Ubiquitin C-Terminal Hydrolase L1: Biochemical and Cellular Characterization of a Covalent Cyanopyrrolidine-Based Inhibitor

Abstract

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

Keywords: covalent inhibitors; deubiquitinase; enzyme inhibition; hydrolases; structural biology.

Figure 1.

Reported UCHL1 inhibitors.

Figure 2.

Proposed mechanism for inhibition of UCHL1 by 1.

Figure 3.

A) IC₅₀ curves for compound 1 against UCHL1 (black) and UCHL3 (red) after 30 minutes of preincubation. B) Progress curves for UCHL1 treated with DMSO (blue) or compound 1 (black) following dilution into substrate.

Figure 4.

HEK293T cell lysate was incubated with 1 for 10 minutes at room temperature before addition of HA-Ub-VME. This was incubated for 10 additional minutes then quenched by the addition of 4x laemlli buffer and heated at 90 °C for 5 minutes. UCHL1 immunoblot shows a reduction of HA-Ub-UCHL1 formation with increased concentration of 1. HA immunoblot shows two bands decrease as the concentration of 1 is increased, denoted by black arrows.

Figure 5.

A) Chemical shift changes ($\sqrt{\left({\left(\Delta \delta \text{HN}\right)}^2+{\left(0.154\Delta \delta \text{N}\right)}^2\right)/2}$) greater than 0.15 ppm are mapped to the structure of UCHL1 (PDB ID: 2ETL). The difference in ¹⁵N-HSQC chemical shifts between unligated UCHL1 and after addition of 1 were measured at a molar ratio [1:1]. The orientation of UCHL1 is approximately the same as Figure 5B. B) Predicted binding pose of compound 1 (magenta sticks) with UCHL1 (grey), supported by 3D NOE NMR experiments. The crossover loop of UCHL1 is shown as a cartoon for clarity. Compound 1 binds to UCHL1 on the same side of the crossover loop as the ubiquitin binding interface of UCHL1 (green surface). Compound 1 forms a covalent bond with cysteine 90 (yellow surface) and has an experimentally determined NOE with alanine 147 (red surface).

Figure 6.

Predicted binding pose of compound 1 (magenta) with UCHL1 (grey), supported by 3D intermolecular-NOE NMR experiments. Constraints provided when docking included formation of a covalent bond with cysteine 90 (yellow), and a distance between 1 and 6 angstroms between ligand proton 3 (H3) and alanine 147 (red sticks). The resulting pose shows hydrogen bonds (black dashed lines) between compound 1 and the oxyanion hole (Q84 and N88) of UCHL1.

Figure 7.

A) Immunoblots of KMS11, KMS12, HEK293, and SW1271 showing UCHL1 expression. B) Compound 1 displays a dose-dependent effect on the viability of SW1271 cells (CC₅₀ = 138.9 nM). C) Compound 1 decreases the rate of proliferation for both KMS11 and KMS12 cells at 25 μM.

Figure 8.

Fluorescence imaged gels from KMS11 and SW1271 cells treated simultaneously with 10 μM of compound 2 and increasing concentrations of compound 1 for 4 hrs then subjected to CuAAC labeling with Cy5 dye show a dose-dependent decrease in intensity for a band near 25 kDa.

Scheme 1.

Reagents and Conditions: a) HBTU (1.25 eq), DIPEA (1.5 eq), THF, 25 °C, 16 hr; b) NaCO₃H (2 eq), Pd(dppf)Cl₂ (0.1 eq), 9:1 DMF:H₂O, 110 °C, 1.5 hr; c) TFA (20 eq), DCM, 25 °C, 2 hr; d) K₂CO₃ (2.2 eq), BrCN (1.0 eq), THF, 0 °C, 30 minutes.

Scheme 2.

Reagents and Conditions: a) K₂CO₃ (2 eq), ACN, 110 °C, 1.5 hr; b) TFA (20 eq), DCM, 25 °C, 2 hr; c) K₂CO₃ (2.2 eq), BrCN (1.0 eq), THF, 0 °C, 30 minutes.