Identification and validation of selective deubiquitinase inhibitors

Abstract

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes.

Keywords: deubiquitinase; high-throughput screening; small-molecule inhibitor.

Figure 1.. Overview of DUB HTS campaign.

A) Schematic of Ub-Rho screening assay. Uninhibited DUB cleaves rhodamine from ubiquitin (top) resulting in a fluorescent signal. In the presence of an inhibitor (bottom), the DUB cannot cleave the substrate, and fluorescence is unchanged. B) DUBs included in the screen along with constructs used (cat denotes catalytic domain, FL denotes full length protein, cat + UBA denotes the catalytic domain plus UBA domain). All proteins are human isoforms except USP28, which is from mouse (mus musculus). C) Summary of druglike properties partition coefficient (LogP) to polar surface area (PSA) and molecular weight (MW) among library members. D) Diagram of screening cascade workflow.

Figure 2.. Primary Screening Data

A) Heatmap of primary screen hits. Maximal inhibition of each compound is plotted for each DUB. Compounds are clustered on the x-axis by structural similarity (Tanimoto score). Patches of blue in each of the rows show compound clusters that demonstrate inhibition of the corresponding DUB. B) UpSet plot summarizing primary screening data. Compounds with activity against at least one DUB, as defined by maximal inhibition ≥ 30%, are clustered by activity against each DUB in the panel. The bar chart represents the number of compounds in a particular cluster, the dot underneath the bar chart correlates that cluster with one or more DUBs (the presence of a single dot correlates to a specific DUB on the left, the presence of multiple dots means those compounds are hits against those particular DUBs). Single DUB hits are colored in red to highlight potentially selective hits in the primary screen for each DUB. The bar chart on the lower left represents the total number of hits for each DUB regardless of selectivity. The top 40 clusters of compounds are shown.

Figure 3.. Dose-response screening data with selectivity and potency filters

A) Number of compounds that qualify as hits for each DUB based on filter criteria listed in the table below. The arrow denotes increasing potency and/or selectivity of hit compounds for each DUB. The * denotes that for UCHL1, 25 μM was the highest tested dose in the dose-response and thus this value was used for calling hits as active against UCHL1. B) The violin plot shows the distribution of IC₅₀s for DUBs that had qualifying hits in section IV in Fig 3A.

Figure 4.. Conformation of screening hits

A) Structures of USP7 and USP8 screening hits prompted further screening of a spiro indolinone expansion set leading to the identification of new USP7 and USP8 hits for validation studies. B) Structures of screening hits for each corresponding DUB that were selected for validation studies. C) Dose-response data for screening hits in Fig 4A and 4B. Compounds were screened in a 12-point dose-response using a kinetic Ub-Rho biochemical assay. Data are represented as mean ± SEM, n ≥ 2.

Figure 5.. Selectivity Profiling

Compounds were assessed for selectivity against an expanded panel of purified recombinant DUBs using DUBProfiler (Ubiquigent). Compounds were tested at 100 μM, except bin-01–07-07 which was tested at 10 μM, and the percent activity of each DUB remaining following compound treatment is reported for each compound.

Figure 6.. Validation of USP28 and USP30 hits by Quantitative Activity Based Protein Profiling

A) Schematic for quantitative activity-based protein profiling. B) Heatmap showing results of screening compounds profiled in ABPP assay. Compounds demonstrate blockage of probe labeling of target DUBs compared to all other DUBs detected in cell lysate. AZ1 is a literature reported USP25/28 inhibitor (Wrigley et al., 2017). XL-188 is a USP7 inhibitor included as a positive control(Lamberto et al., 2017). See also Supplemental Table 2. C) Western blots show blockage of USP28 labeling by covalent probe (Ub-PA/Ub-VME) in cellular lysate after 1 hour pre-treatment with indicated compounds in dose-response.