DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras

Abstract

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

Figure 1.. A cell-based screen to identify electrophilic PROTACs that degrade FKBP12.

A. Schematic depicting the strategy of screening candidate electrophilic PROTACs targeting the FKBP12 protein (either cytosol- or nuclear-localized) fused to a luciferase reporter (Luc-FKBP12). Cells were exposed to compounds at 2 μM for 8 h. Luciferase activity was normalized to cell viability measured by parallel CellTiter-Glo assays (Table S1). B. Heatmap showing the relative abundance of Luc-FKBP12 in cells treated with candidate electrophilic PROTACs compared to DMSO control. Data represent mean values (n = 2 biologically independent experiments). C. Structures of representative active (10-SLF, 21-SLF) and inactive (18-SLF) compounds from the screen. Bar graph (bottom) represents the screening data in B for 18-SLF and 21-SLF. Data are mean ± SD (n = 2). D. Confirmation of the activity of 21-SLF (2 μM, 8 h) by Western blotting of 22Rv1 cells stably expressing cytosolic or nuclear FLAG-tagged FKBP12. The result is representative of three biologically independent experiments. Bar graph (right) represents quantification of the FLAG-FKBP12 protein content. Data are mean values ± SEM (n = 3). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing 21-SLF-treated to DMSO or 18-SLF-treated cells. **P < 0.01.

Figure 2.. 21-SLF promotes proteasomal degradation of cytosolic and nuclear FKBP12 via the action of Cullin-RING ubiquitin ligase(s).

A. 21-SLF-mediated Luc-FKBP12 degradation is blocked by the proteasome inhibitor MG132 and the neddylation inhibitor MLN4924. 22Rv1 cells stably expressing cytosolic or nuclear Luc-FKBP12 were co-treated with 21-SLF (2 μM) and MG132 (10 μM) or MLN4924 (1 μM) for 8 h. Relative Luc-FKBP12 abundance was measured by luciferase signals in comparison to DMSO-treated control cells and normalized to cell viability. Data are mean values ± SEM (n = 4 biologically independent experiments). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing 21-SLF-treated to DMSO-treated cells. ***P < 0.001. B. 21-SLF induces polyubiquitination of cytosolic and nuclear FLAG-FKBP12 in 22Rv1 cells. 22Rv1 cells stably expressing cytosolic or nuclear FLAG-FKBP12 were treated with DMSO or 21-SLF (10 μM) in the presence of the proteasome inhibitor MG132 (10 μM) for 2 h. The result is representative of two biologically independent experiments.

Figure 3.. DCAF11 mediates 21-SLF-induced degradation of FKBP12.

A. Quantitative MS-based proteomics showing 21-SLF/DMSO ratio values of proteins identified in anti-FLAG affinity enrichment experiments, where a high ratio indicates proteins preferentially enriched from cells treated with 21-SLF (10 μM). The maximum 21-SLF/DMSO value was set as 10. 22Rv1 cells stably expressing cytosolic or nuclear FLAG-FKBP12 were treated with DMSO or 21-SLF (10 μM) in the presence of the proteasome inhibitor MG132 (10 μM) for 2 h. The y-axis and x-axis correspond to the average 21-SLF/DMSO ratio and protein number, respectively, from four biologically independent experiments. B. Concentration-dependent degradation of stably expressed cytosolic or nuclear FLAG-FKBP12 in DCAF11-WT and DCAF11-KO 22Rv1 cells following treatment with 21-SLF (1, 2, and 5 μM) for 8 h. The result is representative of three biologically independent experiments. Bar graph (right) represents quantification of the FLAG-FKBP12 protein content. Data are mean ± SEM (n = 3). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing 21-SLF-treated to DMSO-treated cells. *P < 0.05, **P < 0.01, ***P < 0.001. C. Expression of HA-DCAF11 in DCAF11-KO 22Rv1 cells restored 21-SLF-mediated degradation of cytosolic and nuclear FLAG-FKBP12. 22Rv1 DCAF11-KO cells were transiently transfected with HA-DCAF11 or empty pRK5 vector and cytosolic or nuclear FLAG-FKBP12 for 24 h and then treated with 21-SLF (2 μM, 8 h). The result is a representative of three biologically independent experiments. Bar graph (right) represents quantification of the FLAG-FKBP12 protein content. Data are mean ± SEM (n = 3). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing 21-SLF-treated HA-DCAF11-expressing cells to 21-SLF-treated DCAF11-KO cells. **P < 0.01. D. DCAF11 interacted with and mediated polyubiquitination of cytosolic and nuclear FKBP12 in the presence of 21-SLF. HEK293T cells were transiently transfected with HA-DCAF11 and cytosolic or nuclear FLAG-FKBP12 for 24 h and then treated with DMSO or 21-SLF (10 μM, 2 h) in the presence of MG132 (10 μM). The result is representative of two experiments (n = 2 biologically independent experiments). E. Quantitative MS-based proteomics comparing protein abundance profiles of DCAF11-WT and DCAF11-KO 22Rv1 cells treated with DMSO or 21-SLF (10 μM) for 8 h. The y-axis and x-axis correspond to the average relative abundance (21-SLF/DMSO) and coefficient of variation, respectively, from two biologically independent experiments. F. Degradation of stably expressed nuclear FLAG-FKBP12 in DCAF11-WT and DCAF11-KO 22Rv1 cells following treatment with the indicated compounds (2 μM for KB02-SLF and 10 μM for the others, 8 h). The result is a representative of two biologically independent experiments. Bar graph (right) represents quantification of the FLAG-FKBP12 protein content. Data are mean ± SD (n = 4 for DMSO treatment, n = 2 for others). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing DMSO-treated to compound-treated cells. *P < 0.05, **P < 0.01.

Figure 4.. Evaluation of DCAF11 cysteines involved in 21-SLF-induced degradation of FKBP12.

A. 21-SLF-mediated degradation of cytosolic and nuclear Luc-FKBP12 in HEK293T cells co-expressing HA-DCAF11. HEK293T cells were transfected with HA-DCAF11 and cytosolic or nuclear Luc-FKBP12 for 24 h and then co-treated with 21-SLF (10 μM) and DMSO or MLN4924 1 μM) for 8 h. Luc-FKBP12 abundance was measured by luciferase signals in comparison to DMSO-treated control cells and normalized to cell viability. Data are mean ± SEM (n = 3 biologically independent experiments). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing 21-SLF-treated and DMSO-treated HA-DCAF11/Luc-FKBP12-co-expressing cells. ***P < 0.001. B. Concentration-dependent degradation of cytosolic or nuclear Luc-FKBP12 in HEK293T cells co-expressing Luc-FKBP12 and HA-DCAF11. HEK293T cells were transfected with HA-DCAF11 or pRK5 empty vector and cytosolic or nuclear Luc-FKBP12 for 24 h and then treated with 21-SLF (0.2, 0.5, 2, 5 and 10 μM) for 8 h. Luc-FKBP12 abundance was measured as in panel A. Data are mean ± SEM (n = 3 biologically independent experiments). C. Evaluation of DCAF11 cysteines that support 21-SLF-mediated Luc-FKBP12 degradation. HEK293T cells were transfected with the indicated C-to-A mutants of HA-DCAF11 and nuclear Luc-FKBP12 for 24 h and then treated with 21-SLF (10 μM, 8 h). Luc-FKBP12 abundance was measured as in panel A. Data are mean ± SEM (n = 3 biologically independent experiments). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing DCAF11-WT and DCAF11-C460A. ***P < 0.001. D. Cryo-electron microscopy structure of SNRNP40, the U5 subunit of human spliceosome (pdb: 3JCR). C291, S304 and S329 in SNRNP40 correspond to C443, C460 and C485 in DCAF11 based on sequence alignment (Figure S5). Blue highlighted amino acids correspond to the predicted locations of other cysteines in DCAF11. E. Evaluation of DCAF11 cysteines that support 21-SLF-mediated Luc-FKBP12 degradation. HEK293T cells were transfected with the indicated C-to-A mutants of HA-DCAF11 and nuclear Luc-FKBP12 for 24 h and then treated with 21-SLF (10 μM, 8 h). Luc-FKBP12 abundance was measured as in panel A. Data are mean ± SEM (n = 3 biologically independent experiments).

Figure 5.. An electrophilic PROTAC that degrades the androgen receptor (AR) in a DCAF11-dependent manner.

A. Structure of 21-ARL, a candidate AR-directed electrophilic PROTAC. B. Concentration-dependent degradation of AR in DCAF11-WT and DCAF11-KO 22Rv1 cells following treatment with 21-ARL (1–10 μM) for 8 h. The result is representative of three biologically independent experiments. Bar graph (right) represents quantification of the AR content. Data are mean ± SEM (n = 3). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing 21-ARL-treated DCAF11-WT to DCAF11-KO cells at each concentration. *P < 0.05, **P < 0.01.