Jennifer J. Gesell

Flexible lid to the p53-binding domain of human Mdm2: Implications for p53 regulation

The stabilization of p53 against Mdm2-mediated degradation is an important event in DNA damage response. Initial models of p53 stabilization focused on posttranslational modification of p53 that would disrupt the p53–Mdm2 interaction. The N-terminal regions of both p53 and Mdm2 are modified in vivo in response to cellular stress, suggesting that modifications to Mdm2 also may affect the p53–Mdm2 interaction. Our NMR studies of apo-Mdm2 have found that, in addition to Mdm2 residues 25–109 that form the well ordered p53-binding domain that was observed in the p52–Mdm2 complex, Mdm2 residues 16–24 form a lid that closes over the p53-binding site. The Mdm2 lid, which is strictly conserved in mammals, may help to stabilize apo-Mdm2. It also competes weakly with peptidic and nonpeptidic antagonists. Modifications to the Mdm2 lid may disrupt p53–Mdm2 binding leading to p53 stabilization. Mdm2 and Mdm4 possess nearly identical p53-binding domains but different lids suggesting that lid modifications may select for p53 binding.

NMR-Based Screening Applied to Drug Discovery Targets

While conventional bioassay-based high-throughput screening (HTS) remains a mainstream approach for lead discovery, its limitations have driven the development of alternative and complementary tools. In this regard, novel NMR-based approaches that have emerged over the last few years show great promise. We have used NMR-based screening approaches for a variety of drug targets to identify low molecular weight (MW) small molecule hits from customized libraries, which subsequently could be optimized into leads through focused, structure-guided chemistry. Focus was placed on targets for which HTS failed to identify suitable leads. This report discusses different NMR-based screening techniques and follow-up strategies for lead discovery and illustrates their application to the NS3 protease and NS3 helicase domains of the hepatitis C virus (HCV).