Amplified Luminescence Proximity Homogeneous Assay Research Articles

A novel test for type-I allergy based on crosslink formation of immunoglobulin-E receptors by allergen-specific immunoglobulin-E antibodies and an allergen

Detection of allergen-specific immunoglobulin E (IgE) antibodies (Abs) in serum would allow for screening of the causative allergen in patients with type-I allergy. In this study, we developed a new assay method to detect allergen-specific IgE Abs, which involved crosslinking the plural FcεRIα molecules with an allergen and detection using an amplified luminescence proximity homogeneous assay (AlphaCL). First, the allergen concentration, bead concentrations, and incubation time were optimized for the detection of anti-2,4-dinitrophenyl (DNP) IgE Abs in buffer. Under optimal conditions, AlphaCL was able to detect DNP-specific IgE Abs in simulated human serum at levels comparable to those in serum from type-I allergic patients. When AlphaCL was used to detect anti-DNP IgE Abs, no signal counts were obtained with the monovalent allergen 2,4-dinitrophenylated poly-γ-glutamic acid, whereas high signal counts were obtained with the multivalent allergen DNP-BSA. This confirmed that AlphaCL could specifically detect allergen-specific IgE Abs with the ability to crosslink a multivalent allergen. In summary, we have established a new assay model using AlphaCL to detect allergen-specific IgE Abs with FcεRIα crosslinking ability in human serum. This simple and practical assay model may be applied as a new diagnostic tool for patients with type-I allergy.

Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay

To identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections.

Overcoming the Challenges of Detecting GPCR Oligomerization in the Brain.

G protein-coupled receptors (GPCRs) constitute the largest group of membrane receptor proteins controlling brain activity. Accordingly, GPCRs are the main target of commercial drugs for most neurological and neuropsychiatric disorders. One of the mechanisms by which GPCRs regulate neuronal function is by homo- and heteromerization, with the establishment of direct protein-protein interactions between the same and different GPCRs. The occurrence of GPCR homo- and heteromers in artificial systems is generally well accepted, but more specific methods are necessary to address GPCR oligomerization in the brain. Here, we revise some of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay (PLA), resonance energy transfer (RET) between fluorescent ligands and the Amplified Luminescent Proximity Homogeneous Assay (ALPHA). Of note, we use the archetypical GPCR oligomer, the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer as an example to illustrate the implementation of these techniques, which can allow visualizing GPCR oligomers in the human brain under normal and pathological conditions. Indeed, GPCR oligomerization may be involved in the pathophysiology of neurological and neuropsychiatric disorders.

A novel detection method for cross-linking of IgE-receptors by autoantibodies in chronic spontaneous urticaria

BackgroundAutoantibodies (AAbs) against immunoglobulin E (IgE) antibodies (Abs) and their high-affinity receptor alpha subunits (FcεRIα) are key factors in the elicitation of type IIb autoimmune chronic spontaneous urticaria (type IIb aiCSU). In this study, we aimed to develop a new method to detect functional anti-FcεRIα and anti-IgE AAbs, which can crosslink the plural FcεRІα molecules and IgE Abs on the surface of mast cells and basophils, in sera from aiCSU patients using the amplified luminescence proximity homogeneous assay (Alpha). MethodsSera were obtained from 14 aiCSU patients, as diagnosed by recurrent chronic spontaneous urticaria episodes and positive results for the autologous serum skin test and/or histamine release test (HRT). The AAbs to FcεRIα and IgE Abs were determined in sera from aiCSU patients using enzyme-linked immunosorbent assay (ELISA) and Alpha by cross-linking (AlphaCL) of IgE Abs and/or FcεRІα. ResultsSerum anti-FcεRIα and anti-IgE AAb levels were not significantly different between aiCSU patients and healthy subjects in ELISA. Anti-FcεRIα AAbs were detected in 10 of 14 aiCSU patients who displayed positive (5/5) and negative (5/9) results in the HRT for anti-FcεRIα AAbs by AlphaCL, whereas no signals were observed in healthy subjects. Additionally, anti-IgE AAbs were detected in two of four aiCSU patients who displayed positive results in the HRT for anti-IgE AAbs. ConclusionsA new assay method using AlphaCL can detect anti-FcεRIα and anti-IgE AAbs with FcεRIα- and IgE-crosslinking abilities in sera from aiCSU patients. This simple and practical assay method may be available as a diagnostic tool for urticaria patients.

Anti-FIRΔexon2 autoantibody as a novel indicator for better overall survival in gastric cancer.

There is no clinically available biomarker for efficiently indicating the overall survival or therapy response of gastric cancer (GC). The autoantibodies (Abs) in the sera of anti‐far‐upstream element‐binding protein‐interacting repressor‐lacking exon2 (FIRΔexon2), anti‐sorting nexin 15, and anti‐spermatogenesis and oogenesis–specific basic helix–loop–helix 1 were markedly higher in GC patients than in healthy donors (HDs). These Abs were identified by large‐scale serological identification of antigens by recombinant cDNA expression cloning screenings and their expression levels were evaluated by amplified luminescence proximity homogeneous assay. In particular, compared with age‐matched HDs, the level of anti‐FIRΔexon2 Abs in GC patients was significantly higher (P < .001). The Spearman's rank correlation analysis between anti‐FIRΔexon2 Abs and clinically available tumor markers such as carcinoembryonic antigen (CEA) was statistically insignificant, indicating that FIRΔexon2 Abs is an independent biomarker. We performed receiver‐operating curve analysis to evaluate the anti‐FIRΔexon2 Ab as a candidate biomarker with CEA and carbohydrate antigen 19‐9 (CA19‐9). The overall survival of GC patients with high anti‐FIRΔexon2 Abs titer was significantly favorable (P = .04) than that of GC patients who were below detection level of anti‐FIRΔexon2 Abs. However, clinical stages were not apparently correlated with the levels of anti‐FIRΔexon2 Ab, CEA, and CA19‐9. In conclusion, anti‐FIRΔexon2 Abs detected in GC patients is a potential biomarker for monitoring a better prognosis. Hence, anti‐FIRΔexon2 Abs is a promising biomarker for indicating better overall survival of gastric cancer patients.

KN3014, a piperidine-containing small compound, inhibits auto-secretion of IL-1\u03b2 from PBMCs in a patient with Muckle\u2013Wells syndrome

NLRP3, an intracellular pattern recognition receptor, recognizes numerous pathogens and/or its own damage-associated molecules, and forms complexes with the adaptor protein ASC. These complexes constitute the NLRP3 inflammasome, a platform for processing interleukin (IL)-1β and/or IL-18. Several NLRP3 mutations result in constitutive activation of the NLRP3 inflammasome, causing cryopyrin-associated periodic syndrome (CAPS). To the best of our knowledge, small compounds that specifically inhibit inflammasome activation through the pyrin domain (PYD) have not yet been developed. This study describes an attempt to develop small compounds targeting the NLRP3 inflammasome. A core chemical library of 9,600 chemicals was screened against reconstituted NLRP3 inflammasome in a cell-free system with an amplified luminescence proximity homogeneous assay and a cell-based assay by human peripheral blood mononuclear cells (PBMCs). Inflammasome activation was evaluated by ASC-speck formation in human PBMCs, accompanied by IL-1β secretion and processing, and by using IL-1β-based dual operating luciferase (IDOL) mice. The activity of these compounds was evaluated clinically using PBMCs from a patient with Muckle–Wells syndrome (MWS), a type of CAPS, with an R260W mutation in NLRP3. Screening identified KN3014, a piperidine-containing compound targeting the interaction between NLRP3 and ASC through the PYD. KN3014 reduced ASC-speck formation in human PBMCs, luminescence from IDOL mice, and auto-secretion of IL-1β by PBMCs from the patient with MWS. These findings suggest that KN3014 may be an attractive candidate for treatment of MWS, as well as other NLRP3 inflammasomopathies.

Circulating Anti-Sorting Nexins 16 Antibodies as an Emerging Biomarker of Coronary Artery Disease in Patients with Obstructive Sleep Apnea.

Biomarkers are not available for monitoring the onset and progression of coronary artery disease (CAD) in patients with obstructive sleep apnea (OSA), a major risk factor for arteriosclerotic cardiovascular diseases. This study aimed to test for correlation between circulating anti-Sorting Nexins 16 antibody (SNX16-Ab) levels, CAD history and clinical parameters of patients with OSA. Sixty-four healthy donors, 82 adults with OSA, and 96 with acute coronary syndrome (ACS) were studied. Serum samples were collected at diagnostic polysomnography in the OSA group or at the disease onset in the ACS group. Serum SNX16-Ab levels were measured by amplified luminescence proximity homogeneous assay (AlphaLISA), and correlation between SNX16-Ab levels and clinical parameters was analyzed. SNX16-Ab levels and apnea-hypopnea index (AHI) were weakly correlated. Additionally, logistic regression analyses of OSA group identified that elevated SNX16-Ab level associated with the history of CAD. Circulating SNX16-Ab could increase during CAD pathogenesis in patients with OSA. Further prospective studies are required to prove the predictive potential of SNX16-Ab level in CAD onset of patients with OSA.

Anti-FIRΔexon2, a splicing variant form of PUF60, autoantibody is detected in the sera of esophageal squamous cell carcinoma.

Anti‐PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c‐myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large‐scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti‐FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti‐FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti‐FIRΔexon2 Ab had higher sensitivity than anti‐FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti‐FIRΔexon2 Ab as candidate markers such as anti‐p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti‐FIRΔexon2 Ab and anti‐p53 Ab. In summary, our results suggest the use of anti‐FIRΔexon2 Ab in combination with the anti‐p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti‐FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed.

A motif in HSP90 and P23 that links molecular chaperones to efficient estrogen receptor α methylation by the lysine methyltransferase SMYD2

Heat shock protein 90 (HSP90) is a molecular chaperone that supervises folding of cellular signaling proteins such as steroid receptors and many protein kinases. HSP90 relies on ATP hydrolysis for powering a conformational circuit that helps fold the client protein. To that end, HSP90 binds to co-chaperone proteins that regulate ATP hydrolysis rate or interaction with client proteins. Co-chaperones such as P23, cell division cycle 37 (CDC37), or activator of HSP90 ATPase activity 1 (AHA1) interact with the N-terminal or middle domain of HSP90, whereas others, such as HSP70/HSP90-organizing protein (HOP), use tetratricopeptide repeat (TPR) domains to bind the EEVD motif at the very C-terminal end of HSP90. Recently, the lysine methyltransferase SET and MYND domain-containing 2 (SMYD2) has been proposed as an HSP90-binding partner, and interaction analyses indicate that SMYD2 binding to HSP90 is independent of the EEVD motif. Using the amplified luminescence proximity homogeneous assay (Alpha) technique, I identified a new (M/I/L/V)PXL motif at the C termini of HSP90 and P23 that mediates an interaction with SMYD2, and synthetic peptides harboring this motif dissociated this complex. Of note, the HSP90- and P23-dependent client estrogen receptor α (ERα), was a major methylation target of SMYD2. In a reconstituted system in bacteria, I analyzed HSP90/P23-associated, SMYD2-mediated ERα methylation and found that when SMYD2 binds to the molecular chaperones, it considerably increases methylation of Lys-266 in ERα. Because methylation represses ERα activity, the observed complex formation between SMYD2 and HSP90/P23 may contribute to ERα regulation.

Identification of novel inhibitors of histone acetyltransferase hMOF through high throughput screening

The histone acetyltransferases (HATs) in mammals include GCN5 N-acetyltransferases, the MOZ, YBF2, SAS2, and TIP60 proteins, and the orphan HATs. The males absent on the first (MOF) is mainly related to acetylation of histone H4 Lys16 and has influence on downstream genes expression. However, the only inhibitor MG149 presented low activity against MOF. Besides, there was no high throughput screening platform on MOF, which limited the inhibitor discovery and functional study. In our study, we set up a high throughput screening platform based on amplified luminescent proximity homogeneous assay (ALPHA), which led us to a moderate inhibitor DC_M01. By chemical modification, we found DC_M01_7, which was the analog of DC_M01 with an IC50 value of 6 μM. DC_M01_7 significantly inhibited HCT116 cells proliferation and could also inhibit histone 4 lysine 16 acetylation in HCT116 cells. To sum up, our work will probably assist the further development of more potent MOF inhibitors and the functional study of hMOF.

Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea.

ObjectiveAlthough severe obstructive sleep apnea (OSA) is an important risk factor for atherosclerosis-related diseases including coronary artery disease (CAD), there is no reliable biomarker of CAD risks in patients with OSA. This study aimed to test our hypothesis that circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1-Abs) are associated with the prevalence of CAD in patients with OSA.MethodsEighty-two adults diagnosed with OSA by polysomnography, 96 patients with a diagnosis of acute coronary syndrome (ACS) and 64 healthy volunteers (HVs) were consecutively enrolled. Serum samples were collected from patients with OSA at diagnostic polysomnography and from patients with ACS at disease onset. Serum NBL1-Ab level was measured by amplified luminescence proximity homogeneous assay and its association with clinical variables related to atherosclerosis was evaluated.ResultsNBL1-Ab level was significantly elevated in patients with both OSA and ACS compared with HVs. Subgroup analyses showed that NBL1-Ab level was markedly higher in patients with severe OSA and OSA patients with a history of CAD. Weak associations were observed between NBL1-Ab level and apnea-hypopnea index, age, mean SpO2 and arousal index, whereas significantly higher NBL1-Ab levels were observed in OSA patients with a history of CAD than in those without a history of CAD. Sensitivity analysis using a logistic regression model also demonstrated that increased NBL1-Ab levels were associated with the previous history of CAD in patients with OSA.ConclusionsElevated NBL1-Ab levels may be associated with the prevalence of CAD in patients with OSA, which needs to be confirmed further.

Identification of specific and common diagnostic antibody markers for gastrointestinal cancers by SEREX screening using testis cDNA phage library

The present study was planned to identify novel serum antibody markers for digestive organ cancers. We have used screening by phage expression cloning and identified novel fourteen antigens in this experiment. The presence of auto-antibodies against these antigens in serum specimens was confirmed by western blotting. As for auto-antibodies against fourteen antigens, AlphaLISA (amplified luminescence proximity homogeneous assay) assay was performed in the sera of gastrointestinal cancers patients to confirm the results. Serum antibody levels against these fourteen recombinant proteins as antigens between healthy donors (HD) and esophageal squamous cell carcinoma (ESCC) patients, gastric cancer (GC), or colon cancer (CC) were compared. The serum levels of all fourteen auto-antibodies were significantly higher in ESCC and GC than those of HD. Among those auto-antibodies, except ECSA2 and CCNL2, were also detected significantly higher levels in CC than those of HD. Receiver operating curve (ROC) revealed similar results except CCNL2 in CC. AUC values calculated by ROC were higher than 0.7 in auto-antibodies against TPI1, HOOK2, PUF60, PRDX4, HS3ST1, TUBA1B, TACSTD2, AKR1C3, BAMBI, DCAF15 in ESCC, auto-antibodies against TPI1, HOOK2, PUF60, PRDX4, TACSTD2, AKR1C3, BAMBI, DCAF15 in GC, and auto-antibodies against TPI1, HOOK2, PUF60 in CC. AUC of the combination of HOOK2 and anti-p53 antibodies in ESCC was observed to be as high as 0.8228. Higher serum antibody levels against ten antigens could be potential diagnostic tool for ESCC. Higher serum antibody levels against eight antigens could be potential diagnostic tool for GC, and serum antibody levels against three antigens could be potential diagnostic tool for CC.

Identifying Inhibitors of the Hsp90-Aha1 Protein Complex, a Potential Target to Drug Cystic Fibrosis, by Alpha Technology

Deletion of a single phenylalanine residue at position 508 of the protein CFTR (cystic fibrosis transmembrane conductance regulator), a chloride channel in lung epithelium, is the most common cause for cystic fibrosis. As a consequence, folding of the CFTRΔF508 protein and delivery to the cell surface are compromised, resulting in degradation of the polypeptide. Accordingly, decreased surface presence of CFTRΔF508 causes impaired chloride ion conductivity and is associated with mucus accumulation, a hallmark of cystic fibrosis. Molecular chaperones such as Hsp90 and its co-chaperone partner Aha1 are thought to play a key role in targeting folding-deficient CFTRΔF508 for degradation. Thus, pharmacologic manipulation to inhibit Hsp90-Aha1 chaperone complex formation appears beneficial to inhibit proteolysis of CFTRΔF508 and rescue its residual chloride channel activity. Therefore, we have screened a collection of 14,400 druglike chemical compounds for inhibitors of the Hsp90-Aha1 complex by amplified luminescence proximity homogeneous assay (Alpha). We identified two druglike molecules that showed promising results when we tested their ability to restore chloride channel activity in culture cells expressing the mutant CFTRΔF508 protein. The two molecules were most effective in combination with the corrector VX-809 and may therefore serve as a lead compound that can be further developed into a drug to treat cystic fibrosis patients.

Poly (I:C) and hyaluronic acid directly interact with NLRP3, resulting in the assembly of NLRP3 and ASC in a cell-free system

In the NLR family, pyrin domain containing 3 (NLRP3) is an intracellular pattern recognition receptor that activates pro-caspase-1, leading to IL-1β and IL-18 processing and activation in a large complex called the NLRP3 inflammasome. Since various pathogens or endogenous metabolites have been reported to stimulate NLRP3 inflammasome, the interaction between NLRP3 and ASC induced by these stimulants may be an attractive drug target for NLRP3-related diseases, called inflammasomopathies. However, the endogenous ligand that directly interacts with NLRP3, leading to binding to ASC, remains unclear. Therefore, we developed a cell-free system consisting of NLRP3, ASC, and pro-caspase-1 or ASC and NLRP3 with an amplified luminescent proximity homogeneous assay (ALPHA). ALPHA signals of the interaction between NLRP3 and ASC were not enhanced following an incubation without any ligand, whereas strong ALPHA signals for the interaction between NLRP3 and ASC and between NLRP3 and pro-caspase-1 with the adaptor ASC were observed upon an incubation with poly (I:C) and hyaluronic acid (HA). Poly (I:C) and HA both directly interacted with NLRP3 within a specific concentration. These results suggest that NLRP3 directly interacts with intrinsic RNA and HA, which is followed by the activation of NLRP3 inflammasome, and the cell-free system consisting of NLRP3 and ASC, or NLRP3, ASC, and pro-caspase-1 may be a useful tool for elucidating the pathogenesis of inflammasomopathies and developing target therapeutics.

Circulating Anti-Coatomer Protein Complex Subunit Epsilon (COPE) Autoantibodies as a Potential Biomarker for Cardiovascular and Cerebrovascular Events in Patients with Obstructive Sleep Apnea.

Although moderate to severe obstructive sleep apnea (OSA) is an independent risk factor for severe arteriosclerotic diseases such as cardiovascular disease (CVD) and stroke, the development of atherosclerosis-related diseases cannot yet be predicted in patients with OSA. In a pilot study, we identified autoantibodies against the coatomer protein complex, subunit epsilon [circulating anti-coatomer protein complex subunit epsilon autoantibody (COPE-Ab)], a cytosolic complex that mediates protein transport in the Golgi compartment, as a potential novel biomarker of atherosclerosis. This study aimed to evaluate whether COPE-Ab levels had an association with cardiovascular and cerebrovascular events in patients with OSA. Eighty-two adult patients with a diagnosis of OSA via polysomnography and 64 healthy donors were studied. Serum COPE-Ab levels were measured using an amplified luminescence proximity homogeneous assay. Then, clinical factors related to atherosclerosis were evaluated with respect to COPE-Ab levels. Significant differences in COPE-Ab levels were observed in terms of OSA severity. COPE-Ab levels were significantly higher in patients with OSA and also CVD and/or stroke, hypertension, and a high body mass index. Univariate and multivariate logistic regression analyses of patients with OSA identified elevated COPE-Ab level as a significant predictor of CVD and/or stroke. An elevated COPE-Ab level may be a potential predictor of the risks of cardiovascular and cerebrovascular events in patients with OSA. Therefore, patients with higher COPE-Ab levels may require more careful and intensive treatment. A commentary on this article appears in this issue on page 361.

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis.

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

Development of homogeneous luminescence assays for histone demethylase catalysis and binding

Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JumonjiC (JmjC)-containing histone demethylases (HDMs) catalyze the demethylation of methylated lysine residues on histone tails. Here we report the development of homogeneous luminescence-based assay methods for measuring the catalytic activity and the binding affinities of peptides to HDMs. The assays use amplified luminescent proximity homogeneous assay (ALPHA) technology, are sensitive and robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate a correlation between the inhibitor potencies determined by the ALPHA and other types of assays. Although this study focuses on the JMJD2E isoform, the catalytic turnover and binding assays described here can be used in studies on other HDMs. The assays should be useful for the development of small molecule inhibitors selective for HDM isoforms.

Systematic interpretation of cyclic nucleotide binding studies using KinetXBase

Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine-3',5'-cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA-screen), radioligand binding or activity-based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site-specific cAMP analogues could be identified. The software package is free for download by academic users.

High-Throughput Screen for Small Molecule Inhibitors of Mint1-PDZ Domains

Several hundred PDZ (postsynaptic density-95, Drosophila disks-large, ZO-1) domain-containing proteins have been identified in the human genome. PDZ domains play a critical role in organization and function of cellular signaling pathways. Thus, small molecule inhibitors of PDZ domain association with their targets have wide potential applications as research and therapeutic agents. PDZ domains typically bind to a carboxyl-terminal tail of the target protein. Here we describe a high-throughput screening (HTS) assay for small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). The performance of a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) and an amplified luminescent proximity homogeneous assay (ALPHA) were systematically compared in parallel pilot HTS experiments with glutathione S-transferase-Mint1-PDZ1/2 protein and biotinylated NC peptide. Both of the two assays showed similar sensitivities in our target protein assay. Using HTRF-based assay we screened a library of 100,000 small molecule compounds and identified a number of potential "hits." The activity of isolated "hits" was confirmed by ALPHA assay. However, further evaluation revealed that isolated "hits" most likely act as "promiscuous binders," not as specific Mint-PDZ inhibitors, and that additional screening will be required to identify the true Mint-PDZ inhibitors. The assays described provided an example of HTS for a small molecule inhibitor of Mint-PDZ domain that can be easily adapted to other PDZ domain-mediated interactions.

Characterization of binding activity between nuclear factor of activated T cells and calcineurin by amplified luminescent proximity homogeneous assay

For quantitative evaluation of the relationship between biological binding partners, including protein–protein interactions, a novel analyzing system, amplified luminescent proximity homogeneous assay (ALPHA), has been developed. We here employed ALPHA for accurate assessment of the binding properties between nuclear factor of activated T cells 1 (NFAT1) and calcineurin (CN), which is essential for Ca 2+-dependent regulation of immune responses. A recombinant protein of the Ca 2+ regulatory domain (CRD) of NFAT1 was prepared and its binding activity with biotinylated CN was determined by ALPHA ( K d = 0.20 μM). The contribution of each CN-binding component involved in the CRD of NFAT1 to CN/NFAT1 binding was next examined by competitive assay. Not only the whole CRD but also the N- and C-terminal CN-binding regions (CNBR1 and CNBR2, respectively) dose-dependently blocked CN/NFAT1 binding and their potency was CRD ≫ CNBR2 ≥ CNBR1. CN/NFAT1-binding properties were further characterized using short inhibitory peptides derived from NFAT1-CNBR1 as well as NFAT4-CNBR2. In conclusion, ALPHA is a useful system to analyze biological signaling cascades, due to its capability of quantitative evaluation of protein–protein interactions.