Analyses of Ligand Binding to IP3 Receptors Using Fluorescence Polarization.

Fluorescence polarization (FP) can be used to measure binding of a small fluorescent ligand to a larger protein because the ligand rotates more rapidly in its free form than when bound. When excited with plane polarized light, the free fluorescent ligand emits depolarized light, which can be quantified. Upon binding, its rotation is reduced and more of the emitted light remains polarized. This allows FP to be used as a nondestructive assay of ligand binding. Here we describe a fast, high-throughput FP assay to quantify the binding of fluorescently labeled inositol 1,4,5-trisphosphate (IP3) to N-terminal fragments of the IP3 receptor. The assay is fast (1-6 h), it avoids use of radioactive materials and when measurements are performed at different temperatures, it can resolve Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) changes of ligand binding

Novel SMAC-mimetics synergistically stimulate melanoma cell death in combination with TRAIL and Bortezomib

BACKGROUND: XIAP (X-linked inhibitor of apoptosis protein) is an anti-apoptotic protein exerting its activity by binding and suppressing caspases. As XIAP is overexpressed in several tumours, in which it apparently contributes to chemoresistance, and because its activity in vivo is antagonised by second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis-binding protein with low pI, small molecules mimicking SMAC (so called SMAC-mimetics) can potentially overcome tumour resistance by promoting apoptosis. METHODS: Three homodimeric compounds were synthesised tethering a monomeric SMAC-mimetic with different linkers and their affinity binding for the baculoviral inhibitor repeats domains of XIAP measured by fluorescent polarisation assay. The apoptotic activity of these molecules, alone or in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and/or Bortezomib, was tested in melanoma cell lines by MTT viability assays and western blot analysis of activated caspases. RESULTS: We show that in melanoma cell lines, which are typically resistant to chemotherapeutic agents, XIAP knock-down sensitises cells to TRAIL treatment in vitro, also favouring the accumulation of cleaved caspase-8. We also describe a new series of 4-substituted azabicyclo[5.3.0] alkane monomeric and dimeric SMAC-mimetics that target various members of the IAP family and powerfully synergise at submicromolar concentrations with TRAIL in inducing cell death. Finally, we show that the simultaneous administration of newly developed SMAC-mimetics with Bortezomib potently triggers apoptosis in a melanoma cell line resistant to the combined effect of SMAC-mimetics and TRAIL. CONCLUSION: Hence, the newly developed SMAC-mimetics effectively synergise with TRAIL and Bortezomib in inducing cell death. These findings warrant further preclinical studies in vivo to verify the anticancer effectiveness of the combination of these agents

Development of Peptidomimetics Targeting IAPs

Inhibitor of apoptosis proteins (IAPs) such as XIAP subvert apoptosis by binding and inhibiting caspases. Because occupation of the XIAP BIR3 peptide binding pocket by Smac abolishes the XIAP–caspase 9 interaction, it is a proapoptotic event of great therapeutic interest. An assay for pocket binding was developed based on the displacement of Smac 7-mer from BIR3. Through the physical and biochemical analysis of a variety of peptides, we have determined the minimum sequence required for inhibition of the Smac–BIR3 interaction and detailed the dimensions and topology of the BIR3 peptide binding pocket. This work describes the structure–activity relationship (SAR) for peptide inhibitors of Smac-IAP binding

The Apoptosome: Emerging Insights and New Potential Targets for Drug Design

Apoptosis plays a crucial role in tissue homeostasis, development and many diseases. The relevance of Apaf1, the molecular core of apoptosome, has been underlined in mitochondria-dependent apoptosis, which according to a growing body of evidence, is involved in various pathologies where the equilibrium of life-and-death is dysregulated, such as heart attack, stroke, liver failure, cancer and autoimmune diseases. Consequently, great interest has emerged in devising therapeutic strategies for regulating the key molecules involved in the life-and-death decision. Here we review recent progress in apoptosis-based pharmacological therapies and, in particular, we point out a possible role of the apoptosome as an emerging and promising pharmacological target

Antiangiogenic Effect of TW37, a Small-Molecule Inhibitor of Bcl-2

Bcl-2 is an antiapoptotic protein that is up-regulated in several tumor types, and its expression levels have strong correlation, to development of resistance to therapy and poor prognosis. We have shown recently that Bcl-2 also functions as a proangiogenic signaling molecule that activates a nuclear factor-kappa B-mediated pathway resulting in up-regulation of the angiogenic chemokines CXCL1 and CXCL8 by neovascular endothelial cells. Here, we evaluate the antiangiogenic effect of the novel small-molecule inhibitor of Bcl-2 (TW37) developed using a structure-based design strategy. We observed that TW37 has an IC50 of 1.8 mu mol/L for endothelial cells but showed no cytotoxic effects for fibroblasts at concentrations up to 50 mu mol/L. The mechanism of TW37-induced endothelial cell death was apoptosis, in a process mediated by mitochondrial depolarization and activation of caspase-9 and caspase-3. The effect of TW37 on endothelial cell apoptosis was not prevented by coexposure to the growth factor milieu secreted by tumor cells. Inhibition of the angiogenic potential of endothelial cells (i.e., migration and capillary sprouting assays) and expression of the angiogenic chemokines CXCL1 and CXCL8 were accomplished at sub-apoptotic TW37 concentrations (0.005-0.05 mu mol/L). Notably, administration of TW37 i.v. resulted in a decrease in the density of functional human microvessels in the severe combined immunodeficient mouse model of human angiogenesis. In conclusion, we describe functionally separate proapoptotic and antiangiogenic mechanisms for a small-molecule inhibitor of Bcl-2 and show the potential for Bcl-2 inhibition as a target for antiangiogenic therapy