Tool Compounds Robustly Increase Turnover of an Artificial Substrate by Glucocerebrosidase in Human Brain Lysates

Berger, Zdenek; Perkins, Sarah E.; Ambroise, Claude; Oborski, Christine; Calabrese, Matthew F.; Noell, Stephen; Riddell, David; Hirst, Warren D.

doi:10.1371/journal.pone.0119141

Cited by 12 publications

(9 citation statements)

References 27 publications

(42 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…26 In previous studies, high concentrations of taurocholate (4−10 mM) were used to improve the signal in GCase enzyme activity assays. 16,27,28 We found that taurocholate can interfere with our assay results, which was also reported recently by Berger et al; 29 therefore, we excluded taurocholate in our screening assay. Using this approach, we discovered several different scaffolds of GCase inhibitors and activators with moderate activity.…”

Section: ■ Results and Discussionsupporting

confidence: 82%

Design and Synthesis of Potent Quinazolines as Selective β-Glucocerebrosidase Modulators

et al. 2016

View full text Add to dashboard Cite

Gaucher’s disease is a common genetic disease caused by mutations in the β-glucocerebrosidase (GBA1) gene that have been also linked to increased risk of Parkinson’s disease and Lewy Body Dementia. Stabilization of misfolded mutant β-glucocerebrosidase (GCase) represents an important therapeutic strategy in synucleinopathies. Here we report a novel class of GCase quinazoline inhibitors, obtained in a high throughput screening, with moderate potency against wild-type GCase. Rational design and a SAR study of this class of compounds led to a new series of quinazoline derivatives with single digit nanomolar potency. These compounds were shown to selectively stabilize GCase when compared to other lysosomal enzymes and to increase N370S mutant GCase protein concentration and activity in cell assays. To the best of our knowledge, these molecules are the most potent non-iminosugar GCase inhibitors to date that may prove useful for future mechanistic studies and therapeutic approaches in Gaucher’s and Parkinson’s diseases.

show abstract

Section: ■ Results and Discussionsupporting

confidence: 82%

Design and Synthesis of Potent Quinazolines as Selective β-Glucocerebrosidase Modulators

et al. 2016

View full text Add to dashboard Cite

show abstract

“…S1). Isofagomine selectively inhibits GBA1 at low nanomolar concentration, but not the other glucosidases, GBA2 and GBA3 (35,36). This enabled us to study whether inhibition of GBA1 activity directly affects the SC glucosyl (ceramide) composition.…”

mentioning

confidence: 99%

In situ visualization of glucocerebrosidase in human skin tissue: zymography versus activity-based probe labeling

Smeden

Dijkhoff

Helder

et al. 2017

Journal of Lipid Research

View full text Add to dashboard Cite

Epidermal β-glucocerebrosidase (GBA1), an acid β-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis: ) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods using either substrate 4-methylumbelliferyl-β-D-glucopyranoside or resorufin-β-D-glucopyranoside; and) a novel technique to visualize active GBA1 molecules by their specific labeling with a fluorescent activity-based probe (ABP), MDW941. The latter method pro-ved to be more robust and sensitive, provided higher resolution microscopic images, and was less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various β-glucosidases, MDW941 specifically labeled GBA1. We demonstrate that active GBA1 in the epidermis is primarily located in the extracellular lipid matrix at the interface of the viable epidermis and the lower layers of the SC. With ABP-labeling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with the reversible inhibitor, isofagomine, irrespective of GBA expression. This inhibition affected the SC ceramide composition: MS analysis revealed an inhibitor-dependent increase in the glucosylceramide:ceramide ratio.

show abstract

“…As previously shown for GCase modulators there can be a variability in potency across assays, in particular in cell‐free assays [17, 19] . Thus, we developed a cell‐based activity assay to determine the increase in human GCase activity selectively.…”

Section: Resultsmentioning

confidence: 99%

“…As previously shown for GCase modulators there can be avariability in potencyacross assays,inparticular in cell-free assays. [17,19] Thus,w ed eveloped ac ell-based activity assay to determine the increase in human GCase activity selectively. We confirmed afterwards in an orthogonal assay,w hether putative cellular activators had the capability to bind to the enzyme.…”

Section: Resultsmentioning

confidence: 99%

Novel β‐Glucocerebrosidase Activators That Bind to a New Pocket at a Dimer Interface and Induce Dimerization

et al. 2021

View full text Add to dashboard Cite

Genetic,p reclinical and clinical data link Parkinsonsd isease and Gauchersd isease and providearational entry point to disease modification therapyvia enhancement of b-Glucocerebrosidase (GCase) activity.W ed iscoveredanew class of pyrrolo[2,3-b]pyrazine activators effecting both Vmax and Km. They bind to human GCase and increase substrate metabolism in the lysosome in acellular assay. We obtained the first crystal structure for an activator and identified an ovel non-inhibitory binding mode at the interface of ad imer, rationalizing the observed structure-activity relationship (SAR). The compound binds GCase inducing formation of ad imeric state at both endoplasmic reticulum (ER) and lysosomal pHs,asconfirmed by analytical ultracentrifugation. Importantly,the pyrrolo[2,3-b]pyrazines have central nervous system (CNS) drug-like properties.Our findings are important for future drug discovery efforts in the field of GCase activation and provideadeeper mechanistic understanding of the requirements for enzymatic activation, pointing to the relevance of dimerization.

show abstract

Tool Compounds Robustly Increase Turnover of an Artificial Substrate by Glucocerebrosidase in Human Brain Lysates

Cited by 12 publications

References 27 publications

Design and Synthesis of Potent Quinazolines as Selective β-Glucocerebrosidase Modulators

Design and Synthesis of Potent Quinazolines as Selective β-Glucocerebrosidase Modulators

In situ visualization of glucocerebrosidase in human skin tissue: zymography versus activity-based probe labeling

Novel β‐Glucocerebrosidase Activators That Bind to a New Pocket at a Dimer Interface and Induce Dimerization

Contact Info

Product

Resources

About