Structural studies of antitumor compounds that target the RING domain of MDM2

Abstract

Mouse double minute 2 homolog (MDM2) is an E3 ubiquitin-protein ligase that is involved in the transfer of ubiquitin to p53 and other protein substrates. The expression of MDM2 is elevated in cancer cells and inhibitors of MDM2 showed potent anticancer activities. Many inhibitors target the p53 binding domain of MDM2. However, inhibitors such as Inulanolide A and MA242 are found to bind the RING domain of MDM2 to block ubiquitin transfer. In this report, crystal structures of MDM2 RING domain in complex with Inulanolide A and MA242 were solved. These inhibitors primarily bind in a hydrophobic site centered at the sidechain of Tyr489 at the C-terminus of MDM2 RING domain. The C-terminus of MDM2 RING domain, especially residue Tyr489, is required for ubiquitin discharge induced by MDM2. The binding of these inhibitors at Tyr489 may interrupt interactions between the MDM2 RING domain and the E2-Ubiquitin complex to inhibit ubiquitin transfer, regardless of what the substrate is. Our results suggest a new mechanism of inhibition of MDM2 E3 activity for a broad spectrum of substrates.

Keywords: E3 ubiquitin ligase; RING finger protein; crystal structure; drug design; inhibition mechanism.

FIGURE 1

Structure of MDM2 RING‐InuA complex. (a) Binding of InuA in the MDM2 RING dimer. The A subunit is in green and the B subunit is in cyan. The structure of InuA is shown as a stick model colored by elements: carbon in yellow and oxygen in red. Zn²⁺ ions are shown as spheres. N and C termini are labeled. The one‐turn α‐helix in the A subunit is indicated by residues 432–435. (b) A close‐up view of the binding site of InuA. The sidechains of residues that interact with InuA are labeled with one letter code and the residue number and shown as stick models. (c) A diagram describing the interactions between InuA and the protein. The mainchain nitrogen of Glu490 may form a hydrogen bond with O5 in InuA. Other contact information is listed in Table 2

FIGURE 2

Structure of MDM2 RING‐MA242 complex. (a) Binding of MA242 in the MDM2 RING dimer. The A subunit is in green and the B subunit is in cyan. The structure of MA242 is shown as a stick model colored by elements: carbon in yellow, nitrogen in blue, oxygen in red, sulfur in brown, and chlorine in green. Zn²⁺ ions are shown as spheres. N and C termini are labeled. The one‐turn α‐helix in the A subunit is indicated by residues 432–435. (b) A close‐up view of the binding site of MA242. The sidechains of residues that interact with MA242 are labeled with one letter code and the residue number and shown as stick models. (c) A diagram describing the interactions between MA242 and the protein. The mainchain of residue Phe490 is in the proximity of MA242 but has no direct interactions

FIGURE 3

Autoubiquitination in the presence of InuA and MA242. (a) Reduced SDS‐PAGE showing auto‐Flr‐ubiquitination of GST‐MDM2‐422‐C‐S429E/G443T‐491 in the presence of DMSO (control) and inhibitor compounds at 50 μM concentration. Monoubiquitination bands appear at 43.5 kDa with di‐ and polyubiquitination appearing directly above as laddered bands. Fluorescein‐labeled ubiquitin conjugation yields an additional 9.5 kDa per conjugation. (b) Relative ubiquitination following peak integration in ImageJ at 3 min reaction time. Reactions were performed in triplicates

FIGURE 4

Tripartite complex of MDM2 RING domain, Ub, and E2. (a) The structure of the tripartite complex (PDB 6SQS). The dimer of MDM2 RING domain is colored as in Figure 1, and N or C termini are labeled. Ub is in red and E2 UbcH5B in blue. (b) Superposition of InuA bound with the MDM2 RING domain in the tripartite complex. The stick model of InuA is colored by elements. (c) Superposition of MA242 bound with the MDM2 RING domain in the tripartite complex. The stick model of MA242 is colored by elements. (d) A close‐up of the bound InuA superimposed in the tripartite complex. Residues of the InuA binding site in the MDM2 RING domain are colored by elements. Key residues are labeled. (e) A close‐up of the bound MA242 superimposed in the tripartite complex. Residues of the MA242 binding site in the MDM2 RING domain are colored by elements. Key residues are labeled