RESUMO
The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic ß-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects. Inspired by the hypothesis that structural changes that are associated with events initiating unfolding in DBD are likely to present opportunities for inhibition, we investigate the dynamics of the wild type (WT) and some aggregating mutants through extensive all atom explicit solvent MD simulations. Simulations reveal differential conformational sampling between the WT and the mutants of a turn region (S6-S7) that is contiguous to a known aggregation-prone region (APR). The conformational properties of the S6-S7 turn appear to be modulated by a network of interacting residues. We speculate that changes that take place in this network as a result of the mutational stress result in the events that destabilize the DBD and initiate unfolding. These perturbations also result in the emergence of a novel pocket that appears to have druggable characteristics. FDA approved drugs are computationally screened against this pocket.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Mutantes/química , Bibliotecas de Moléculas Pequenas/química , Proteína Supressora de Tumor p53/química , Proteínas de Ligação a DNA/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/genética , Mutação/genética , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/genética , Desdobramento de Proteína/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
We have previously developed a sensitive and modular homogenous biosensor system using peptides to detect target ligands. By transposing the basic mechanistic principle of the nuclease protection assay into this biosensor framework, we have developed the protease exclusion (PE) assay which can discern antagonists of protein-protein interactions in a rapid, single-step format. We demonstrate the concept with multiple protein-peptide pairs and validate the method by successfully screening a small molecule library for compounds capable of inhibiting the therapeutically relevant p53-Mdm2 interaction. The Protease Exclusion method adds to the compendium of assays available for rapid analyte detection and is particularly suited for drug screening applications.