The fluorescent two-hybrid assay for live-cell profiling of androgen receptor modulators.

The androgen receptor (AR) is an important target for drug therapies combating prostate cancer. However, various acquired mutations within the AR sequence often render this receptor resistant to treatment. Ligand-induced interaction between the N- and C-termini of the AR marks the initial step in the AR signaling cascade and can thus serve as an early read-out for analysis of potential antagonists of wt and mutant AR.

Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides.

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.

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human 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.

The fluorescent two-hybrid assay to screen for protein-protein interaction inhibitors in live cells: targeting the interaction of p53 with Mdm2 and Mdm4.

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.

Inhibition of nutlin-resistant HDM2 mutants by stapled peptides.

Pharmacological modulation of p53 activity is an attractive therapeutic strategy in cancers with wild-type p53. Presently in clinical trials, the small molecule Nutlin-3A competitively binds to HDM2, a key negative regulator of p53 and blocks its activity. We have described resistance mutations in HDM2 that selectively reduce affinity for Nutlin but not p53.

In vitro selection of mutant HDM2 resistant to Nutlin inhibition.

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.

Stapled peptides with improved potency and specificity that activate p53.

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.

Self-segregation of myelin membrane lipids in model 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.

A size barrier limits protein diffusion at the cell surface to generate lipid-rich myelin-membrane sheets.

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.

Central nervous system myelin: structure, synthesis and assembly.

The wrapping of multiple layers of myelin membrane sheets around an axon is of fundamental importance for the function of the nervous system. In the central nervous system (CNS) oligodendrocytes synthesize tremendous amounts of cellular membrane to form multiple myelin internodes of highly stable membranes with a specific set of tightly packed lipids and proteins. In recent years, mouse mutants have allowed great advances in our understanding of the functional and structural role of many of the major components of myelin.

Generation of knockout mice expressing a GFP-reporter under the control of the Lmx1a locus.

Lmx1a is a member of the LIM homeodomain containing transcription factors and plays an important role during embryonic development. Specifically, it is required for the proper formation of several structures in the central nervous system, such as the roof plate, the cerebellum, and the inner ear. All these defects may contribute to the neurological phenotype observed in dreher mice, lacking functional Lmx1a protein.