PDBsum entry 1rv1

Ligase

PDB id: 1rv1

Contents

Protein chains

85 a.a. *

Ligands

IMZ ×5

Waters ×31

* Residue conservation analysis

PDB id:

1rv1

Name:

Ligase

Title:

Crystal structure of human mdm2 with an imidazoline inhibitor

Structure:

Ubiquitin-protein ligase e3 mdm2. Chain: a, b, c. Fragment: residues 25-109. Synonym: p53-binding protein mdm2, oncoprotein mdm2, double minute 2 protein, hdm2. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: mdm2. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Biol. unit:

Trimer (from PQS)

Resolution:

2.30Å R-factor: 0.266 R-free: 0.322

Authors:

C.Lukacs,U.Kammlott,B.Graves

Key ref:

L.T.Vassilev et al. (2004). In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science, 303, 844-848. PubMed id: 14704432 DOI: 10.1126/science.1092472

Date:

12-Dec-03 Release date: 20-Jan-04

Protein chains

Q00987  (MDM2_HUMAN) -  E3 ubiquitin-protein ligase Mdm2 from Homo sapiens

Seq: 491 a.a.

Struc: 85 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.3.2.27 - RING-type E3 ubiquitin transferase.
• Reaction: S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [acceptor protein]-L-lysine = [E2 ubiquitin-conjugating enzyme]-L-cysteine + N₆- ubiquitinyl-[acceptor protein]-L-lysine

DOI no: 10.1126/science.1092472

Science 303:844-848 (2004)

PubMed id: 14704432

In vivo activation of the p53 pathway by small-molecule antagonists of MDM2.

L.T.Vassilev, B.T.Vu, B.Graves, D.Carvajal, F.Podlaski, Z.Filipovic, N.Kong, U.Kammlott, C.Lukacs, C.Klein, N.Fotouhi, E.A.Liu.

ABSTRACT

MDM2 binds the p53 tumor suppressor protein with high affinity and negatively modulates its transcriptional activity and stability. Overexpression of MDM2, found in many human tumors, effectively impairs p53 function. Inhibition of MDM2-p53 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. Here, we identify potent and selective small-molecule antagonists of MDM2 and confirm their mode of action through the crystal structures of complexes. These compounds bind MDM2 in the p53-binding pocket and activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice.

Selected figure(s)

Figure 2.
Fig. 2. Inhibition of MDM2-p53 binding by Nutlin-1 activates the p53 pathway in cells with wild-type p53. (A) SW480 (mutant p53) and HCT116 (wild-type p53) cells were incubated with the indicated concentrations of Nutlin-1 for 8 hours and p53, MDM2, and p21 proteins were analyzed in the cell lysates by Western blotting. (B) Nutlin-1 treatment induces the expression of the p21 gene but not the p53 gene. Cells with wild-type p53 (HCT116, RKO, and H460a) were treated with Nutlin-1 for 8 hours, and the change in the level of transcription was measured by quantitative PCR and expressed as fold induction compared with the untreated control. (C) Nutlin-1 arrests cell cycle in the G[1] and G[2] phases. HCT116 and SJSA-1 cells were incubated with 4 µM Nutlin-1 or an equivalent amount of solvent for 22 hours and an additional 2 hours with 10 µM BrdU, and cell cycle distribution was analyzed after propidium iodide/fluorescein isothiocyanate-antibody to BrdU staining (30). Cells within the rectangles are in S phase. (D) Antiproliferative and cytotoxic activity of Nutlin-1. Exponentially growing cancer cells with wild-type p53 (HCT116, RKO, and SJSA-1) or mutant p53 (MDA-MB-435 and SW480) were incubated with a range of concentrations for 5 days and the cell mass and viability were measured by the MT T assay. (E) Inhibition of clonogenic cell growth. Cancer cells with wild-type p53 (HCT116 and RKO) or mutant p53 (MDA-MB-435, SW480, and PC3) were seeded at a low cell density and their ability to form colonies was measured after 5 days of incubation with Nutlin-1. (F) p53 activation by Nutlin-1 does not involve Ser15 phosphorylation on p53. Cancer cells were treated with doxorubicin (1 µM), etoposide (10 µM), or Nutlin-1 (6 µM) for 20 hours, and the amount of total p53 and p53 phosphorylated on Ser15 was determined by Western blottingin aliquots of cell lysates normalized for total protein.

Figure 4.
Fig. 4. In vivo antitumor activity of MDM2 inhibitors. Nude mice (10 animals per dose group) bearing subcutaneous human cancer xenografts (SJSA-1) with mean volumes of 185 mm3 received 200 mg/kg of an oral dose of Nutlin-3 (racemic) twice daily or 10 mg/kg of intravenous doxorubicin once a week for 3 weeks. The tumor volumes were measured and recorded periodically duringthe course of the study. P < 0.001 for Nutlin-3 and doxorubicin compared with corresponding vehicle controls. Error bars show SEM.

The above figures are reprinted by permission from the AAAs: Science (2004, 303, 844-848) copyright 2004.

Figures were selected by an automated process.