Structural basis for specific Inhibition of the Deubiquitinase UCHL1
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Date
2023
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Abstract
Highly enriched in neurons, the DUB UCHL1 accounts for up to 2% of the total soluble protein in the human brain. Loss of UCHL1 is associated with neurodegenerative diseases, and its dysregulation contributes to the promotion of cancer metastasis and invasive behaviour. Despite its multiple associations with diseases and disease-related pathways, the precise functions of UCHL1 remain poorly understood. A comprehensive understanding of the role of UCHL1 in these pathways is currently hampered by an insufficiently defined set of UCHL1 substrate proteins and a lack of suitable pharmacological tools to specifically inhibit UCHL1 activity. Efforts to improve the specificity and efficacy of DUB inhibitors face significant challenges due to limited structural information about inhibitor:DUB complexes.
This dissertation reports on the design, synthesis and target validation of an activity-based probe library derived from putative covalent DUB inhibitors. This led to the discovery of a chemogenomic pair of 3-carboxy-cyanopyrrolidine-based compounds GK13S and GK16S that enable the selective investigation of UCHL1 activity in a cellular setting. Detailed biochemical characterisation of the lead probe GK13S revealed nanomolar and stereoselective binding to UCHL1. The crystal structure of UCHL1 in complex with GK13S bound to the catalytic cysteine revealed that the enzyme is trapped in a hybrid conformation between apo- and ubiquitin-bound states, explaining its exquisite UCHL1 specificity relative to other members of the UCH family. Consistent with neural tissue from UCHL1 mutant mice, inhibition of UCHL1 in glioblastoma but not epithelial cells resulted in a probe-dependent decrease in mono-Ubiquitin levels. In vitro analyses of GK13S and GK16S in combination with activity-based protein profiling revealed PARK7 as a major off-target protein of 3-carboxy-cyanopyrrolidines. A crystal structure of PARK7 in co-complex with the minimal probe GK16S revealed differences in the binding site compared to UCHL1. Based on these findings, repeated cycles of chemical synthesis as well as cellular and biochemical analyses led to the discovery of piperazine-based compounds that did not target PARK7. CG341 and CG306 were found to be non-toxic, nanomolar potent and selective inhibitors of UCHL1, overcoming the limitations of previously reported UCHL1-targeting probes. In addition, this dissertation introduces a novel “dual-functional warhead“ strategy that offers an initial framework for efficiently creating extensive and varied libraries of activity-based probes. These libraries have the potential to facilitate the discovery of novel and precise probes targeting specific DUB enzymes.
In conclusion, this dissertation advances the knowledge of DUB biology and inhibitor development, offering selective tools to study UCHL1 in cellular contexts. The work delves into the structural underpinnings of UCHL1 inhibition, validates the selectivity of the developed probes, and proposes their utility in investigating intricate disease-related pathways. This approach holds promise for advancing drug development efforts and devising diagnostic tools, thereby contributing to a comprehensive understanding of DUB function in the context of various diseases.
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Keywords
UCHL1, DUB, Deubiquitinase, ABPP, Activity based probe, ABP, Pull down, Protein, Ligand, Xtal, Crystal structure, Crystal, Crystallisation, Crystallization
Subjects based on RSWK
Protein, Enzyminhibitor