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

Abstract

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.

LRRK2 kinase activity regulates GCase level and enzymatic activity differently depending on cell type in Parkinson’s disease

“…When exploiting the in-cells protocol previously used in fibroblasts (Fig. 3e ) 24 , we found a non-significant decrease in GCase in KO cells, but when treating LRRK2 WT cells with 100 nM Mli-2 there was a significant decrease in the GCase activity in the lysosomes of LRRK2 WT cells (Fig. 6e ), further corroborating the previous results (Fig.…”

“…To evaluate GCase activity also in the cellular context, we employed a method following the protocol by Benz et al (2021) 24 based on the use of the GCase fluorescence substrate PFB-FDGlu. The GCase substrate was given to the cells 6 h before the assay, it specifically accumulates in lysosomes and it becomes fluorescence proportionally to the GCase activity in the organelles.…”

GCase Enhancers: A Potential Therapeutic Option for Gaucher Disease and Other Neurological Disorders

“…Even more interestingly, the authors characterized (by native mass spectrometry and X-ray crystallography) an allosteric binding site for these modulators by conducting a covalent modification of a lysine residue close to a properly modified ligand. Through transmission electron microscopy (TEM) experiments, they also provided insight into the mechanism of action of 103, suggesting that GCase stabilization is due to the ability of the ligand to induce GCase dimerization [91].…”

Design, Synthesis and Structural Analysis of Glucocerebrosidase Imaging Agents

“…In combination with the previously reported structure of rhGBA in complex with a Cy5 tagged ABP 9, [27] this ABP 5 co-complex provides further evidence for a unique binding mode of this hydrophobic cavity, which has recently been exploited for the binding of a novel class of pyrrozopyrazine activators with chaperoning potential. [38] GBA is notable for its tolerance, indeed preference, for O6substituted reagents which exhibit increased specificity for GBA over other β-glucosidases, including non-lysosomal GBA (GBA2) and generic GH1 β-glucosidases. [27] GBA also favours iminosugar inhibitors and cyclophellitol aziridines extended at the aziridine nitrogen position.…”

“… [31] Later studies have investigated GBA at the 3D level to obtain insight into GD mutations and potential molecular chaperone binding motifs. [ 32 , 33 , 34 , 35 ] For example, active site directed quinazoline modulators,[ 21 , 36 ] competitive 3,4,5,6‐tetra‐hydroxyazepane inhibitors [37] and uncompetitive pyrrolo[2,3‐b]pyrazines inhibitors [38] have recently been reported.…”