Discovery of New Binders for DCAF1, an Emerging Ligase Target in the Targeted Protein Degradation Field

Abstract

In this study, we describe the rapid identification of potent binders for the WD40 repeat domain (WDR) of DCAF1. This was achieved by two rounds of iterative focused screening of a small set of compounds selected on the basis of internal WDR domain knowledge followed by hit expansion. Subsequent structure-based design led to nanomolar potency binders with a clear exit vector enabling DCAF1-based bifunctional degrader exploration.

Figure 1

(a) Top view and (b) side view of the crystal structure (PDB ID: 4CC9) of the human E3 ligase substrate adaptor DCAF1 extracted from the ternary complex with the viral accessory protein X (Vpx) and the carboxy-terminal region of human SAMHD1 (not shown). The “top” and “bottom” surfaces are displayed on the right and left of panel (b), respectively. Residues 1315–1327 are not visible in the crystal structure. The Cα atom of the last visible residues, 1314 and 1328 belonging to blade 6, are marked with a green sphere. The white and black spheres indicate the two pocket locations as identified by SiteMap and fpocket. The white spheres correspond to the large ligandable donut hole pocket. The black spheres indicate the location of the blade region, an additional less druggable cavity that lies between blade 1 (colored in blue) and blade 2 (in pink). Each blade (numbered in the picture from 1 to 7) is constituted by a four-stranded antiparallel β-sheet. Strands are labeled from (a) to (d) from the inside toward the outside of the propeller.

Figure 2

Overlay of the methyl region of 2D [¹³C,¹H]-HMQC spectra of selectively ¹³C^ε-methionine labeled DCAF1 in the absence (blue) and in the presence of the compound 1 (red). The concentrations of the protein and the compound were 15 μM and 480 μM, respectively.

Figure 3

(a) Compound 1 binds to the central donut cavity of DCAF1 in two orientations. In both crystal structures, the protonated imidazoline moiety makes a hydrogen bond, with the carbonyl of T1097 in pose 1 (PDB ID: 8OG6, cyan) and with the carboxyl moiety of D1356 side chain in pose 2 (PDB ID: 8OG5, green). The hydrogen bonds are shown as dashed black lines. (b) Overlay of crystal structure of 1 bound to DCAF1 (PDB ID: 8OG6, cyan) and to EED (PDB ID: 5H25, orange). The three residues forming the aromatic cages in EED are labeled.

Figure 4

Crystal structures of 2 bound to the blade region of DCAF1 (PDB ID: 8OG7, cyan) and of EED (orange). Only the most buried portion of the ligand is built in the density in both crystal structures. The two crystal structures are overlaid with the DCAF1-Vpx-SAMHD1 crystal structure (PDB ID: 4CC9), from which only the Vpx protein is shown in violet, and with the EED-1-EZH2 crystal structure (PDB ID: 5H25), from which only the EZH2 protein is shown in yellow.

Scheme 1. Analogues of Hit 1

Figure 5

Crystal structure of 3 (PDB ID: 8OG8, blue) and 4 (PDB ID: 8OG9, purple) in (a) the donut hole cavity and in (b) the blade region. For 3 only one of the blade poses is shown. The other pose has the tricyclic core flipped by 180°. The DCAF1 protein is shown in gray, and the hydrogen bonds are shown as dashed black lines.

Scheme 2. Structure-Based Design Strategies (K_D Values from SPR)

Figure 6

(a) DCAF1 crystal structure in complex with 8 (PDB ID: 8OGB, purple) overlaid with the DCAF1 crystal structure in complex with 1 (PDB ID: 8OG6, cyan). (b) DCAF1 crystal structure in complex with 11 (PDB ID: 8OGC, yellow) overlaid with the DCAF1 crystal structure in complex with 8 (purple), of which only the ligand is shown. The DCAF1 protein surface is shown in gray, and the hydrogen bonds are shown as dashed black lines.

Scheme 3. Exploration of the Benzylic Position (K_D Values from SPR)

Figure 7

DCAF1 crystal structures in complex with (a) compound 8 (PDB ID: 8OGB) and (b) compound 26e of reference (18) (PDB ID: 8F8E). (c) DCAF1 crystal structure deposited in PDB under the code 7SSE. Hydrogen bonds are shown as dashed black lines, and water molecules are shown as red spheres.