Discovery of neddylation E2s inhibitors with therapeutic activity

Abstract

Neddylation is the writing of monomers or polymers of neural precursor cells expressed developmentally down-regulated 8 (NEDD8) to substrate. For neddylation to occur, three enzymes are required: activators (E1), conjugators (E2), and ligators (E3). However, the central role is played by the ubiquitin-conjugating enzymes E2M (UBE2M) and E2F (UBE2F), which are part of the E2 enzyme family. Recent understanding of the structure and mechanism of these two proteins provides insight into their physiological effects on apoptosis, cell cycle arrest and genome stability. To treat cancer, it is therefore appealing to develop novel inhibitors against UBE2M or UBE2F interactions with either E1 or E3. In this evaluation, we summarized the existing understanding of E2 interaction with E1 and E3 and reviewed the prospective of using neddylation E2 as a pharmacological target for evolving new anti-cancer remedies.

Fig. 1. Canonical neddylation pathway.

Precursor NEDD8 with hydrolase is the first process before NEDD8 reacts with NAE. By actions of ATP and Mg²⁺, NAE is first loaded with one NEDD8 molecule. Following the same procedure, the second NEDD8 is attached with NAE. Double NEDD8 loaded NAE allows conjugating enzyme E2 binding and transferring NEDD8 from NAE’s active cysteine to E2’s cysteine. Next, by coordinating with E3 ligase, NEDD8 moves to the target protein. Finally, through the deneddylation process, NEDD8 is removed from the substrates.

Fig. 2. Structural features of UBE2M and UBE2F.

A Classification of E2 conjugating enzyme based on N and C terminal extensions. B The crystal structure of UBE2M in green (PDB: 1Y8X) and UBE2F in the sky (PDB: 3FN1). C Virtually identified V30, F56, and C116 pockets of UBE2F for inhibitor binding. D Principle of developing DI-591 and (E) Proposed idea of developing E2 targeting inhibitor.

Fig. 3. Structural analysis of E1-E2 binding pattern.

A Interaction of E2 with E1. Appropriate interaction is necessary for NEDD8 transfer to E2. N-extension= N-terminal 26 residues of E2 (UBE2M). Improper docking of the E2 core domain or N-terminal extension prevented NEDD8 transfer from E1 to E2. B, C UBE2M’s N-terminal (purple) interaction with the hydrophobic pocket of UBA3 (cyan) and interacted amino acids, respectively (PDB:1TT5).

Fig. 4. Inhibitors reported against various interaction sites of UBE2M.

The picture represents the inhibitors reported regarding E2 interaction inhibition with E1 or E3, “*” indicate the IC₅₀ measurement according to western blot band observation.

Fig. 5. UBE2M and DCN1 interaction.

Acetylated-UBE2M N-terminal (purple) binding in the pocket of DCN1 (left, green) and interacted amino acids, right (PDB:3TDU).

Fig. 6. E2-based neddylation inhibitor discovery method.

Schematic presentation of TR-FRET based HTRF method for drug discovery.