The structure of an MDM2-Nutlin-3a complex solved by the use of a validated MDM2 surface-entropy reduction mutant.

The p53-binding site of MDM2 holds great promise as a target for therapeutic intervention in MDM2-amplified p53 wild-type forms of cancer. Despite the extensive validation of this strategy, there are relatively few crystallographically determined co-complex structures for small-molecular inhibitors of the MDM2-p53 interaction available in the PDB. Here, a surface-entropy reduction mutant of the N-terminal domain of MDM2 that has been designed to enhance crystallogenesis is presented. This mutant has been validated by comparative ligand-binding studies using differential scanning fluorimetry and fluorescence polarization anisotropy and by cocrystallization with a peptide derived from p53. Using this mutant, the cocrystal structure of MDM2 with the benchmark inhibitor Nutlin-3a has been determined, revealing subtle differences from the previously described co-complex of MDM2 with Nutlin-2.

Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation.

The ubiquitin-proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein-protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

Analysis of the components of cancer risk perception and links with intention and behaviour: A UK-based study.

Risk perception refers to how individuals interpret their susceptibility to threats, and has been hypothesised as an important predictor of intentions and behaviour in many theories of health behaviour change. However, its components, optimal measurement, and effects are not yet fully understood. The TRIRISK model, developed in the US, conceptualises risk perception as deliberative, affective and experiential components. In this study, we aimed to assess the replicability of the TRIRISK model in a UK sample by confirmatory factor analysis (CFA), explore the inherent factor structure of risk perception in the UK sample by exploratory factor analysis (EFA), and assess the associations of EFA-based factors with intentions to change behaviour and subsequent behaviour change. Data were derived from an online randomised controlled trial assessing cancer risk perception using the TRIRISK instrument and intention and lifestyle measures before and after communication of cancer risk. In the CFA analysis, the TRIRISK model of risk perception did not provide a good fit for the UK data. A revised model developed using EFA consisted of two separate "numerical" and "self-reflective" factors of deliberative risk perception, and a third factor combining affective with a subset of experiential items. This model provided a better fit to the data when cross-validated. Using multivariable regression analysis, we found that the self-reflective and affective-experiential factors of the model identified in this study were reliable predictors of intentions to prevent cancer. There were no associations of any of the risk perception factors with behaviour change. This study confirms that risk perception is clearly a multidimensional construct, having identified self-reflective risk perception as a new distinct component with predictive validity for intention. Furthermore, we highlight the practical implications of our findings for the design of interventions incorporating risk perception aimed at behaviour change in the context of cancer prevention.

Structure of Rpn10 and its interactions with polyubiquitin chains and the proteasome subunit Rpn12.

Schizosaccharomyces pombe Rpn10 (SpRpn10) is a proteasomal ubiquitin (Ub) receptor located within the 19 S regulatory particle where it binds to subunits of both the base and lid subparticles. We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR. We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys(48)-linked diUb, which it binds selectively over monoUb and Lys(63)-linked diUb. We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys(48)-linked diUb. This is the first observation of a UIM binding other than a Ub fold and suggests that SpRpn12 could modulate the activity of SpRpn10 as a proteasomal Ub receptor.

MDM2-p53 protein-protein interaction inhibitors: a-ring substituted isoindolinones.

Structure-activity relationships for the MDM2-p53 inhibitory activity of a series of A-ring substituted 2-N-benzyl-3-(4-chlorophenyl)-3-(1-(hydroxymethyl)cyclopropyl)methoxy)isoindolinones have been investigated, giving rise to compounds with improved potency over their unsubstituted counterparts. Isoindolinone A-ring substitution with a 4-chloro group for the 4-nitrobenzyl, 4-bromobenzyl and 4-cyanobenzyl derivatives (10a-c) and substitution with a 6-tert-butyl group for the 4-nitrobenzyl derivative (10j) were found to confer additional potency. Resolution of the enantiomers of 10a showed that potent MDM2-p53 activity resided in the (-)-enantiomer ((-)-10a; IC(50)=44 ± 6 nM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compounds 10a and (-)-10a increase p53 protein levels, activate p53-dependent MDM2 and p21 transcription in MDM2 amplified cells, and show improved selectivity for growth inhibition in wild type p53 cell lines over the parent compound.

The impact of information about different absolute benefits and harms on intention to participate in colorectal cancer screening: A think-aloud study and online randomised experiment.

BACKGROUND: There is considerable heterogeneity in individuals' risk of disease and thus the absolute benefits and harms of population-wide screening programmes. Using colorectal cancer (CRC) screening as an exemplar, we explored how people make decisions about screening when presented with information about absolute benefits and harms, and how those preferences vary with baseline risk, between screening tests and between individuals. METHOD: We conducted two linked studies with members of the public: a think-aloud study exploring decision making in-depth and an online randomised experiment quantifying preferences. In both, participants completed a web-based survey including information about three screening tests (colonoscopy, sigmoidoscopy, and faecal immunochemical testing) and then up to nine scenarios comparing screening to no screening for three levels of baseline risk (1%, 3% and 5% over 15 years) and the three screening tests. Participants reported, after each scenario, whether they would opt for screening (yes/no). RESULTS: Of the 20 participants in the think-aloud study 13 did not consider absolute benefits or harms when making decisions concerning CRC screening. In the online experiment (n = 978), 60% expressed intention to attend at 1% risk of CRC, 70% at 3% and 77% at 5%, with no differences between screening tests. At an individual level, 535 (54.7%) would attend at all three risk levels and 178 (18.2%) at none. The 27% whose intention varied by baseline risk were more likely to be younger, without a family history of CRC, and without a prior history of screening. CONCLUSIONS: Most people in our population were not influenced by the range of absolute benefits and harms associated with CRC screening presented. For an appreciable minority, however, magnitude of benefit was important.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Analysis of chemical shift changes reveals the binding modes of isoindolinone inhibitors of the MDM2-p53 interaction.

In this study we present a method for defining the binding modes of a set of structurally related isoindolinone inhibitors of the MDM2-p53 interaction. This approach derives the location and orientation of isoindolinone binding, based on an analysis of the patterns of magnitude and direction of chemical shift perturbations for a series of inhibitors of the MDM2-p53 interaction. The MDM2-p53 complex is an attractive target for therapeutic intervention in cancer cells with intact tumor suppressor p53, as it offers the possibility of releasing p53 by blocking the MDM2-p53 binding site with a small molecule antagonist to promote apoptosis. Isoindolinones are a novel class of MDM2-antagonists of moderate affinity, which still require the development of more potent candidates for clinical applications. As the applicability of conventional structural methods to this system is limited by a number of fundamental factors, the exploitation of the information contained in chemical shift perturbations has offered a useful route to obtaining structural information to guide the development of more potent compounds. For a set of 12 structurally related isoindolinones, the data suggests 4 different orientations of binding, caused by subtle changes in the chemical structure of the inhibitors.

Understanding small-molecule binding to MDM2: insights into structural effects of isoindolinone inhibitors from NMR spectroscopy.

The interaction between murine double minute (MDM2) and p53 is a major target in anticancer drug design. Several potent compound series, including the nutlins and spirooxindoles, have previously been established as high-affinity antagonists of MDM2. In this paper, we describe the interaction of isoindolinone inhibitors with MDM2, as characterized by nuclear magnetic resonance spectroscopy. Isoindolinone inhibitors bind specifically to the MDM2 p53 binding site and exploit all sub-pockets used by p53, nutlins and spirooxindoles. Furthermore, isoindolinones bind with low micromolar to high nanomolar affinities, with the best compound approaching the potency of nutlin-3.

Isoindolinone inhibitors of the murine double minute 2 (MDM2)-p53 protein-protein interaction: structure-activity studies leading to improved potency.

Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 µM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 µM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.

Inhibitors of MDM2 and MDMX: a structural perspective.

p53 is a potent tumor suppressor with a crucial role in preventing uncontrolled cell proliferation and is therefore frequently deleted or mutated in cancer. For tumors with wild-type p53, its function can be overcome by overactive cellular antagonists, such as the ubiquitin ligase murine double minute clone 2 (MDM2). Restoring p53 activity by inhibiting MDM2 in such cancers can eradicate tumors. Consequently, the MDM2-p53 interaction has been extensively targeted for inhibition by small molecules. In recent years, MDM2-like protein (MDMX), another key downregulator of p53, has gained increasing importance as an additional target for drug development, in order to provide a complementary approach to MDM2 inhibition. In this review, we describe how detailed structural knowledge of the MDM2-p53 interface and, more recently, of the MDMX-p53 interaction have helped advance the development of inhibitors against the two targets. We present a summary of the functional biochemistry of MDM2, MDMX and p53 as well as their interactions and examine recent progress in the development of inhibitors of MDM2 and MDMX.

All change: protein conformation and the ubiquitination reaction cascade.

The structures of enzymes that collectively modify proteins by covalent addition of ubiquitin-like protein moieties have provided significant insights into the regulatory pathways they compose and have highlighted the importance of protein flexibility for the mechanism and regulation of the ubiquitination reaction.