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 modality that leverages the endogenous cellular protein degradation machinery to degrade selected proteins. Recently, we validated CUL3^KLHL20 E3 ligase as a new actionable E3 ligase for TPD application by developing a synthetic macrocycle ligand to engage KLHL20. Linking the KLHL20 ligand to JQ1, we created the PROTAC molecule BTR2004, which exhibited potent degradation of BET family proteins BRD 2, 3, and 4. As CUL3^KLHL20 is new to the TPD field, here we report the first temporal and spatial characterization of CUL3^KLHL20-driven TPD with BTR2004. Our study revealed 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 into the design of future macrocyclic PROTAC molecules.

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

(A) Chemical structure of BTR2004. (B) Western blot of BRD2/4 degradation in U2OS T-REx cells treated with BTR2004 (8μM), MG132 (10μM), MLN4924 (3μM), and DMSO. Each treatment group shown in the western blot are cell lysates of biological duplicates. For densitometry analysis of Western Blot, see Figure S9. (C) Immunofluorescent staining of BRD4 (Red) in U2OS T-REx cells treated with BTR2004 or DMSO, with or without MG132 or MLN4924 co-treatment. (Scale bar = 100μm)

Figure 2.. BTR2004 degradation kinetics.

(A) Western blot of BRD2 degradation with different BTR2004 concentrations. Each treatment group shown in the western blot are cell lysates of biological duplicates and each concentration condition included accompanied DMSO negative controls. For densitometry analysis of Western Blot, see Figure S9. (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 (Western blots used for quantification shown in Figure S4 & S5). (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 fraction at maximum degradation.

Figure 3.. Degradability, persistence, and bioactivity onset and 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. Each treatment group shown in the western blot are cell lysates of biological duplicates. (D) Schematic diagram of modified BTR2004 washout experiment design. U2OS T-REx cells were 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). (E) BRD2 protein level at 135 minutes total incubation endpoint (pink) vs. BRD2 protein level at the end of BTR2004 compound incubation (blue). (* P-value = 0.0001; ** P-value = 0.0001; ** P-value = 0.0018; N.S.= no significance)

Figure 4.. 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. (Scale bar = 30μm) (B) HA-KLHL20 and Strep-KLHL20 (green) showed similar localization patterns. (Scale bar = 100μm) (C) BTR2004 did not change BRD4 (red) subcellular localization. DAPI nuclear staining in blue. (Scale bar = 100μm) (D) BTR2004 did not increase KLHL20 (green) nuclear localization. DAPI nuclear staining in blue. (Scale bar = 20μm) (E) BTR2004 mediated ternary complex formation revealed by Proximity Ligation Assay (PLA) using confocal microscopy imaging. (Scale bar = 20μm) (F) Quantified 4E PLA signals (using Imaris program) showed ternary complex primarily formed in the nucleus. (* P-value = 0.00001; ** P-value = 0.00004; N.S.= no significance) (G) Exportin 1 inhibitor KPT276 did not prevent BRD2 degradation. Each treatment group shown in the western blot are cell lysates of biological duplicates.