Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

Nikolovska‐Coleska, Zaneta; Wang, Renxiao; Fang, Xueliang; Pan, Hongna; Tomita, York; Li, Peng; Roller, Peter P.; Krajewski, Krzysztof; Saito, Naoyuki G.; Stuckey, Jeanne A.; Wang, Shaomeng

doi:10.1016/j.ab.2004.05.055

Cited by 502 publications

(550 citation statements)

References 33 publications

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“…All K d and IC 50 values were calculated using Prism5 using a one-site binding hyperbola (Y ϭ B max ϫ X/K d ϩ X). For competition experiments, K i values were calculated from IC 50 values using an equation derived by Nikolovska-Coleska et al (38).…”

Section: Methodsmentioning

confidence: 99%

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Potent and Selective Peptide-based Inhibition of the G Protein Gαq

Charpentier

Waldo²,

Lowery-Gionta³

et al. 2016

Journal of Biological Chemistry

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Edited by Norma AllewellIn contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active G␣ q binds its two major classes of effectors, the phospholipase C (PLC)-␤ isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages G␣ q within its canonical binding site consisting of a groove formed between switch II and helix ␣3. This information was exploited to synthesize peptides that bound active G␣ q in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of G␣ q . A representative peptide was specific for active G␣ q because it did not bind inactive G␣ q or other classes of active G␣ subunits and did not inhibit the activation of PLC-␤3 by G␤ 1 ␥ 2 . In contrast, the peptide robustly prevented activation of PLC-␤3 or p63RhoGEF by G␣ q ; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of G␣ q in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited G␣ q -dependent activation of PLC-␤3 at least as effectively as a dominant-negative form of full-length PLC-␤3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active G␣ q in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of G␣ q in cells.

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Section: Methodsmentioning

confidence: 99%

“…2). The capacity of unlabeled 27-mer(I860A) to interact with (38). The sequence alignment is annotated as in Fig.…”

Section: Canonical Effector Interactions With G␣ Q Drive Complexmentioning

confidence: 99%

Potent and Selective Peptide-based Inhibition of the G Protein Gαq

Charpentier

Waldo²,

Lowery-Gionta³

et al. 2016

Journal of Biological Chemistry

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“…As dimeric SMAC-mimetics have been shown to be more potent than their monomeric counterparts in activating apoptosis , we synthesised three homodimeric compounds by coupling two SMAC067 units with different linkers (Supplementary Figure S2). The affinity of the compounds for XIAP BIR3 and lkBIR2/BIR3, measured by fluorescent polarisation as described (Nikolovska-Coleska et al, 2004 revealed that all three dimeric compounds had a lower affinity for BIR3 than the monomers ( Table 1), suggesting that the linker region slightly hinders the interaction with BIR3 in vitro. However, the interaction of the homodimers with the lkBIR2/BIR3 was 10-fold higher compared with the monomer.…”

Section: Bortezomib Sensitises Melanoma Cells Without Affecting Caspamentioning

confidence: 99%

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

et al. 2010

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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 apoptosisbinding 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.

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“…The concentration (IC 50 ) of the inhibitor, which is required for 50% displacement of peptide (2) from NCoA-1, could be determined from the dependence of this ratio on inhibitor concentration. Finally, the dissociation constant (K D ) of the [NCoA-1-cyclopeptide] complex could be calculated from the IC 50 values using the general Cheng-Prusoff equation described by Nikolovska-Coleska [23].…”

Section: Competition Assay Using Myoglobin As Internal Standardmentioning

confidence: 99%

How to deal with weak interactions in noncovalent complexes analyzed by electrospray mass spectrometry: Cyclopeptidic inhibitors of the nuclear receptor coactivator 1-STAT6

Touboul

Maillard

Grässlin

et al. 2009

J. Am. Soc. Mass Spectrom.

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Mass spectrometry, and especially electrospray ionization, is now an efficient tool to study noncovalent interactions between proteins and inhibitors. It is used here to study the interaction of some weak inhibitors with the NCoA-1/STAT6 protein with K D values in the M range. High signal intensities corresponding to some nonspecific electrostatic interactions between NCoA-1 and the oppositely charged inhibitors were observed by nanoelectrospray mass spectrometry, due to the use of high ligand concentrations. Diverse strategies have already been developed to deal with nonspecific interactions, such as controlled dissociation in the gas phase, mathematical modeling, or the use of a reference protein to monitor the appearance of nonspecific complexes. We demonstrate here that this last methodology, validated only in the case of neutral sugar-protein interactions, i.e., where dipole-dipole interactions are crucial, is not relevant in the case of strong electrostatic interactions. Thus, we developed a novel strategy based on half-maximal inhibitory concentration (IC 50 ) measurements in a competitive assay with readout by nanoelectrospray mass spectrometry. T here is a strong interest in the study of noncovalent complexes between biomolecules, which are playing key roles in life. Numerous solutionphase analytical techniques were developed to determine the specificity and the strength of these types of interactions [1]. Mass spectrometry (MS), and especially electrospray ionization (ESI) [2], has become an efficient tool to study specific noncovalent complexes between various species (protein-protein, protein-small molecules, protein-DNA, DNA-DNA . . .) [3][4][5][6][7][8]. In fact, ESI is a very soft ionization technique, i.e., noncovalent complexes can be transferred intact from solution into the gas phase. Quantitative information such as stoichiometry, binding constants, or reaction kinetics can be obtained by ESI-MS, and values are often in good agreement with data coming from well-established solution phase techniques. Nevertheless, the study of noncovalent [protein-ligand] complexes require careful control of experimental parameters. Buffer, pH, pressure, and voltages applied to the different stages of the mass spectrometer have great influence on spectral characteristics and on the information gained. Moreover, electrochemical reactions and desolvation/ionization mechanisms involved in ESI can also complicate the analysis, thus giving rise to the so-called nonspecific interactions (i.e., interactions with nonspecific binding sites) that alter the solution phase stoichiometry. To study weak complexes with dissociation constants (K D ) in the M range or higher in solution, high ligand concentrations are employed, leading to an increase of nonspecific complex ions signals and to underestimate K D values, which might not reflect the solution-phase equilibria anymore [5, 9 -16].Three strategies have been developed to determine affinities of weak [protein-ligand] complexes by ESI-MS, even when nonspecific gas-pha...

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Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

Cited by 502 publications

References 33 publications

Potent and Selective Peptide-based Inhibition of the G Protein Gαq

Potent and Selective Peptide-based Inhibition of the G Protein Gαq

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

How to deal with weak interactions in noncovalent complexes analyzed by electrospray mass spectrometry: Cyclopeptidic inhibitors of the nuclear receptor coactivator 1-STAT6

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