pharmacological interventions on GlcCer metabolism in individual larvae. Activity-based probes and quantification of relevant glycolipid metabolites confirmed enzyme deficiency. GlcSph increased in gba1-/- larvae (0.09 pmol/fish) but did not increase more in gba1-/-:gba2-/- larvae. GlcCer was comparable in gba1-/- and WT larvae but increased in gba2-/- and gba1-/-:gba2-/- larvae. Independent of Gba1 status, GlcChol was low in all gba2-/- larvae (0.05 vs. 0.18 pmol/fish in WT). Pharmacologic inactivation of zebrafish Gba1 comparably increased GlcSph. Inhibition of GlcCer synthase (GCS) in Gba1-deficient larvae reduced GlcCer and GlcSph, and concomitant inhibition of GCS and Gba2 with iminosugars also reduced excessive GlcChol. Finally, overexpression of human GBA1 and injection of recombinant GBA1 both decreased GlcSph. We determined that zebrafish larvae offer an attractive model to study glucosidase actions in glycosphingolipid metabolism in vivo, and we identified distinguishing characteristics of zebrafish Gba2 deficiency.

Reweighting of the sensory inputs for postural control in patients with cervical spondylotic myelopathy after surgery.

BACKGROUND: Cervical spondylotic myelopathy (CSM) is a degenerative cervical disease in which the spinal cord is compressed. Patients with CSM experience balance disturbance because of impaired proprioception. The weighting of the sensory inputs for postural control in patients with CSM is unclear. Therefore, this study investigated the weighting of sensory systems in patients with CSM. METHOD: Twenty-four individuals with CSM (CSM group) and 24 age-matched healthy adults (healthy control group) were analyzed in this observational study. The functional outcomes (modified Japanese Orthopaedic Association Scale [mJOA], Japanese Orthopaedic Association Cervical Myelopathy Questionnaire [JOACMEQ], Nurick scale) and static balance (eyes-open and eyes-closed conditions) were assessed for individuals with CSM before surgery, 3 and 6 months after surgery. Time-domain and time-frequency-domain variables of the center of pressure (COP) were analyzed to examine the weighting of the sensory systems. RESULTS: In the CSM group, lower extremity function of mJOA and Nurick scale significantly improved 3 and 6 months after surgery. Before surgery, the COP mean velocity and total energy were significantly higher in the CSM group than in the control group for both vision conditions. Compared with the control group, the CSM group exhibited lower energy content in the moderate-frequency band (i.e., proprioception) and higher energy content in the low-frequency band (i.e., cerebellar, vestibular, and visual systems) under the eyes-open condition. The COP mean velocity of the CSM group significantly decreased 3 months after surgery. The energy content in the low-frequency band (i.e., visual and vestibular systems) of the CSM group was closed to that of the control group 6 months after surgery under the eyes-open condition. CONCLUSION: Before surgery, the patients with CSM may have had compensatory sensory weighting for postural control, with decreased weighting on proprioception and increased weighting on the other three sensory inputs. After surgery, the postural control of the patients with CSM improved, with decreased compensation for the proprioceptive system from the visual and vestibular inputs. However, the improvement remained insufficient because the patients with CSM still had lower weighting on proprioception than the healthy adults did. Therefore, patients with CSM may require balance training and posture education after surgery. TRIAL REGISTRATION: Trial Registration number: NCT03396055 Name of the registry: ClinicalTrials.gov Date of registration: January 10, 2018 - Retrospectively registered Date of enrolment of the first participant to the trial: October 19, 2015.

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 ß-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. Here we demonstrate that cyclophellitol derivatives carrying a bulky hydrophobic substituent at C8 are potent and selective GBA inhibitors and that an unambiguous Gaucher animal model can be readily generated by treatment of zebrafish with these.

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

Glucocerebrosidase (GBA) is a lysosomal ß-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). Conduritol B epoxide (CBE), a mechanism-based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity-based probes to visualize catalytic pocket occupancy of candidate off-target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α-glucosidase were identified as major off-targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD-like models. ENZYMES: Glucocerebrosidase (EC 3.2.1.45), nonlysosomal ß-glucocerebrosidase (EC 3.2.1.45); cytosolic ß-glucosidase (EC 3.2.1.21); α-glucosidases (EC 3.2.1.20); ß-glucuronidase (EC 3.2.1.31).

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® ). The structures of IDUA when complexed with the inhibitors in a non-covalent transition state mimicking form and a covalent enzyme-bound form provide insights into its conformational itinerary. Inhibitors 1-3 adopt a half-chair conformation in solution (4 H3 and 3 H4 ), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1-3 may need to overcome an energy barrier in order to switch from the 4 H3 conformation to the transition state (2, 5 B) binding conformation before reacting and adopting a covalent 5 S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.

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 ß-glucosidase (glucocerebrosidase, GBA) and resulting in pathological lysosomal storage of glucosylceramide (glucocerebroside) in tissue macrophages. GBA is a retaining glucosidase with a characteristic glycosyl-enzyme intermediate formed during catalysis. Using the natural suicide inhibitor cyclophellitol as a lead, we developed mechanism-based irreversible inhibitors of GBA equipped with a fluorescent reporter. These reagents covalently link to the catalytic nucleophile residue of GBA and permit specific and sensitive visualization of active enzyme molecules. The amphiphilic activity-based probes (ABPs) allow in situ detection of active GBA in cells and organisms. Furthermore, they may be used to biochemically confirm the diagnosis of Gaucher disease and they might assist in screening for small compounds interacting with the catalytic pocket. While the focus of this chapter is ABPs for ß-glucosidases and Gaucher disease, the described concept has meanwhile been extended to other retaining glycosidases and related disease conditions as well.

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 ß-glucosidases and ß-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. A focused library is generated based on leads from the FluoPol-ABPP screen and assessed on GBA2 selectivity offset against the other glucosylceramide metabolizing enzymes, glucosylceramide synthase (GCS), lysosomal glucosylceramidase (GBA), and the cytosolic retaining ß-glucosidase, GBA3. Our work, yielding potent and selective GBA2 inhibitors, also provides a roadmap for the development of high-throughput assays for identifying retaining glycosidase inhibitors by FluoPol-ABPP on cell extracts containing recombinant, overexpressed glycosidase as the easily accessible enzyme source.