A fluorescence polarization-based screen identifies ligands of the Kelch domain of KLHCD2.

A) Structure of the Kelch domain of KLHDC2 from PDB 6DO3 bound to PPPMAGG, C terminal peptide from SelK (left). Interface of SelK peptide (teal) bound to KLHDC2 (right) with TAMRA labeled peptide used in this work above. B) Fluorescence polarization signal of TAMRA-SelK peptide in response to increasing concentrations of GST-KLHDC2. C) Fluorescence polarization signal from KLHDC2 and KEAP1 assays in response to increasing concentrations of unlabeled SelK peptide. Fluorescence polarization signals in Z’ based determination assays with and without unlabeled SelK peptide (1 µM) in 384-well (D) and 1536-well (E) assays. F) Corrected fluorescence polarization signal from the primary screening campaign with hits noted in teal. G) Screening funnel depicting the high throughput screening campaign.

Tetrahydroquinoline-based KLHDC2 ligands.

Structures and summary of activities of compounds (A) and 2 (B). C) Fluorescence polarization signal from KLHDC2 and KEAP1 assays in response to increasing concentrations of 1 (C) and 2 (D). Representative SPR sensorgrams of KLHDC2 binding from the indicated concentrations of 1 (E) and 2 (F).

Optimization of tetrahydroquinolines as KLHDC2 ligands.

A) Heatmap of fluorescence polarization signal in response to dose responses of the 54 related compounds. Structures and summary of activities of compounds 5 (B) and 6 (C). C) Fluorescence polarization signal from KLHDC2 and KEAP1 assays in response to increasing concentrations of 5 (D) and 6 (E). Representative SPR sensorgrams of KLHDC2 binding from the indicated concentrations of 5 (F) and 6 (G).

KLHDC2 ligands can be coopted for targeted degradation of BRD4.

A) Binding pose of compound 6 to KHLDC2 (PDB 6DO3). B) Structure and summary of activities of the indicated PROTAC molecules. Relative BRD4 levels at 4 (C) or 24 hours (D) as measured by luminescence from HEK293T cells expressing a HiBiT-tagged BRD4 transgene (n=3; mean and s.e.m.). E) Representative Western blot and densitometry-based quantification of BRD4 levels from SK-BR-3 in response to 24-hour treatment with the indicated concentrations of 8.

A fluorescence polarization-based control assay with Kelch domain containing protein KEAP1.

A) Fluorescence polarization signal of TAMRA-conjugated NRF2 peptide in response to increasing concentrations of KEAP1. B) Structure of KEAP1 Kelch domain ligand KI696. C) Fluorescence polarization signal in response to the indicated concentrations of KI696 in the KEAP1 assay. N=3, mean and s.e.m.

A 2-amino thiazole-based KLHDC2 ligand scaffold.

Structures of compounds 3 (A) and 4 (B). Fluorescence polarization signal from KLHDC2 and KEAP1 assays from the indicated concentrations of 3 (C) and 4 (D) (n=3; mean and s.e.m.).