Temporal and Spatial Characterization of CUL3KLHL20-driven Targeted Degradation of BET family, BRD Proteins by the Macrocycle-based Degrader BTR2004

Abstract

Targeted protein degradation (TPD) is a promising new therapeutic modality that leverages the endogenous cellular protein degradation machinery of the ubiquitin-proteasome system (UPS) to degrade selected proteins. Recently, we developed a synthetic macrocycle ligand to recruit CUL3^KLHL20 E3 ligase for TPD. Using this KLHL20 ligand, we constructed the PROTAC BTR2004, which demonstrated potent degradation of BET family proteins BRD 2, 3, and 4. As the TPD field expands, it is important to understand the cellular and biochemical properties of all utilized E3 ligases. Herein we report the temporal and spatial processes of BTR2004-facilitated BET family protein degradation by KLHL20: The target protein degradation kinetics, BTR2004 intracellular activity half-life, and the onset of BTR2004 cell permeabilization. Employing proximity ligation and confocal microscopy techniques, we also illustrate the subcellular location of the ternary complex assembly upon BTR2004 treatment. These characterizations provide further insight into the processes that govern TPD and features that could be incorporated when designing future PROTAC molecules.

Keywords: CUL3; E3 ligase; KLHL20; PROTAC; macrocycle; targeted protein degradation.

Figure 1.. BTR2004-facilitated targeted protein degradation is ubiquitin-proteasomal pathway dependent.

(A) Chemical structure of BET family protein PROTAC: BTR2004. (B) Western blot of BRD2/4 degradation in U20S T-REx cells treated with BTR2004 (8μM), MG132 (10μM), MLN4924 (3μM), and DMSO. MG132 and MLN4924 prevented BTR2004 facilitated BRD2 and BRD4 degradation. (C) Immunofluorescent staining of BRD4 (Red) in U20S T-REx cells treated with BTR2004 or DMSO with or without MG132 or MLN4924 co-treatment.

Figure 2.. BTR2004 degradation kinetics.

(A) Western blot of BRD2 degradation with different BTR2004 concentrations. (B) BRD2 degradation by 4μM BTR2004 with different treatment time. (C) Quantitative BRD2 degradation time course with 2 μM and 4 μM BTR2004 treatment showed different kinetics. (D) Figure 2C curve fitting parameters: The constant “n,” is proportional to the slope of the line and therefore related to BRD2 degradation rate (normalized protein/min). The maximum rate of degradation is equal to n(1−m)/4. The constant “k” is the time (minutes) at which protein is being maximally degraded and refers to the time at which half of the protein has been attenuated. The constant “m” is equal to remaining protein % at maximum degradation.

Figure 3.. Degradability persistence and bioactivity half-life of BTR2004.

(A) Schematic diagram of compound washout experiment design. U2OS T-REx cells were first incubated with 4 μM BTR2004 for 2 hours (highlighted in blue) followed by compound washout and continued incubation for varying periods of time (highlighted in pink) in compound-free culture medium. (B) Cellular BRD2 protein recovery level over time after BTR2004 washout. Protein level measured by western blot quantification. (C) BRD2 protein remained degraded for 25 hours with constant BTR2004 incubation.

Figure 4.. BTR2004 bioactivity onset.

(A) Schematic diagram of modified BTR2004 washout experiment design. U2OS T-REx cells were first incubated with BTR2004 for varied amounts of time (highlighted in blue) followed by compound washout and continued incubation in compound-free culture medium until 135 minutes total incubation endpoint (highlighted in pink). (B) BRD2 protein level at 135 minutes total incubation endpoint (pink) vs. BRD2 protein level at the end of BTR2004 treatment (blue).

Figure 5.. Subcellular localization of ternary complex formation

(A) BRD4 (red) is exclusively localized to the nucleus whereas most HA-KLHL20 (green) is cytosolic. (Left) Merged image of BRD4 (red) and HA-KLHL20 (green) channels. (Right) Merged image with DAPI staining. (B) Strep-KLHL20 and HA -KLHL20 (green) showed similar localization patterns. (C) BTR2004 did not change BRD4 (red) subcellular localization. DAPI nuclear staining in blue. (D) BTR2004 did not change KLHL20 (green) subcellular localization. DAPI nuclear staining in blue. (E) BTR2004 mediated ternary complex formation revealed by Proximity Ligation Assay (PLA) using confocal microscopy imaging. (F) Quantified PLA signals (using Imaris program) showed ternary complex primarily formed in the nucleus. (G) Exportin 1 inhibitor KPT276 did not prevent BRD2 degradation, suggesting nuclear proteasomal degradation of the nuclear formed ternary complex.