By using a phage display derived peptide as an initial template, compounds have been developed that are highly specific against Mdm2/Mdm4. These compounds exhibit greater potency in p53 activation and protein-protein interaction assays than a compound derived from the p53 wild-type sequence. Unlike Nutlin, a small molecule inhibitor of Mdm2/Mdm4, the phage derived compounds can arrest cells resistant to p53 induced apoptosis over a wide concentration range without cellular toxicity, suggesting they are highly suitable for cyclotherapy.

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The insulating layers of myelin membrane wrapped around axons by oligodendrocytes are essential for the rapid conduction of nerve impulses in the central nervous system. To fulfill this function as an electrical insulator, myelin requires a unique lipid and protein composition. Here we show that oligodendrocytes employ a barrier that functions as a physical filter to generate the lipid-rich myelin-membrane sheets. Myelin basic protein (MBP) forms this molecular sieve and restricts the diffusion of proteins with large cytoplasmic domains into myelin. The barrier is generated from MBP molecules that line the entire sheet and is, thus, intimately intertwined with the biogenesis of the polarized cell surface. This system might have evolved in oligodendrocytes in order to generate an anisotropic membrane organization that facilitates the assembly of highly insulating lipid-rich membranes.

Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.

Protein–protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53–Mdm2 inhibitors. However, none exhibited intracellular activity on p53–Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53–Mdm2 interaction as well as p53–Mdm4 interaction. Here, we show that the F2H assays enable side-by-side analysis of substances’ dual Mdm2–Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

HDM2 binds to the p53 tumour suppressor and targets it for proteosomal degradation. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2 and abrogates its repressive function. Using a novel in vitro selection methodology, we simulated the emergence of resistance by evolving HDM2 mutants capable of binding p53 in the presence of Nutlin concentrations that inhibit the wild-type HDM2-p53 interaction. The in vitro phenotypes were recapitulated in ex vivo assays measuring both p53 transactivation function and the direct p53-HDM2 interaction in the presence of Nutlin. Mutations conferring drug resistance were not confined to the N-terminal p53/Nutlin–binding domain, and were additionally seen in the acidic, zinc finger and RING domains. Mechanistic insights gleaned from this broad spectrum of mutations will aid in future drug design and further our understanding of the complex p53-HDM2 interaction.

Cancer drugs often fail due to the emergence of clinical resistance. This can manifest through mutations in target proteins that selectively exclude drug binding whilst retaining aberrant function. A priori knowledge of resistance-inducing mutations is therefore important for both drug design and clinical surveillance. Stapled peptides represent a novel class of antagonists capable of inhibiting therapeutically relevant protein-protein interactions. Here, we address the important question of potential resistance to stapled peptide inhibitors. HDM2 is the critical negative regulator of p53, and is often overexpressed in cancers that retain wild-type p53 function. Interrogation of a large collection of randomly mutated HDM2 proteins failed to identify point mutations that could selectively abrogate binding by a stapled peptide inhibitor (PM2). In contrast, the same interrogation methodology has previously uncovered point mutations that selectively inhibit binding by Nutlin, the prototypical small molecule inhibitor of HDM2. Our results demonstrate both the high level of structural p53 mimicry employed by PM2 to engage HDM2, and the potential resilience of stapled peptide antagonists to mutations in target proteins. This inherent feature could reduce clinical resistance should this class of drugs enter the clinic.