Periodic Reporting for period 1 - TargetDUBs (Targeting ubiquitin processing in cancer and fibrosis: novel probes for the Ubiquitin Carboxy-Terminal Hydrolases)
Periodo di rendicontazione: 2019-08-01 al 2021-07-31
Ubiquitin specific peptidase 30 (USP30) is a member of the USP family of deubiquitinases (DUBs) and the only DUB present in mitochondria, where it deubiquitylates mitochondrial proteins and antagonizesmitophagy. USP30 overexpression is strongly associated with drug resistant lymphoma, leukaemia and multiple myeloma, in which apoptotic pathways are dysregulated through altered expression of BCL-2. USP30 depletion sensitizes cancer cells to BH3-mimetics (e.g. ABT-737), making it a potential target for cancer therapy, however, its endogenous substrates and regulation remain poorly understood. Similarly, Ubiquitin Carboxy-Terminal Hydrolase L1 (UCHL1) is a member of the UCH family of deubiquitinases (DUBs), and is the most abundant protein in the brain. UCHL1 dysregulation has been shown to be associated with neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, various types of cancers (colorectal, breast, prostate, ovarian, and lung cancers), and liver fibrosis which could be related to lung and kidney fibrosis. While UCHL1 was shown to exert diverse Ub-associated activities (hydrolase, ligase, mono-Ub stabilizing), its actual functions, endogenous substrates, and how its activity is regulated in vivo, both in pathological and healthy tissues, remain poorly understood.
To overcome these limitations, we planed to develop a USP30/UCHL1 activity-based probe (ABP) and apply them to the identification and quantification of USP enzyme activity and selective USP30/UCHL1 inhibitors in cancers and neurodegenerative diseases, using activity-based protein profiling.
To overcome these limitations, we planed to develop a USP30/UCHL1 activity-based probe (ABP) and apply them to the identification and quantification of USP enzyme activity and selective USP30/UCHL1 inhibitors in cancers and neurodegenerative diseases, using activity-based protein profiling.
To design the Activity-based probe (Fig. 1A) we have analysed various irreversible inhibitor designs (active inhibitors, Fig. 1B) with low-nanomolar potency containing a nitrile warhead discovered recently by our collaborators, Mission Therapeutics (Pat. Appl. WO2018060689). From these data we have designed activity-based probe (ABP) 1 (Fig. 2C) with a terminal alkyne based on two key properties: potential for nanomolar covalent inhibitory potency against USP30, and compatibility with bioorthognal click chemistry, to form an ABP (Fig. 1C). We have also designed a structurally similar negative control probe (Fig. 1D, compound 2). These compounds were synthesized at 20 mg scale by adapting a short synthetic route. We have confirmed that compound 1 retains inhibitory potency toward USP30 by a fluorescence polarization assay using a Ub-TAMRA substrate, and against a broad panel of DUBs (available through the Tate lab’s collaboration with Mission Therapeutics) to validate selectivity. Formation of a covalent adduct between recombinant USP30 and ABP 1 were assayed by mass spectrometry, and by in-gel fluorescence, according to our previous work on UCHL1 DUBs. A suitable concentration of probe for USP30 labelling studies were determined in a small panel of patient-derived myeloma cells and myeloma cell lines endogenously expressing USP30 and/or BCL-2 as well as in neuroblastoma cell to ensure sufficient labelling for competitive ABPP. The optimal probe concentration were identified using bioorthogonal ligation of probe-labelled proteins to fluorescent capture agents recently developed in the Tate group (Storck et al., Nature Chemistry 2019, 552), e.g. azido−TAMRA−biotin (AzTB, Fig. 1C). Targets labelled by 1 were visualized by in-gel fluorescence imaging, and an optimal probe concentration determined. Activity-based proteome profiling (ABPP) were performed to establish ABP 1 target identification in multiple myeloma and neuroblastoma cells, and targets quantified using ultra high resolution nanoLC-MS/MS proteomics following ligation to capture reagent and enrichment on neutravidin resin. Enriched proteins were analysed by ultra-high resolution nanoLC-MS/MS on an in-house Q Exactive platform. We used 10-plex Tandem Mass Tagging (TMT) labelling technology to rapidly and unbiasedly confirm target selectivity for up to ten different probe conditions and controls in one analysis run. This validation path will facilitate rapid optimisation and establish the robustness of our technology platform for further applications in more complex pre-clinical models and patient samples.
ABPs specific for USP family members have not been reported to date, and our work will enable USP30 activity profiling and dynamic changes of USP30 activity (e.g. due to inhibition) in any cancer cell line or system of interest for the first time. These probes will be valuable chemical tools for the study of USP30 activity and regulation in live cells, and for identification of USP30 substrates and specific and potent USP30 inhibitors as a novel therapeutic approach in multiple myeloma. Identification of the first specific USP30 activity probe opens up multiple novel lines of future research including profiling and imaging of changes in USP30 activity in xenograft models, and in clinical samples to identify USP30 activity as a potential biomarker, coupled to flow cytometry. Multiple myelomatherapeutic options are now very complex and expensive (1% of all cancers, >20% of cancer drug costs in the UK), and there is a desperate need for novel clinically useful biomarkers. This work also provides proof of principle for development of novel probes for many other DUBs emerging as potential therapeutic targets in cancer (e.g. USP7).