1,4,5-Trisubstituted Imidazole-Based p53-MDM2/MDMX Antagonists with Aliphatic Linkers for Conjugation with Biological Carriers.

The tumor suppressor protein p53, the "guardian of the genome", is inactivated in nearly all cancer types by mutations in the TP53 gene or by overexpression of its negative regulators, oncoproteins MDM2/MDMX. Recovery of p53 function by disrupting the p53-MDM2/MDMX interaction using small-molecule antagonists could provide an efficient nongenotoxic anticancer therapy. Here we present the syntheses, activities, and crystal structures of the p53-MDM2/MDMX inhibitors based on the 1,4,5-trisubstituted imidazole scaffold which are appended with aliphatic linkers that enable coupling to bioactive carriers. The compounds have favorable properties at both biochemical and cellular levels. The most effective compound 19 is a tight binder of MDM2 and activates p53 in cancer cells that express the wild-type p53, leading to cell cycle arrest and growth inhibition. Crystal structures reveal that compound 19 induces MDM2 dimerization via the aliphatic linker. This unique dimerization-binding mode opens new prospects for the optimization of the p53-MDM2/MDMX inhibitors and conjugation with bioactive carriers.

Side-binding proteins modulate actin filament dynamics.

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics.

Transient protein states in designing inhibitors of the MDM2-p53 interaction.

Reactivation of p53 by release of the functional protein from its inhibition by MDM2 provides an efficient, nongenotoxic approach to a wide variety of cancers. We present the cocrystal structures of two complexes of MDM2 with inhibitors based on 6-chloroindole scaffolds. Both molecules bound to a distinct conformational state of MDM2 with nM-µM affinities. In contrast to other structurally characterized antagonists, which mimic three amino acids of p53 (Phe19, Trp23, and Leu26), the compounds induced an additional hydrophobic pocket on the MDM2 surface and unveiled a four-point binding mode. The enlarged interaction interface of the inhibitors resulted in extension of small molecules binding toward the "lid" segment of MDM2 (residues 19-23)--a nascent element that interferes with p53 binding. As supported by protein engineering and molecular dynamics studies, employing these unstable elements of MDM2 provides an efficient and yet unexplored alternative in development of MDM2-p53 association inhibitors.

NMR screening for lead compounds using tryptophan-mutated proteins.

NMR-based drug screening methods provide the most reliable characterization of binding propensities of ligands to their target proteins. They are, however, one of the least effective methods in terms of the amount of protein required and the time needed for acquiring an NMR experiment. We show here that the introduction of tryptophan to proteins permits rapid screening by monitoring a simple 1D proton NMR signal of the NH side chain ((N)H(epsilon)) of the tryptophan. The method could also provide quantitative characterization of the antagonist-protein and antagonist-protein-protein interactions in the form of KDs and fractions of the released proteins from their mutual binding. We illustrate the method with the lead compounds that block the Mdm2-p53 interaction and by studying inhibitors that bind to cyclin-dependent kinase 2 (CDK2).