Targeting TRIM Proteins: A Quest towards Drugging an Emerging Protein Class

Abstract

The ubiquitylation machinery regulates several fundamental biological processes from protein homeostasis to a wide variety of cellular signaling pathways. As a consequence, its dysregulation is linked to diseases including cancer, neurodegeneration, and autoimmunity. With this review, we aim to highlight the therapeutic potential of targeting E3 ligases, with a special focus on an emerging class of RING ligases, named tri-partite motif (TRIM) proteins, whose role as targets for drug development is currently gaining pharmaceutical attention. TRIM proteins exert their catalytic activity as scaffolds involved in many protein-protein interactions, whose multidomains and adapter-like nature make their druggability very challenging. Herein, we give an overview of the current understanding of this class of single polypeptide RING E3 ligases and discuss potential targeting options.

Keywords: high-throughput screening; inhibitors; ligases.

Figure 1

Mechanism of ubiquitin transfer. A) Ub forms a thioester intermediate with the E1 and is then transferred to the E2 through a trans‐thiolation reaction. B) Next, E3 ligases (RING, HECT or RBR) catalyze the transfer of Ub onto the substrate directly or by intermediacy of a Ub‐thioester. SBD: substrate binding domain; CUL: cullin protein; A: adaptor; R: substrate‐receptor; C: C‐terminal domain, N: N‐terminal domain; R1: RING domain 1, B: in‐between domain, R2: RING domain 2.

Figure 2

Classification of TRIM/RBCC proteins. The N‐terminal domain (N) or RBCC motif is mostly conserved in TRIM family members and includes RING, BB1 (B‐box 1), BB2 (B‐box 2), and CC (coiled‐coil domain). A variable C‐terminal domain (C) classifies TRIMs into 12 different classes and includes COS (COS box motif), FN3 (FibroNectin type III motif), PRY, SPRY (SPla and the RYanodine receptor), PHD (Plant Homeodomain), BRD (bromo domain), FILAMIN, NHL (NCL‐ 1/HT2A/LIN‐41), MATH (meprin and TRAF homology domain), ARF (ADP ribosylation factor)/SAR, TM (transmembrane motif), and a variable domain. The presence of certain domains can vary even among members of the same class, as indicated by brackets.

Figure 3

TRIM structural determinants. I. RING domain. A) Schematic representation of the RING domain: the coordination of zinc ions is tetrahedral and is based upon the position and the number of Cys and His residues. B) TRIM28 RING domain (PDB ID: 6I9H) coordination of the zinc ions. II. E2‐RING interaction. A) TRIM21^RING and the isopeptide‐linked Ube2N (Ubc13)‐Ub (PDB ID: 6S53): Glu12 and Asp21 interact with Ube2N via its positively charged residues Arg6, Arg7, Lys10 and Arg14, present on helix 1 of Ube2N, facing the TRIM^RING surface. The number of aromatic side chains at the E2 : E3^RING interface mediates hydrophobic and conserved H‐bond interactions. B) The TRIM^RING protomer interacts with Ub by forming H‐bonds between its Asn71 and Lys33. III. B‐Boxes. Schematic representations of A) B‐box 1 and B) B‐box 2. IV. Coiled Coil domain. TRIM25 coiled‐coil domain (PDB ID: 4LTB): within the symmetrical dimer each subunit shows a hairpin‐like folding forming long (L1) and short arms (L2). V. PRYSPRY domain. A) Schematic representation of the PRYSPRY domain, consisting of a PRY domain at its the N terminus, including 3 antiparallel β‐strands, and a SPRY domain that forms the remaining 10 strands towards its C terminus. B) PRYSPRY domain of TRIM5α (PDB ID: 2FBE): a compact module folded to acquire a jellyroll β‐sandwich tertiary conformation. VI. PHD‐BROMO domain. A) TRIM24 PHD‐BRD (PDB ID: 5H1U): The PHD is a zinc finger where C4HC3 cross braces to form antiparallel‐β‐sheets. The BRD folds with four left‐handed α‐helices (αZ, αA, αB, and αC) that form a large central cavity. The loop regions (ZA and BC loops) constitute the rim of the acetyl‐Lys binding pocket. B) Phylogenetic representation of the human BRD family, including 8 clusters (I‐VIII). ^[112] TRIM proteins are non‐BET BRDs: TRIM24, TRIM33, and TRIM66 belong to subfamily V; TRIM28 is member of subfamily VI. VII. FILAMIN‐NHL domain (PDB ID: 6FPT). A) In TRIM proteins, the NHL domain is associated with the Filamin‐type immunoglobulin domain (FN3) to form a compact structure. The overall tertiary conformation of the NHL domain results in a β‐propeller barrel with a canonical toroidal shape. ^[116] B) The NHL domain is present as multiple repetition units arranged in four to six symmetrical blade‐shaped β‐sheets.[ ²⁸ , ⁷² ]

Figure 4

Schematic representation of potential targeting options for TRIM proteins. Inhibition strategies could be primarily achieved by targeting the N‐terminal conserved RBCC motif represented by I) inhibition of E2 binding; II) exploitation of the RING interactions with Ub to be transferred; III) exploration of the putative allosteric docking of unbound ubiquitin molecules; IV) interfering with coiled‐coil‐mediated self‐association or by targeting the variable C‐ter‐ minal‐mediated substrate binding domain (SBD). The most promising targeting strategy is represented by the latter (V). Inhibition of substrate recruitment with a special focus on three structural motifs found in TRIM proteins: PHD‐BRD cassette, PRYSPRY domains, and NHL repeats. Future research might also define a role and targeting mode for B‐box domain–mediated protein–protein interactions (PPIs).

Figure 5

Published TRIM BRD inhibitors. A) Bromosporine, a broad‐spectrum BRD inhibitor (left); general formula of a patented TRIM33 BRD inhibitor (right); B) TRIM24 BRD inhibitors: Compound 34 ^[157] and IACS‐9571 ^[175] (left). Both compounds are based on a 1,3‐dimethylbenzimidazolone scaffold decorated with a sulfonamide in position 5 and a substituted phenyl in R¹. In IACS‐9571 the introduction of a N,N‐dimethyl amino group on R² with interposition of a short linker, promotes an additional ionic interaction; Co‐crystal structure of TRIM24 BRD and IACS‐9571 ^[175] (PDB ID: 4YC9) (right) showing the conserved folding of the BRD and the ligand interacting with two key residues: N980 (H‐bond between the K^Ac mimicry carbonyl moiety and the NH₂ of the conserved Asn residue) in the BC loop and D926 (ionic interaction between the positively charged tertiary amine in R² and the negatively charged carboxylic moiety of the Asp side chain) in the ZA loop. C) PROTAC for TRIM24 (dTrim24), made up of a PEG linker connecting a derivative of IACS‐9571 and a known VHL binder (VH032).