Small Molecule Screen Identifies Non-catalytic USP3 Chemical Handle

Abstract

Zinc-finger ubiquitin-binding domains (ZnF-UBDs) are noncatalytic domains mostly found in deubiquitylases (DUBs) such as USP3. They represent an underexplored opportunity for the development of deubiquitylase-targeting chimeras (DUBTACs) to pharmacologically induce the deubiquitylation of target proteins. We previously showed that ZnF-UBDs are ligandable domains. Here, a focused small molecule library screen against a panel of 11 ZnF-UBDs led to the identification of compound 59, a ligand engaging the ZnF-UBD of USP3 with a K_D of 14 μM. The compound binds the expected C-terminal ubiquitin binding pocket of USP3 as shown by hydrogen-deuterium exchange mass spectrometry experiments and does not inhibit the cleavage of K48-linked diubiquitin by USP3. As such, this molecule is a chemical starting point toward chemical tools that could be used to interrogate the function of the USP3 Znf-UBD and the consequences of recruiting USP3 to ubiquitylated proteins.

Figure 1

Human ZnF-UBDs: (A) (Left) Dendrogram of full-length human proteins that contain a ZnF-UBD. The dendrogram was created by first doing a multiple sequence alignment using Clustal Omega, followed by tree generation with iTol. Reported ZnF-UBD ligands are highlighted with green circles. (B) Sequence alignment of human ZnF-UBDs. Conserved residues are indicated with shading. USP5 residues that interact with Ub are indicated with arrowheads and boxes (red: expected to be essential for Ub binding; black: not essential). (C) Co-crystal structures of HDAC6 and USP5 ZnF-UBD in complex with ligands (PDB: 6CED, 7MS7).

Figure 2

Small molecule screen identifies ZnF-UBD hits. (A) Heat map showing binding of 64 ligands to 11 ZnF-UBDs by SPR. A 4-fold 6-point dilution series beginning at 200 μM was used for K_D determination with N = 1. (B) Summary of binding data for compound 59 by SPR, N ≥ 3. (C) Representative SPR binding curve from steady-state fit analysis and sensorgrams for USP3 ZnF-UBD and 59. A K_D of 14 ± 4 μM was obtained from the average of seven independent measurements.

Figure 3

59 does not inhibit USP3 catalytic activity. (A) UbRho110 assay (N = 2). Fluorescence signal is normalized to the control [no compound, dimethyl sulfoxide (DMSO) only]. (B) Cleavage of Ub2K48 in the absence or presence of 59 (70 μM final concentration).

Figure 4

Mapping of the ligand binding site by HDX-MS. Top: the difference in deuterium fractional uptake (%) between USP3 ZnF-UBD + 59 (1:10) and USP3 ZnF-UBD is plotted as a function of each time point per peptide. The time points are 15 s (dark blue), 60 s (light blue), and 600 s (light grey). The error (3 times the propagated error) is shown as a dark gray area. If the bars exceed this gray area, they are statistically significant. Bars pointing down indicate a decrease in the level of deuterium uptake in the presence of the ligand. Bottom: HDX signals observed at 15, 60, and 600 s (same color-coding as above) are highlighted as spheres on an AlphaFold prediction of the USP3 ZnF-UBD structure (center). A crystal structure of the homologous USP5 ZnF-UBD bound to a ligand (PDB ID: 7MS7) (left) and a model of 59 docked to the USP3 ZnF-UBD AlphaFold model prediction (right) are shown as references.