A synthetic KLHL20 ligand to validate CUL3KLHL20 as a potent E3 ligase for targeted protein degradation

Abstract

Targeted protein degradation (TPD) has risen as a promising therapeutic modality. Leveraging the catalytic nature of the ubiquitin-proteasome enzymatic machinery, TPD exhibits higher potency to eliminate disease-causing target proteins such as oncogenic transcription factors that may otherwise be difficult to abrogate by conventional inhibitors. However, there are challenges that remain. Currently, nearly all degraders engage CUL4^CRBN or CUL2^VHL as the E3 ligase for target ubiquitination. While their immediate efficacies are evident, the narrowed E3 ligase options make TPD vulnerable to potential drug resistance. In addition, E3 ligases show differential tissue expression and have intrinsic limitations in accessing varying types of disease-relevant targets. As the success of TPD is closely associated with the ability of E3 ligases to efficiently polyubiquitinate the target of interest, the long-term outlook of TPD drug development will depend on whether E3 ligases such as CUL4^CRBN and CUL2^VHL are accessible to the targets of interest. To overcome these potential caveats, a broad collection of actionable E3 ligases is required. Here, we designed a macrocyclic degrader engaging CUL3^KLHL20 for targeting BET proteins and validated CUL3^KLHL20 as an E3 ligase system suitable for TPD. This work thus contributes to the expansion of usable E3 ligases for potential drug development.

Keywords: CUL3; KLHL20; PROTAC; macrocycles; targeted protein degradation.

Figure 1.

Developing the CUL^KLHL20 E3 ligase TPD system. The CUL^KLHL20-based TPD molecules are comprised of three parts: (1) the synthetic KLHL20 ligand for E3 ligase engagement, (2) the target protein-engaging ligand for specific target protein recruitment, and (3) a linker moiety that connects both ligand types. The design of target-selective degraders is highly modular, with the synthetic KLHL20 ligand serving as the constant part to engage the E3 ligase and an exchangeable part to target the protein of choice (e.g., a transcription factor in this study).

Figure 2.

Cyclization of a linear DAPK1-derived peptide motif provides increased affinity for the KLHL20 Kelch binding pocket. (A) Crystal structure of the KLHL20 Kelch domain bound to the DAPK-derived peptide fragment ¹³³⁴LGLPDLVAK¹³⁴² (PDB 6GY5). (B) Cyclization scheme. Free sulfhydryls from cysteine residues react with 1,3-bis(bromomethyl)benzene to form two thioether bonds, yielding macrocycles. (C) Fluorescence polarization (FP) binding curves of FITC-labeled peptides incubated with serial dilutions of the KLHL20 Kelch domain. Peptide LAAAV was used as a negative control. Error bars show standard deviation (SD; N = 3). Data are background-corrected, and curves were fitted using a three-parameter dose response model. (D) Crystal structure of the KLHL20 Kelch domain, as in A. Residues mutated for BTR2000 binding studies are highlighted in red. (E) FP curves, as in C, showing binding of FITC-labeled BTR2000 to KLHL20 Kelch wild type (WT) or mutants. (F) Volcano plot showing proteomics data of the pull-down experiment comparing BTR2000 with cyclized CPGAC. The −log₁₀ P-value (N = 3) is plotted against the log₂ fold change BTR2000/CPGAC. KLHL20 showed the highest enrichment, with a log₂ value of 11.

Figure 3.

BTR2003 promotes ternary complex formation between KLHL20 and BRD4 BD1. (A) Structure of the bifunctional molecule BTR2003. The BET bromodomain inhibitor JQ1 (red) was conjugated to BTR2000 (blue) via a four-glycine linker (black). (B) BRD4 BD1 pull-down experiment with the KLHL20 or KLHL7 Kelch domains immobilized to beads in the presence of different BTR2003 concentrations. Bound His-BRD4 BD1 was detected by Western blotting using an anti-His antibody. (C) Binding model of the KLHL20 Kelch domain to the BRD4 BD1 in the presence of BTR2003. Shown are the five best results of a docking experiment by Rosetta docking as judged by the free energy. (D) NMR TROSY spectra of ¹⁵N-labeled BRD4 BD1. Shown excerpts of full spectral overlays are of the domain alone (red) or in the presence of BTR2003 (blue) or BTR2003 and KLHL20 Kelch domain (green). The asterisks indicate signals exclusive to the trimeric complex. (E) The KLHL20 Kelch binding epitope on the BTR2003-bound BRD4 BD1 based on the NMR experiment. Residues highlighted in red showed significant reduction in signal intensities relative to the BTR2003-bound domain after addition of the KLHL20 Kelch domain. BTR2003's position is based on the docking experiment.

Figure 4.

Linker length and composition have effects on trifunctional complex formation and affinity. (A) Structure of 10 different linker variants of BTR2003 derivatives (BTR2004–BTR2013). (B) Pull-down experiment of full-length BRD2, BRD3, and BRD4 from PC3 lysates using immobilized KLHL20 and KLHL7 Kelch in the presence of different BTR2003 derivatives or negative controls. Bound protein was detected by Western blotting using antibodies against the respective BET proteins. (C) ELISA binding curves of T7-BRD4 BD1 to the immobilized KLHL20 Kelch domain in the presence of serial dilutions of BTR2003–BTR2013. Error bars show SD (N = 2). Data were normalized over the full concentration range of 0–1 µM, and data below the hook point were fitted to a three-parameter hyperbolic model. Plots of the full concentration range are shown in Supplemental Figure S5A. (D) Heat map illustrating the ELISA EC₅₀ values of six different BET bromodomains binding to the KLHL20 Kelch domain in the presence of BTR compounds.

Figure 5.

Degradation of BET family proteins in cancer cells. (A) PC3 cells treated with 10 μM BTR compounds for 4 h. Cell lysates from biological duplicates were loaded and probed by Western blotting using the respective BET protein antibodies. α-Tubulin was used as a loading control. (B) BRD3 protein level in PC3 nuclear fraction. Histone-H3 was used as a nuclear loading control. (C) Experiments were as in A using MDA-MB-231, HCT116, and U-2 OS cell lines treated with 10 μM BTR compounds. BRD3 was detected by Western blotting. (D) Quantitation of BRD2, BRD3, and BRD4 protein degradation in PC3 cells treated with varying concentrations of BTR2004 for 4 h. Values are Western blot signal intensities relative to the DMSO negative control. (E) Protein levels of BRD2, BRD3, and BRD4 after 4-h treatment of PC3 cells with 5 μM BTR2004, A1874, MZ-1, and dBET1. DMSO negative control was used as a reference. (F) PC3 cells treated with different concentrations of BTR2004, MZ-1, and dBET1 for 24, 72, and 96 h. DMSO was used as a negative control. Cell titer after treatment was quantitated via luminescence measurement by using CellTiter-Glo 2.0. Error bars show standard deviation (SD; N = 3).