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1.
Anal Biochem ; 555: 67-72, 2018 08 15.
Article in English | MEDLINE | ID: mdl-29733811

ABSTRACT

Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time.


Subject(s)
Biological Assay/methods , Cullin Proteins/metabolism , NEDD8 Protein/metabolism , Protein Processing, Post-Translational , Cullin Proteins/genetics , HCT116 Cells , HEK293 Cells , Humans , NEDD8 Protein/genetics
2.
Anal Biochem ; 509: 73-78, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27387056

ABSTRACT

A new technology from Quanterix called SiMoA (single molecule array) which employs a fully automated system capable of ultrasensitive sandwich based ELISA detection was explored. Our studies focused upon the inhibition of the autophagy initiating kinase ULK1 by measuring the both total Atg13 and the phosphorylation of Atg13(pSer(318)) from control and following compound treatment in either overexpressing or wild type tissue culture samples. The results show linear protein concentration dependence over two orders of magnitude and provide an assay window of 8- to 100-fold signal to background for inhibition of phosphorylation for both wild type and overexpressed samples, respectively. Moreover, overexpressed samples displayed 17-fold pSer(318)-Atg13 above wild type levels of with no apparent differences in compound potency. Lastly, the inhibition of ULK1 from mouse derived wild type xenografts also demonstrated loss of pSer(318)-Atg13 upon ULK1 inhibitor treatment that compared favorably to Western blot. These results show that the SiMoA technology can detect quantitatively low levels of endogenous biomarkers with the ability to detect the loss of pSer(318)-Atg13 upon ULK1 inhibition.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy-Related Protein-1 Homolog/metabolism , Autophagy-Related Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Array Analysis/methods , Animals , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay/methods , Heterografts , Humans , Mice , Neoplasm Transplantation , Phosphorylation
3.
Anal Biochem ; 439(2): 109-15, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-23624319

ABSTRACT

Cellular effects of a Nedd8-activating enzyme (NAE) inhibitor, MLN4924, using the AlphaScreen format were explored. MLN4924 acts as a substrate-assisted inhibitor of NAE by forming a tight binding Nedd8-MLN4924 adduct. The inhibited enzyme can no longer transfer Nedd8 downstream to modify and activate the E3 cullin-RING ligases. This results in the stabilization of proteins regulated by the proteasome, leading to cell death. These studies monitored the endogenous cellular changes to NAE∼Nedd8 thioester, the formation of the Nedd8-MLN4924 adduct, and the reduction in the Cul1-Nedd8. Lysates derived from MLN4924-treated HCT116 cells showed that whereas the ß-subunit of NAE remained constant, reductions of both NAE∼Nedd8 thioester and Cul1-Nedd8 levels occurred with a concomitant rise of the adduct. Moreover, the formation of the Nedd8-MLN4924 adduct was approximately stoichiometric with the concentration of NAEß. Higher density 384-well cell-based assays illustrated the kinetics of enzyme inactivation across a wider range of MLN4924 concentrations, showing a rapid loss of NAE∼Nedd8 thioester and Cul1-Nedd8. The reduction of NAE∼Nedd8 thioester precedes the loss of Cul1-Nedd8 at twice the rate. Finally, these results clearly demonstrate the utility of the homogeneous assay for quantitative assessment of these endogenous cellular components in a 384-well plate in response to inhibition of NAE by MLN4924.


Subject(s)
Cyclopentanes/pharmacology , Gene Expression Regulation/drug effects , Pyrimidines/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , HCT116 Cells , Humans , Protein Binding , Protein Subunits , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism
4.
Anal Biochem ; 408(2): 321-7, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-20868646

ABSTRACT

The utility of antibody reagents for the detection of specific cellular targets for both research and diagnostic applications is widespread and continually expanding. Often it is useful to develop specific antibodies as reagent pairs that distinguish different epitopes of the target such that sandwich enzyme-linked immunosorbent assay can be used for selective and specific detection. However, the identification of pairing antibodies is often cumbersome and labor-intensive even with the use of designed peptide-specific epitopes as antigens. We have developed a robust and high-throughput method for identifying pairing complementary antibodies derived either from commercial sources or during a rabbit hybridoma monoclonal screening and selection process using protein A capture with the AlphaScreen bead-based assay format. We demonstrate the value and effectiveness of this assay with three protein targets: Akt2, ATF3, and NAEß (the ß-subunit of the neddylation activation enzyme).


Subject(s)
Antibodies/chemistry , Immunoassay/methods , Staphylococcal Protein A/chemistry , Activating Transcription Factor 3/chemistry , Activating Transcription Factor 3/immunology , Animals , Antibodies/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Epitopes/immunology , Hybridomas/metabolism , Peptides/immunology , Proto-Oncogene Proteins c-akt/chemistry , Proto-Oncogene Proteins c-akt/immunology , Rabbits
5.
Anal Biochem ; 410(1): 13-8, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21078283

ABSTRACT

Pharmacodynamic responses to drug treatment are often used to confirm drug-on-target biological responses. Methods ranging from mass spectrometry to immunohistochemistry exist for such analyses. By far, the most extensively used methodologies employ antigen-specific antibodies for detection (at a minimum) and, in some cases, target quantitation as well. Using a novel frequency-modulating technology from BioScale called acoustic micro magnetic particle (AMMP) detection, two pathway biomarkers were chosen for pharmacodynamic analysis and compared with either AlphaScreen or LI-COR Western blot assays. For these studies, pharmacodynamic biomarkers for both proteasome and phosphoinositol 3-kinase inhibition were used. Our results show clearly that the BioScale technology is a robust and rapid method for measuring recombinant standards or endogenously derived proteins from both tissue culture and mouse xenograft tumor lysates. Moreover, the sensitivity obtained with the BioScale platform compares favorably with LI-COR Western blot and AlphaScreen technologies. Furthermore, the use of the ViBE Bioanalyzer eliminates the labor-intensive effort of Western blot analysis and is devoid of the optical and other endogenous interfering substances derived from lysates of xenograft tumors typically observed with AlphaScreen.


Subject(s)
Acoustics , Biosensing Techniques/instrumentation , Magnetics , Animals , Biomarkers/metabolism , Cell Line, Tumor , Humans , Mice , Protein Kinase Inhibitors/pharmacology , Protein Phosphatase 1/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Xenograft Model Antitumor Assays
6.
Assay Drug Dev Technol ; 4(6): 661-9, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17199504

ABSTRACT

Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.


Subject(s)
Adenine Phosphoribosyltransferase/metabolism , Scintillation Counting/methods , Adenine/metabolism , Adenine Phosphoribosyltransferase/antagonists & inhibitors , Adenylate Kinase/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , NAD/metabolism , Pyruvate Kinase/metabolism , Recombinant Proteins/metabolism , Tritium
7.
Assay Drug Dev Technol ; 3(5): 533-41, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16305310

ABSTRACT

NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.


Subject(s)
Amide Synthases/analysis , Amide Synthases/chemistry , Biological Assay/methods , Drug Evaluation, Preclinical/methods , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Spectrometry, Fluorescence/methods , Amide Synthases/genetics , Enzyme Activation , Fluorescent Dyes , Humans , Protein Engineering/methods , Staining and Labeling/methods
8.
Assay Drug Dev Technol ; 2(3): 300-7, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15285911

ABSTRACT

Fatty acyl coenzyme A (CoA) synthetases are a group of enzymes responsible for the activation of fatty acids through ligated high-energy CoA thioester bonds. Ultimately these fatty acyl-CoA conjugates are routed toward either anabolic or catabolic pathways. Long-chain-fatty-acid-CoA ligase 5 (LACS 5) utilizes a wide range of saturated fatty acids with a substrate preference for C16-C18 unsaturated fatty acids. This enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a radiometric homogeneous measurement of the enzymatic reaction by employing ionic capture of the reaction product onto YSi scintillation proximity assay (SPA) beads. We present kinetic and inhibition data for LACS 5 using this SPA format. Our results show that the assay method is both robust and well suited for this class of lipid-metabolizing enzymes.


Subject(s)
Acetate-CoA Ligase/metabolism , Recombinant Proteins/metabolism , Acetate-CoA Ligase/antagonists & inhibitors , Acyl Coenzyme A/analysis , Acyl Coenzyme A/metabolism , Coenzyme A Ligases/antagonists & inhibitors , Coenzyme A Ligases/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Recombinant Proteins/antagonists & inhibitors , Scintillation Counting , Substrate Specificity
9.
Assay Drug Dev Technol ; 1(3): 435-43, 2003 Jun.
Article in English | MEDLINE | ID: mdl-15090180

ABSTRACT

Inositol-specific PLCs comprise a family of enzymes that utilize phosphoinositide substrates, e.g., PIP(2), to generate intracellular second messengers for the regulation of cellular responses. In the past, monitoring this reaction has been difficult due to the need for radiolabeled substrates, separation of the reaction products by organic-phase extraction, and finally radiometric measurements of the segregated products. In this report, we have studied the enzymatic characteristics of two novel PLCs that were derived from functional genomic analyses using a phospholipid-modified solid scintillating support. This method allows for the hydrophobic capture of the [(3)H]phosphoinositide substrate on a well defined scintillation surface and the homogenous measurement of the enzymatic hydrolysis of the substrate by proximity effects. Our results show that the assay format is robust and well suited for this class of lipid-metabolizing enzymes.


Subject(s)
Inositol/chemistry , Type C Phospholipases/chemistry , Animals , Cattle , Humans , Isoenzymes/chemistry , Mice , Phosphatidylinositols/chemistry , Reproducibility of Results , Scintillation Counting/methods , Swine , Time Factors , Tritium
10.
Assay Drug Dev Technol ; 1(4): 555-63, 2003 Aug.
Article in English | MEDLINE | ID: mdl-15090252

ABSTRACT

We have characterized a recombinantly expressed N-terminally tagged GST fusion of the tyrosine kinase domain of human EphB3. The EphB3 kinase domain was shown to phosphorylate a group of synthetic tyrosine-containing peptides derived from a proprietary biotinylated kinase-biased peptide substrate library. In addition, the enzyme activity was stimulated by the divalent cation, manganese, and inhibited by addition of magnesium. The most active tyrosine-containing peptide, a biotinylated 49-mer, displayed saturation kinetics with an apparent K(m) of approximately 0.4 microM. The apparent K(m) for ATP was determined to be approximately 3 microM. The kinetics of the reaction was linear from concentrations of enzyme of 0.5 to 2 nM, and at or below the K(m) concentrations of the two substrates for at least 2 h at room temperature. Moreover, the tryrosine kinase inhibitor, PP2, produced an IC(50) of roughly 0.8 microM. In addition, the enzyme tolerated the solvent DMSO and was stable to multiple freeze/thaw cycles. Stability of the enzyme at 4 degrees C storage was seen out to 6 h with an approximately 50% reduction of activity by 24 h. Formatting the assay in a 384-well microtiter plate produced good uniformity of signal at 100% inhibition, 50% inhibition, and no inhibition. The coefficient of variance was at or below 10% with a signal-to-background ratio of approximately 24 and a z value of 0.72. Collectively, these results showed the ability to configure a robust HTS for a truncated recombinantly expressed family member of the Ephrin tyrosine kinases.


Subject(s)
Protein-Tyrosine Kinases/chemistry , Receptor, EphB3/chemistry , Scintillation Counting/methods , Ephrin-B3/chemistry , Humans , Receptor, EphB3/genetics
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