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Osteosarcoma remains the most common primary bone tumour in dogs with half of affected dogs unable to survive 1 year beyond diagnosis. New therapeutic options are needed to improve outcomes for this disease. Recent investigations into potential therapeutic targets have focused on cell surface molecules with little clear therapeutic benefit. Transcription factors and protein interactions represent underdeveloped areas of therapeutic drug development. We have utilized allosteric inhibitors of the core binding factor transcriptional complex, comprised of core binding factor beta (CBFβ) and RUNX2, in four canine osteosarcoma cell lines Active inhibitor compounds demonstrate anti-tumour activities with concentrations demonstrated to be achievable in vivo while an inactive, structural analogue has no activity. We show that CBFβ inhibitors are capable of inducing apoptosis, inhibiting clonogenic cell growth, altering cell cycle progression and impeding migration and invasion in a cell line-dependent manner. These effects coincide with a reduced interaction between RUNX2 and CBFβ and alterations in expression of RUNX2 target genes. We also show that addition of CBFβ inhibitors to the commonly used cytotoxic chemotherapeutic drugs doxorubicin and carboplatin leads to additive and/or synergistic anti-proliferative effects in canine osteosarcoma cell lines. Taken together, we have identified the interaction between components of the core binding factor transcriptional complex, RUNX2 and CBFβ, as a potential novel therapeutic target in canine osteosarcoma and provide justification for further investigations into the anti-tumour activities we describe here.

The vital role of residual dipolar couplings (RDCs) in refinement of structures with sparse restraint data such as integral membrane proteins is emphasized. The results have indicated that dipolar couplings are critical for obtaining accurate structures of membrane proteins.

A method of parallel spin-labeling with paramagnetic and diamagnetic labels is introduced and it shows that distances in the range 15-24 A can be readily determined. The protein was labeled at 11 water-exposed and lipid-covered sites, and 320 paramagnetic relaxation enhancement (PRE) distance restraints were measured and the addition of these restraints resulted in significant improvement of the calculated backbone structure of outer membrane protein A (OmpA).

Heteronuclear NMR and single crystal X-ray diffraction are used to show how peptides with different sequences, including those corresponding to the C-termini of syndecan, neurexin, and ephrin B, can simultaneously bind to both PDZ domains of the scaffolding protein syntenin. The data help to explain how syntenin might preferentially bind to dimeric and clustered targets, and provide a mechanistic explanation for the reported cooperative ligand binding by syntenin's two PDZ domains.

A study was conducted to examine ribonucleotide reductase B1 C-terminal peptides functioning as substrates for glutaredoxin. Nuclear magnetic resonance samples were dissolved in a 50 mM potassium phosphate solution while computations were carried out using the QUANTA96 software. Results indicated that the electrostatic charge distribution of the protein was positive and supported a good match for the highly negatively charge peptide.

Inverse-detection two dimensional N-H nuclear magnetic resonance relaxation spectroscopy was used to compare the backbone dynamics of reduced and oxidized Eschericia coli glutaredoxin-1. Data revealed that the reduced protein has higher mobility compared to the oxidized form owing to its proximity to the active site and more residues requiring an R9(sub)ex exchange term. The oxidized form however has more stability.

The Cbfb gene, the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF), is essential for embryogenesis. Specifically, it is found to be required for CBF(alpha)2 function in vivo. Homozygous mutation of the Cbfb gene is found to result in the same range of abnormalities observed in Cbfa2-deficient mice, including embryonic lethality at midgestation due to hemorrhaging in the central nervous system and inhibition of fetal liver hematopoiesis.

The slowing of inversion at nitrogen and the slowing of isolated bridgehead phenyl rotation disrupt the 1H NMR spectra of 10-benzyl-9,11-diphenyl-10-azatetracyclo(6.3.0.0.(super 4,11)0.(super 5,9))undecane and 10-methyl-9,11-diphenyl-10-azatetracyclo(6.3.0.0.(super 4,11)0.(super 5,9))undecane. Molecular mechanics calculations indicate that the close passage of an o-phenyl proton and a methyl (or benzylmethylene) proton in the transition state give rise to the high phenyl rotation barriers.

Cys14-Ser mutant of Escherichia coli glutaredoxin and its mixed disulfide with glutathione (GSH) were synthesized and characterized. 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate is retained in the Cys14-Ser mutant. In addition, nuclear magnetic resonance (NMR) spectroscopy and 15N-labeling of the protein confirm its covalent structure. Furthermore, the established data allows the identification with NMR techniques the glutathione binding site on glutaredoxin and elucidation of the interactions responsible for this binding.