NMR structure of a complex between MDM2 and a small molecule inhibitor

MDM2 is a regulator of cell growth processes that acts by binding to the tumor suppressor protein p53 and ultimately restraining its activity. While inactivation of p53 by mutation is commonly observed in human cancers, a substantial percentage of tumors express wild type p53. In many of these cases, MDM2 is overexpressed, and it is believed that suppression of MDM2 activity could yield therapeutic benefits. Therefore, we have been focusing on the p53-MDM2 interaction as the basis of a drug discovery program and have been able to develop a series of small molecule inhibitors. We herein report a high resolution NMR structure of a complex between the p53-binding domain of MDM2 and one of these inhibitors. The form of MDM2 utilized was an engineered hybrid between the human and Xenopus sequences, which provided a favorable combination of relevancy and stability. The inhibitor is found to bind in the same site as does a highly potent peptide fragment of p53. The inhibitor is able to successfully mimic the peptide by duplicating interactions in three subpockets normally made by amino acid sidechains, and by utilizing a scaffold that presents substituents with rigidity and spatial orientation comparable to that provided by the alpha helical backbone of the peptide. The structure also suggests opportunities for modifying the inhibitor to increase its potency.

[1]  R. Kriwacki,et al.  Structural basis for LFA-1 inhibition upon lovastatin binding to the CD11a I-domain. , 1999, Journal of molecular biology.

[2]  P. Furet,et al.  Discovery of potent antagonists of the interaction between human double minute 2 and tumor suppressor p53. , 2000, Journal of medicinal chemistry.

[3]  P. Toogood Inhibition of protein-protein association by small molecules: approaches and progress. , 2002, Journal of medicinal chemistry.

[4]  A. Levine,et al.  Structure of the MDM2 Oncoprotein Bound to the p53 Tumor Suppressor Transactivation Domain , 1996, Science.

[5]  Paul A. Keifer,et al.  Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity , 1992 .

[6]  K. Nagayama Four-quadrant pure-phase representation of two-dimensional spectra with time reversal or frequency inversion , 1986 .

[7]  A. Gronenborn,et al.  Determination of three‐dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms Circumventing problems associated with folding , 1988, FEBS letters.

[8]  A. Hamilton,et al.  Toward proteomimetics: terphenyl derivatives as structural and functional mimics of extended regions of an alpha-helix. , 2001, Journal of the American Chemical Society.

[9]  E B Reilly,et al.  Novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intracellular adhesion molecule-1 interaction. 2. Mechanism of inhibition and structure-based improvement of pharmaceutical properties. , 2001, Journal of medicinal chemistry.

[10]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[11]  P. Meltzer,et al.  Amplification of a gene encoding a p53-associated protein in human sarcomas , 1992, Nature.

[12]  D. Lane,et al.  The p53-Mdm2 pathway: targets for the development of new anticancer therapeutics. , 2003, Mini reviews in medicinal chemistry.

[13]  M. Oren,et al.  Mdm2 promotes the rapid degradation of p53 , 1997, Nature.

[14]  S. Stass,et al.  The human MDM-2 oncogene is overexpressed in leukemias. , 1993, Blood.

[15]  C. Humblet,et al.  Structure-Based Design of a Novel Series of Nonpeptide Ligands that Bind to the Pp60Src Sh2 Domain , 1997 .

[16]  M. Imamura,et al.  p53 mutation, murine double minute 2 amplification, and human papillomavirus infection are frequently involved but not associated with each other in esophageal squamous cell carcinoma. , 1995, Clinical cancer research : an official journal of the American Association for Cancer Research.

[17]  A. Levine p53, the Cellular Gatekeeper for Growth and Division , 1997, Cell.

[18]  D. Lane,et al.  p53 protein stability in tumour cells is not determined by mutation but is dependent on Mdm2 binding , 1997, Oncogene.

[19]  D. Fry,et al.  Solution structure of the Ras-binding domain of c-Raf-1 and identification of its Ras interaction surface. , 1995, Biochemistry.

[20]  G. Powers,et al.  Identification of a Small Molecule Inhibitor of the IL-2/IL-2Rα Receptor Interaction Which Binds to IL-2 , 1997 .

[21]  M. Hatada,et al.  X-Ray structure of citrate bound to Src SH2 leads to a high-affinity, bone-targeted Src SH2 inhibitor. , 2001, Journal of medicinal chemistry.

[22]  D. Fry,et al.  NMR characterization of interleukin‐2 in complexes with the IL‐2Rα receptor component, and with low molecular weight compounds that inhibit the IL‐2/IL‐Rα interaction , 2003, Protein science : a publication of the Protein Society.

[23]  Michelle R Arkin,et al.  Discovery of a potent small molecule IL-2 inhibitor through fragment assembly. , 2003, Journal of the American Chemical Society.

[24]  A. J. Shaka,et al.  Iterative schemes for bilinear operators; application to spin decoupling , 1988 .

[25]  A B Smith,et al.  An orally bioavailable pyrrolinone inhibitor of HIV-1 protease: computational analysis and X-ray crystal structure of the enzyme complex. , 1997, Journal of medicinal chemistry.

[26]  John W. Bean,et al.  Direct design of a potent non-peptide fibrinogen receptor antagonist based on the structure and conformation of a highly constrained cyclic RGD peptide , 1993 .

[27]  R. Campbell,et al.  N-acyl-L-phenylalanine derivatives as potent VLA-4 antagonists that mimic a cyclic peptide conformation. , 2002, Bioorganic & medicinal chemistry letters.

[28]  C. Renner,et al.  Chalcone derivatives antagonize interactions between the human oncoprotein MDM2 and p53. , 2001, Biochemistry.

[29]  L. Mueller,et al.  Selective shaped pulse decoupling in NMR: homonuclear [carbon-13]carbonyl decoupling , 1992 .

[30]  E. R. Andrew,et al.  Nuclear Magnetic Resonance , 1955 .

[31]  F. Vögtle,et al.  Die Diaza‐Cope‐Umlagerung , 1976 .

[32]  Thomas Rawson,et al.  From Peptide to Non-Peptide. 2. The de Novo Design of Potent, Non-peptidal Inhibitors of Platelet Aggregation Based on a Benzodiazepinedione Scaffold , 1994 .

[33]  L. Presta,et al.  Generation of an LFA-1 antagonist by the transfer of the ICAM-1 immunoregulatory epitope to a small molecule. , 2002, Science.

[34]  L. Frye,et al.  Binding site elucidation of hydantoin-based antagonists of LFA-1 using multidisciplinary technologies: evidence for the allosteric inhibition of a protein--protein interaction. , 2001, Journal of the American Chemical Society.

[35]  T. Clackson,et al.  A hot spot of binding energy in a hormone-receptor interface , 1995, Science.

[36]  R. Saiki,et al.  A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. , 1988, Nucleic acids research.

[37]  Ad Bax,et al.  Isotope-filtered 2D NMR of a protein-peptide complex: study of a skeletal muscle myosin light chain kinase fragment bound to calmodulin , 1992 .

[38]  A Gene-Expression Inhibitor that Targets an α-Helix-Mediated Protein Interaction , 2003 .

[39]  B. Vogelstein,et al.  p53 mutations in human cancers. , 1991, Science.

[40]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.

[41]  G. Marius Clore,et al.  1H1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins , 1990 .

[42]  A. Levine,et al.  Mapping of the p53 and mdm-2 interaction domains. , 1993, Molecular and cellular biology.

[43]  G. Reifenberger,et al.  Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. , 1993, Cancer research.

[44]  U. Brinkmann,et al.  High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. , 1989, Gene.

[45]  Stephen N. Jones,et al.  Regulation of p53 stability by Mdm2 , 1997, Nature.