Year: 2009
Type: dissertation
Abstract: Chapter 1 while the last C-terminus regulatory domain takes part in binding to single stranded DNA and RNA (Tidow et al., 2007).The quaternary structure model of the full p53 and different domains constructs in their free form and bound to a specific DNA sequences was recently obtained from NMR, X-ray scattering (SAXS) in solution, and electron microscopy (EM) of Chapter 1 1987).The Mdm2 gene was found as overexpressed in 30% of osteosarcomas and soft tissue tumor (Oliner et al., 1992).This finding was correlated with discovery that Mdm2 gene product co-precipitate with p53 (Momand et al., 1992;Barak and Oren 1992).Mdm2 acts as an ubiquitin ligase (it is a member of the RING E3 ubiquitin ligase family), promoting ubiquitination of p53 for the proteasome degradation will follow (Haupt et al., 1997;Kubbutat et al., 1997).As Mdm2 gene is regulated by p53 pathway, the level of 23 , and Leu 26 , which insert deep into the Hdm2 cleft.Thus the p53 amino acid triad comprises the "hot spot" of the p53/Hdm2 interaction.Noteworthy is the cross section dimension of the p53 binding site in Hdm2 of 18 Å (from C(-Tyr67 to C(-Tyr100), which is the size of small organic molecules thus indicating the possibility of small molecule interference.Mdmx, also known as Mdm4, has only recently emerged as a critical, non-redundant regulator of p53 activation (Toledo and Wahl, 2006;Laurie et al., 2006;Marine et al., 2007).The protein composed of 490 residues was first isolated from the mouse cDNA library (Shvarts et al., 1996).The same group in 1997 identified the human analogue.Independently, Tanimura et al. (1999) showed that Mdmx is associated with Mdm2 in hybrid yeast.Sequence aligment shows that the wild type human Mdmx is highly homoloques to human Mdm2 (identity 31.3%, similarity 50.1% for the full length proteins and identity 51.5%, similarity 69.2% in the N-terminal region comprising p53 binding domain).Another highly conserved region-common for both proteins is located at the C terminus (the RING domain, with 42.9% identity and 66.1% of similarity) and enables heterodimerization with Mdm2.In Chapter 1 Glioblastoma 4 Colon 19 Lung 18 Breast 19 Melanoama 14 Hepatocellular carcinoma 50 Head and neck 50 Sarcoma 22 Bladder cancer 25 All cell lines 40 Mi-219 3-imidazolyl-indoles Preclinical models of human cancer retaining wildtype p53 (Patent WO 2008/119741).Novartis 101 Fragment-based drug discovery and the role of NMR in fragment screeningFragment-based drug design has become an important and powerful approach in structurebased lead discovery (reviewed in: Rees et al., 2004;Schade and Oschkinat, 2005; Jahnke and Erlanson, 2006;Erlanson, 2006;Hajduk and Greer, 2007;Hubbard et al., 2007).Compared with traditional screening hits, the starting fragments have considerably lower Chapter 1Rothweiler et al., 2008a), named AIDA (for the Antagonist Induced Dissociation Assay) NMR, provides unambiguous information on whether an antagonist of a protein-protein interaction is strong enough to dissociate the complex and whether its action is through denaturation, precipitation, or release of a protein in its functional folded state.This method was checked for several lead compounds that have been reported in the literature to inhibit the p53-Mdm2 interaction, only one of them, nutlin-3, turned out to be a potent inhibitor of this interaction in vitro (D´Silva et al., 2005;Krajewski et al., 2005; Popowicz et al., 2007).15 N labeled Mdm2 (each amino acid gives a cross peak for the N-H pair, the side chain N-H resonances are observed at around 7 ppm 1 H and at 120 ppm 15 N chemical shifts).B: The cross-peaks disappear on addition of a large fragment of the p53 protein (ca.35 kDa) that forms a complex with the smaller one.Due to increase in the rotational correlation time of the protein-protein complex, the HSQC signals get broadened and fall into the noise level.C: Addition of an effective antagonist of the protein-protein interaction results in release of the small 15 N-labeled protein from the complex and the HSQC signal recovery.D: A weak inhibitor does not dissociate the complex.Figure 10.1D NMR version of the AIDA assay.A: The 1D proton NMR spectrum of the side chains of tryptophans (W) of free p53 (residues 1-321).The N-terminal domain of p53 has three tryptophan residues: W23, W53, and W91 (the N-terminal domain encompasses residues 1-94; the p53 domain
Author: Anna Czarna
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2748327252