Protocol for a Steady-State FRET Assay in Cancer Chemoprevention.

Cancer chemoprevention is an important strategy to prevent, reverse, or suppress the development of cancer. One of the target pathways that has emerged in recent years is the Keap1-Nrf2-ARE system that regulates the protection of cells against various carcinogens and their metabolites. Increased concentrations of the redox transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces the activation of antioxidant and phase 2 detoxifying genes. Nrf2 is regulated by substrate adaptor protein Kelch-like ECH-associated protein 1 (Keap1) that can target Nrf2 for ubiquitination and degradation by the proteasome. The interaction between Nrf2 and Keap1 can be disrupted at the protein-protein interface in order to increase Nrf2 activity for potential therapeutic purposes. This chapter describes a protocol for a steady-state fluorescence or Förster resonance energy transfer (FRET) assay to examine the Keap1-Nrf2 protein-protein interaction (PPI), to investigate the effects of Nrf2 mutations on Keap1 binding and finally to identify potential inhibitors of this PPI. In the assay system Keap1 is conjugated to an YFP protein at the N-terminus whereas an Nrf2-derived 16-mer peptide containing a high-affinity "ETGE" motif is conjugated to a CFP protein at the N-terminus.

Investigation of the protein alkylation sites of the STAT3:STAT3 inhibitor Stattic by mass spectrometry.

STAT3 (Signal Transducer and Activator of Transcription factor 3) is constitutively active in a wide range of human tumours. Stattic is one of the first non-peptidic small molecules reported to inhibit formation of the STAT3:STAT3 protein dimer complex. A mass spectrometry method has been developed to investigate the binding of Stattic to the un-phosphorylated STAT3ßtc (U-STAT3) protein. Alkylation of four cysteine residues has been observed with possible reaction at a fifth which could account for the mechanism of action.

Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography.

The STAT3 transcription factor plays a central role in a wide range of cancer types where it is over-expressed. Previously, phosphorylation of this protein was thought to be a prerequisite for direct binding to DNA. However, we have now shown complete binding of a purified unphosphorylated STAT3 (uSTAT3) core directly to M67 DNA, the high affinity STAT3 target DNA sequence, by a protein electrophoretic mobility shift assay (PEMSA). Binding to M67 DNA was inhibited by addition of increasing concentrations of a phosphotyrosyl peptide. X-ray crystallography demonstrates one mode of binding that is similar to that known for the STAT3 core phosphorylated at Y705.

A novel small-molecule inhibitor of IL-6 signalling.

A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.

Solid-phase synthesis of chlorofusin analogues.

We report an efficient and versatile solid-phase synthesis through which two series of chlorofusin analogues, one bearing varying chromophores and the other with various amino acid substitutions in the cyclic peptide, were synthesized. These peptides were prepared using a strategy involving side-chain immobilization, on-resin cyclization, and postcyclization modification. The success of these syntheses demonstrates the broad utility of the method. Both series of analogues were evaluated for their inhibitory activity against the p53/MDM2 interaction but were shown to be inactive in the concentration range tested. This suggests that the full chromophore structure may be required for activity.

Protein transduction by lipidic peptide dendrimers.

We investigated the potential of a new family of lipidic peptide dendrimers in protein transduction into cultured cells. Dendrimer-protein interaction was determined by gel retardation assays using purified recombinant protein. To assess intracellular protein delivery, two marker proteins were used: recombinant firefly luciferase and a Cy3-labeled monoclonal antibody to the c-myc proto-oncogene. Protein delivery was determined by luciferase assays and fluorescence microscopy, respectively. While there was minimal delivery of luciferase or antibody in the absence of the dendrimers, the latter increased protein delivery substantially. Luciferase delivery was concentration and cell type-dependent; the efficiency of delivery also varied with the number of terminal amino groups on the dendrimers. In previous reports, we showed that these dendrimers could be used for gene and drug delivery; the data we report herein suggest that they may also be capable of intracellular protein delivery. This finding has important implications for the use of these dendrimers in protein therapeutics and vaccinology.

Versatile peptide dendrimers for nucleic acid delivery.

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function.