Validation of a highly sensitive HaloTag-based assay to evaluate the potency of a novel class of allosteric β-Galactosidase correctors

Rudinskiy, Mikhail; Pons-Vizcarra, Maria; Soldà, Tatiana; Fregno, Ilaria; Bergmann, Timothy Jan; Ruano, Ana; Delgado, Aida; Morales, Sara; Barril, Xavier; Bellotto, Manolo; Cubero, Elena; García-Collazo, Ana María; Pérez-Carmona, Natalia; Molinari, Maurizio

doi:10.1371/journal.pone.0294437

Cited by 5 publications

References 53 publications

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Use of the Novel Site-Directed Enzyme Enhancement Therapy (SEE-Tx) Drug Discovery Platform to Identify Pharmacological Chaperones for Glutaric Acidemia Type 1

Barroso,

Puchwein-Schwepcke,

Buettner

et al. 2024

J. Med. Chem.

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Allosteric regulators acting as pharmacological chaperones hold promise for innovative therapeutics since they target noncatalytic sites and stabilize the folded protein without competing with the natural substrate, resulting in a net gain of function. Exogenous allosteric regulators are typically more selective than active site inhibitors and can be more potent than competitive inhibitors when the natural substrate levels are high. To identify novel structure-targeted allosteric regulators (STARs) that bind to and stabilize the mitochondrial enzyme glutaryl-CoA dehydrogenase (GCDH), the computational site-directed enzyme enhancement therapy (SEE-Tx) technology was applied. SEE-Tx is an innovative drug discovery platform with the potential to identify drugs for treating protein misfolding disorders, such as glutaric acidemia type 1 (GA1) disease. Putative allosteric regulators were discovered using structure-and ligand-based virtual screening methods and validated using orthogonal biophysical and biochemical assays. The computational approach presented here could be used to discover allosteric regulators of other protein misfolding disorders.

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Use of the Novel Site-Directed Enzyme Enhancement Therapy (SEE-Tx) Drug Discovery Platform to Identify Pharmacological Chaperones for Glutaric Acidemia Type 1

Barroso,

Puchwein-Schwepcke,

Buettner

et al. 2024

J. Med. Chem.

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Pharmacological GCase Activity Enhancement Inhibits Tau Accumulation

Ciccaldo,

Pérez-Carmona,

Piovesana

et al. 2024

Preprint

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A slow decline in the autophagy-lysosomal pathway is a hallmark of the normal aging brain. Yet, an acceleration of this cellular function may propel neurodegenerative events. In fact, mutations in genes associated with the autophagy-lysosomal pathway can lead to Parkinson’s disease. Also, amyloidogenic protein deposition is observed in lysosomal storage disorders, which are caused by genetic mutations representing risk factors for Parkinson’s disease. For example, Gaucher’s diseaseGBA1mutations leading to defects in lysosomal sphingolipid metabolism cause α-synuclein accumulation. We observed that increased lysosomal Tau accumulation is found in human dermal fibroblasts engineered for inducible Tau expression. Inhibition of theGBA1product GCase augmented Tau-dependent lysosomal stress and Tau accumulation. Here, we show increased Tau seed-induced Tau accumulation in Gaucher’s fibroblasts carryingGBA1mutations when compared to normal fibroblasts. Pharmacological enhancement of GCase reversed this effect, notably, also in normal fibroblasts. This suggests that boosting GCase activity may represent a therapeutic strategy to slow down aging-dependent lysosomal deficits and brain protein deposition.

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A novel class of allosteric glucosylceramidase beta 1 correctors that reduce cellular stress and enhance lysosomal function

Fregno,

Pérez-Carmona,

Rudinskiy

et al. 2024

Preprint

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Mutations in glucosylceramidase beta 1 (GCase) disrupt the protein conformational maturation in the endoplasmic reticulum (ER) and hinder its transport to the lysosome. The intralysosomal accumulation of glucocerebrosides, which are substrates of the GCase enzyme, impairs lysosomal function and is linked to Gaucher disease (GD). GCase mutations also increase the risk of Parkinson disease (PD) and Dementia with Lewy Bodies. We used Site-directed Enzyme Enhancement Therapy (SEE-Tx) technology to design two structurally targeted allosteric regulators (STARs) of GCase. Administration of GT-02287 and GT-02329 to cultured GD patient-derived primary human fibroblasts enhances folding and protects the two most common disease-causing GCase variants, GCaseAsn370Ser and GCaseLeu444Pro, from proteasomal degradation. Mechanistically, these treatments facilitate the lysosomal delivery of enzymatically active forms of mutant GCase, leading to improved lysosomal function and reduced cellular stress in GD patient-derived fibroblasts. The findings suggest that the allosteric pharmacologic regulators GT-02287 and GT-02329 hold promise for further development as potential therapeutic agents for GCase-related disorders, including GD, PD and Dementia with Lewy Bodies.

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Validation of a highly sensitive HaloTag-based assay to evaluate the potency of a novel class of allosteric β-Galactosidase correctors

Cited by 5 publications

References 53 publications

Use of the Novel Site-Directed Enzyme Enhancement Therapy (SEE-Tx) Drug Discovery Platform to Identify Pharmacological Chaperones for Glutaric Acidemia Type 1

Use of the Novel Site-Directed Enzyme Enhancement Therapy (SEE-Tx) Drug Discovery Platform to Identify Pharmacological Chaperones for Glutaric Acidemia Type 1

Pharmacological GCase Activity Enhancement Inhibits Tau Accumulation

A novel class of allosteric glucosylceramidase beta 1 correctors that reduce cellular stress and enhance lysosomal function

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