Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase

Abstract

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

Figure 1

Cartoon representation of the domain architecture of human TRIM58. Numbers below the ring, B-box, coiled-coil, and PRY-SPRY domains represent residue positions of domain boundaries according to Uniprot entry Q8NG06. Lines below the PRY-SPRY domain indicate the boundaries of the proteins used in this study: TRIM58(251–466), TRIM58(251–466)C277S,C278S, and TRIM58(279–466).

Figure 2

Hit finding and hit validation. A) Shift in transition temperature (ΔT_m) of TRIM58(251–466) observed by DSF experiments with compounds from the hit cluster featuring 107 similar compounds, including TRIM-473. The primary hit for this cluster (compound 1, green star) and TRIM-473 are highlighted. Compounds that lead to a stabilization of >+0.5 °C are labeled in green, compounds that lead to a destabilization/stabilization of −1.0 to +0.5 °C are labeled in gray, and compounds that lead to a destabilization of <−1 °C are marked in red. B) DSF protein melting curve of TRIM58(251–466) in the presence (orange) and absence (black) of TRIM-473. The dotted curves in gray and orange represent the first derivative of the corresponding protein melting curve. The curve inflection point displays the protein T_m. C) Overlay of the methyl region of 2D [¹³C,¹H]-HMQC spectra of uniformly ¹³C,¹⁵N-labeled TRIM58(251–466)C277S,C278S in the absence (black) and in the presence of TRIM-473 (orange), recorded at concentrations of the protein and the compound of 30 and 600 μM, respectively. D) SPR binding experiment for the interaction of TRIM58(251–466)C277S,C278S-Avi with TRIM-473. The equilibrium fit is shown. E) Chemical structures of compounds 1 and TRIM-473.

Figure 3

Identification of a TRIM58 binding peptide. Based on the N-terminus of the dynein intermediate chain (top sequence, helical secondary structure prediction in green below), four overlapping peptides (P1–P4) were tested for binding in a protein-observed NMR binding assay. Peptides with confirmed binding were subsequently measured by SPR. To further narrow the minimal binding sequence, three additional peptides (P5–P7) were tested by NMR and SPR.

Figure 4

A) Apo crystal structure of TRIM58(251–466)C277S,C278S (PDB ID: 8PD4). Chains A and B are shown in gray and cyan, respectively. B) Close-up of interaction of the N-terminal portion of chain B with chain A; TRIM-473 is overlaid for illustration purposes and colored by atom type. C) Sequence alignment between DIC(1–22) and TRIM58(258–278) colored by similarity; Q12 (DIC peptide fluorophore attachment) is highlighted in green.

Figure 5

A) X-ray co-crystal structure of TRIM58(251–466)C277S,C278S (grayish) and TRIM-473 (orange sticks), PDB ID: 8PD6. Side chains of residues D315 and E357 that interact with the ligand are shown as sticks. B) TRIM-473 (orange) engages via apolar π–π interactions (yellow dotted lines) and polar interactions (blue dotted lines) with several TRIM58 residues (grayish side chains as sticks). A putative chloro ion present in the ligand binding site is highlighted as a green sphere, and water molecules are shown as red spheres. C) TRIM-473 (orange) binds in a shallow pocket lined with hydrophobic residues (L395, M393, L387). Potential exit vectors for bifunctional degrader design are indicated with arrows.

Figure 6

Comparison of the TRIM58^PRY-SPRY/TRIM-473 complex structure with those of other TRIM^PRY-SPRY domains. A) List of aligned PRY-SPRY domains from 14 human TRIM family proteins. TRIM-like proteins are denoted with an asterisk. B) An overlay of the 14 structures shows that the β-sandwich of two antiparallel β-sheets superimposes well (where TRIM-473 (orange sticks) binds TRIM58), while the loops flanking the substrate binding region diverge significantly. C) Superimposition of the TRIM58^PRY-SPRY/TRIM473 complex (gray/orange) with the helical recognition motive of RIG-I (yellow) from the cryo-EM structure of RIG-I:dsRNA filament in complex with RIPLET^PRY-SPRY domain (PDG ID: 7JL3). D) Superimposition of the TRIM58^PRY-SPRY/TRIM473 complex (gray/orange) with the FC domain of IgG (residues 420–443 shown in yellow, PDB ID: 2IWG).