ADP Assay Research Articles

Determination of clopidogrel effect in cats using point-of-care Plateletworks ADP and shipped samples for PFA-200 analysis in a clinical practice setting.

Clopidogrel is the recommended first-line antithrombotic in cats for a variety of conditions; however, it is ineffective in 15-20% of cats. The determination of clopidogrel effectiveness with platelet function assays has historically been limited to specialty centers; however, recent work has suggested that in-hospital or shipped analyses of samples may be feasible. The aim of the present study was to investigate the utility of an in-house analysis and shipping of blood samples collected in primary practices for the determination of clopidogrel effectiveness. Citrated blood samples were collected from cats receiving clopidogrel therapy by veterinarians in clinical practices across Canada, a median of 304.4 km from the reference laboratory (range 8-4425). Samples were analyzed in-house using Plateletworks ADP and shipped for remote analysis using PFA-200 P2Y and COL/ADP cartridges. A total of 30 samples were collected from 25 cats. Of these, the percentage of samples analyzable for the presence or absence of the clopidogrel effect was 86% for Plateletworks ADP, 90% for PFA-200 P2Y and 87% for PFA-200 COL/ADP. There was no significant difference in the number of samples unable to be analyzed by each modality (P = 0.689) due to flow obstruction or other sample characteristics. The prevalence of absence of clopidogrel effectiveness on platelet function assays was 8% with the PFA-200 COL/ADP assay, 25% with the PFA-200 P2Y assay and 30% with the Plateletworks ADP assay. The results of this study confirm that samples of feline blood can be collected in clinical practices and shipped to a reference laboratory for PFA-200 analysis with a high rate of success, comparable to point-of-care analysis.

Clinical validation of AggreGuide A-100 ADP, a novel assay for assessing the antiplatelet effect of oral P2Y12 antagonists

In this prospective, 3-arm, repeated-measure multicenter investigation in 280 patients with cardiovascular risk factors, platelet aggregation was measured with the novel AggreGuide A-100 ADP (A-100 ADP) and VerifyNow (VN)-PRU assays at baseline, and after United States Food and Drug Administration approved loading and 7days maintenance doses of clopidogrel (n = 94), prasugrel (n = 43) or ticagrelor, (n = 143). Based on the predetermined cutoff values of < 4.7 platelet activity index with A-100 ADP assay to indicate antiplatelet response, more than 91% of patients met the criteria following loading and maintenance doses of prasugrel and more than 84% patients met the criteria following loading and maintenance doses of ticagrelor whereas only 32% and 51% of patients met the criteria following loading and maintenance doses of clopidogrel, respectively. The total percent agreement between the A-100 ADP and VN-PRU assays was 89%. The A-100 ADP assay, which includes whole blood in motion, performs comparably to the VN-PRU assay in a study of patients with cardiovascular risk factors treated with P2Y12 inhibitors possessing known differences in antiplatelet potencies. Trial registration ClinicalTrials.gov Identifier: NCT3111420.

Aspirin Resistance in Children with Congenital Heart Disease and Its Relationship with Laboratory and Clinical Parameters

Background: Patients with congenital heart disease (CHD) are more prone to thromboembolism. Aspirin is the most commonly used medication around the world to prevent thrombosis in cardiovascular diseases. Objectives: This study aimed to define the frequency of aspirin resistance in pediatric patients with CHD and to evaluate the correlation of clinical and laboratory parameters with aspirin resistance. Methods: The study population consisted of 103 patients using aspirin, including 53 cases of cyanotic CHD and 50 cases of non-cyanotic CHD. Platelet aggregation was measured by the AggreGuide A-100 ADP Assay. Results: The prevalence of aspirin resistance was 36.9% in children with CHD. Although aspirin resistance in cyanotic CHD patients (41.5%) was higher than in non-cyanotic patients (32%), the difference was not statistically significant (P = 0.414). There was no significant association between aspirin responsiveness or resistance and the patient’s sex, age, duration of aspirin use, and concomitant medication use. Comparison of the laboratory data of aspirin-responsive and -resistant patients showed no significant difference between these groups, except for albumin (P = 0.032) and serum fibrinogen (P = 0.0001) levels. The fibrinogen level and thromboembolism history were independent risk factors for aspirin resistance. Also, there was a significant correlation between platelet aggregation in peripheral blood smears and aspirin resistance (P = 0.0001). Conclusions: Our findings suggest that measurement of the serum fibrinogen level and platelet aggregation in blood smears may be the first step to predict aspirin resistance.

The Influence of Prostaglandin E1 and Use of Inhibitor Percentage on the Correlation between the Multiplate and VerifyNow in Patients on Dual Antiplatelet Therapy

Platelet function tests (PFT), such as the Multiple Electrode Analyzer (Multiplate) and VerifyNow, show little concordance in patients using antiplatelet drugs. A major difference between these tests is the use of prostaglandin E1 (PGE1) to inhibit P2Y1-platelet-receptor activation in VerifyNow and is proposed to be of influence in the discrepancy between these tests. We aimed to investigate whether the presence of PGE1 could provide an explanation for the moderate correlation and concordance between Multiplate and VerifyNow by adding PGE1 to the Multiplate ADP assay, also known as the ADP-high sensitivity (ADP-HS) assay. We also aimed to investigate whether the difference in baseline platelet function as measured by the VerifyNow and Multiplate could (partly) explain the moderate correlation between the tests, by plotting ADP assay results against baseline function as measured by the corresponding device, which is expressed as the ‘inhibitor percentage.’ Fifty-one patients who underwent percutaneous coronary intervention (PCI) received dual antiplatelet therapy and were considered to have a high risk of ischemic or bleeding complications were included. The addition of 20 µl PGE1 in the Multiplate resulted in a significant reduction in Arbitrary Aggregation Units, but did not improve correlation with the VerifyNow. The correlation between VerifyNow and Multiplate inhibitor percentage was moderate. Based on these results, we concluded that neither PGE1 nor the calculation of the inhibitor percentage greatly influenced the correlation between PFTs.

Comparison of factors affecting platelet reactivity in various platelet function tests.

Previous studies have reported that various factors affect ADP-induced platelet reactivity during clopidogrel therapy. The aim of this study was to determine whether clinical and laboratory variables for platelet reactivity during dual antiplatelet therapy (DAPT) are dependent on the assay used. We enrolled 904 patients receiving DAPT following coronary intervention. Platelet reactivity was measured using three methods: the VerifyNow P2Y12 assay, multiple electrode aggregometry (MEA) ADP assay, and the light transmittance aggregometry (LTA) ADP assay at 24-48h following coronary intervention. The VerifyNow results demonstrated a significant inverse correlation with hematocrit value (r=-0.268, p<0.0001); however, MEA results had no such correlation with hematocrit (r=0.044, p=0.188). There was a positive correlation between the MEA results and platelet count (r=0.255, p<0.0001). LTA was weakly influenced by hematocrit (r=-0.064, p=0.057) and platelet count (r=0.069, p=0.040). Gender (odds ratio 1.53, 95% CI 1.10-2.14, p=0.013) and hematocrit (odds ratio 0.91,95% CI 0.88-0.94, p<0.0001) were the independent variables for HPR by VerifyNow. Smoking (odds ratio 0.38, 95% CI 0.16-0.94, p=0.036) and platelet count (odds ratio 1.01, 95% CI 1.00-1.01, p<0.0001) were independent factors for HPR when using the MEA assay, whereas platelet count (odds ratio 1.00, 95% CI 1.00-1.01, p=0.006) was identified as the only independent variable for HPR when using LTA. The incidence of HPR and the influencing variables involved are dependent on the platelet function test used.

High On-Treatment Platelet Reactivity Determinants on Dual Antiplatelet Therapy in Patients With Ischemic Heart Disease Before Elective Percutaneous Coronary Intervention.

to determine impact of different laboratory and genetic factors on high on-treatment platelet reactivity (HOPR) during dual antiplatelet therapy (DAPT). We included in this study 94 patients with stable ischemic heart disease (mean age 59±9.67 years). All patients underwent elective PCI with implantation of drug eluting stents at the background of dual antiplatelet therapy (DAPT) with aspirin and clopidogrel. Platelet reactivity was assessed using light transmission aggregometry with 5 μmol/L ADP (LTA 5ADP) and VerifyNow assay before PCI. All patients underwent genotyping to detect CYP2C19 polymorphism. In 74 patients at baseline examination we determined levels of high-sensitivity C-reactive protein (hsCPR), soluble platelet-selectin (sP-selectin), soluble CD40-ligand (sCD40L), interleukin-6 (IL-6), plasminogen activator inhibitor (PAI-1) and activity of von Willebrand factor. Incidence of HOPR according to LTA-5ADP was 16% and VerifyNow - 24.5%. Univariate regression analysis showed that the following factors were significantly associated with HPR determined by LTA-5ADP: body mass index (BMI) (p=0.02), levels of total cholesterol (CH) (p=0.0l), low density lipoprotein CH (p=0.004), and sP-selectin (p=0.009), activity of von Willebrand factor (p=0.04). Carriage of CYP2C19*2 allele was also associated with HOPR (p=0.006). According to multivariate regression analysis body mass index and level of sP-selectin were independent predictors of HOPR during DAPT. HOPR determined by LTA was significantly associated with high BMI, levels of total and LDL CH, carriage of CYP2C19*2 allele, levels of hsCRP and sP-selectin. Independent factors significantly related to HORP were BMI and sP-selectin level.

Analysis of Primary Hemostasis Alterations Induced By Ibrutinib Assessed By Shutter Speed and Aggregometry Impedance

Introduction:Ibrutinib is a covalent inhibitor of Bruton Tyrosine Kinase (BTK), approved for the treatment of Mantle Cell Lymphoma (MCL), Chronic Lymphocytic Leukemia (CLL) and Waldenstrom's Macroglobulinemia (WM). BTK is expressed in platelets and is believed to have a central role in platelet activation through GPIb and GPIV pathway. However, the clinical significance of inhibition of BTK is unclear in terms of bleeding and it is known that patients with congenitally deficient BTK do not exhibit increased risk of bleeding.Objectives:The main objectives were: (1) identify possible alterations in hemostasis induced by Ibrutinib in patients who start taking the drug, and (2) to monitor the development of hemorrhagic adverse effects in patients who start taking Ibrutinib.Methods:From August 31, 2015 and January 31, 2016 we conducted a prospective study including all consecutive patients with the diagnosis of MCL, CLL or WM treated in 2nd line or more, with Ibrutinib as single-drug therapy. The following parameters were analyzed on day 0 (baseline), 10 and 28 after starting treatment with Ibrutinib: APTT, PT, platelet count, PFA-100 Collagen/Epinephrine and Collagen/ADP, aggregometry impedance in whole blood by multiplate assay with AA, ADP, Ristocetin and TRAP as agonists. Von Willebrand factor antigen (vWF:Ag), Ristocetin cofactor (vWF:RCo) and coagulant factor VIII were also analyzed. We collected prospectively bleeding adverse events.Results:In the period of study, we enrolled 11 patients, 7 were males and 4 females, with a median age of 63 years old (range, 53-88). Five patients had the diagnosis of CLL, 5 patients had MCL and 1 patient had WM. After a median follow-up of 6 months, 3 out of 11 (27.3%) developed grade 1 bleeding adverse events. Of these, 1 patient was under anticoagulant treatment with intermediate dose of enoxaparin, and another patient had low levels of vWF:Ag and vWF:RCo, suggesting a possible acquired von Willebrand disease. Five out of 11 (45.5%) of patients had a prolonged shutter speed Collagen/Epinephrine at baseline, with a median of 175 sec (range, 94-270). After Ibrutinib exposure, median shutter speed Collagen/epinephrine at 10 days was 231 sec (range, 108-287), p=0.02; and at 30 days was 142 sec (range, 79-300), p=n.s. Four out of 11 patients (36.4%) had a prolonged shutter speed Collagen/ADP at base line, with a median of 105 sec (range, 63-190). After Ibrutinib exposure, median shutter speed Collagen/ADP at 10 days was 107 sec (range, 66-192), p=n.s; and at 30 days was 78 sec (range, 75-149), p=n.s. Regarding platelet aggregometry impedance, 2 patients had abnormal platelet aggregation baseline at TRAP assay, 6 at ADP assay, 3 at AA assay and 4 at Ristocetin assay. After Ibrutinib exposure, there was a worsening of platelet aggregation in ADP analysis at 10 days of treatment but became normal after 30 days of treatment. There was an improvement with TRAP at both 10 and 30 days of treatment. Of notice, all 3 patients with minor bleeding had abnormal hemostasis studies baseline before starting with Ibrutinib.Conclusions:Almost half of patients showed an altered haemostasis baseline studies before starting with Ibrutinib. The biology of the disease appears to cause in subjects primary hemostasis changes that result in a basal bleeding risk regardless of the treatment used. Only minor bleeding complications were observed under Ibrutinib treatment, all with baseline altered platelet function; 2 of the three patients who developed bleeding complications had other bleeding risk factors associated. PFA-100 Collagen/Epinephrine is prolonged in almost 50% of patients after Ibrutinib is started compared with baseline study. Aggregometry impedance only worsened with ADP at 10 days, but went into normal level at 30 days. There is an interindividual variability in alterations of haemostasis and hemorrhagic clinical signs. DisclosuresCordoba:Janssen: Research Funding, Speakers Bureau.

Validation of a P2Y12-receptor specific whole blood platelet aggregation assay

Testing of P2Y12-receptor antagonist effects can support clinical decision-making. However, most platelet function assays use only ADP as agonist which is not P2Y12-receptor specific. For this reason P2Y12-receptor specific assays have been developed by adding prostaglandin E1 (PGE1) to reduce ADP-induced platelet activation via the P2Y1-receptor. The present study sought to evaluate a P2Y12-receptor specific assay for determination of pharmacodynamic and clinical outcomes. This study enrolled 400 patients undergoing coronary stenting after loading with clopidogrel or prasugrel. ADP-induced platelet reactivity was assessed by whole blood aggregometry at multiple time points with a standard ADP assay (ADPtest) and a P2Y12-receptor specific assay (ADPtest HS, both run on Multiplate Analyzer, Roche Diagnostics). Patients were clinically followed for 1 month and all events adjudicated by an independent committee. In total, 2084 pairs of test results of ADPtest and ADPtest HS were available showing a strong correlation between results of both assays (r = 0.96, p < 0.001). These findings prevailed in multiple prespecified subgroups (e.g., age; body mass index; diabetes). Calculated cutoffs for ADPtest HS and the established cutoffs of ADPtest showed a substantial agreement for prediction of ischemic and hemorrhagic events with a Cohen’s κ of 0.66 and 0.66, respectively. The P2Y12-receptor specific ADPtest HS assay appears similarly predictive for pharmacodynamic and clinical outcomes as compared to the established ADPtest assay indicating its applicability for clinical use. Further evaluation in large cohorts is needed to determine if P2Y12-receptor specific testing offers any advantage for prediction of clinical outcome.

157 Approaches for Pharmacological Inhibition of Cardiac Cell Death: MAP4K4 as a Therapeutic Target

IntroductionCell death plays a causal role in heart failure, and its inhibition poses a promising approach that has not been thoroughly explored. In past approaches to target discovery, clinical failures...

Optimisation and Validation of a High Throughput Screening Compatible Assay to Identify Inhibitors of the Plasma Membrane Calcium ATPase Pump - a Novel Therapeutic Target for Contraception and Malaria

ATPases, which constitute a major category of ion transporters in the human body, have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. We have recently found that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) results in infertility in male mice due to a selective defect in sperm motility. In addition, recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determines the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimise and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF® Transcreener® ADP (TR-FRET) assay to screen a drug library. PMCA4 membrane microsomes were prepared from HEK293 cells overexpressing PMCA4. Western blot quantification revealed nearly nine-fold increased expression of PMCA4 compared to LacZ (control virus)-infected cells. Maximal PMCA4 microsomal activity was achieved in the TR-FRET assay with 15ng/μl microsomal concentration, 30-minute pre-incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1μM free-calcium. Finally a dose-response curve for carboxyeosin (a non-specific PMCA inhibitor) under optimised conditions showed significant PMCA4 inhibition. Upon confirmation that the assay was suitable for high-throughput screening, we have screened the ChemBioNet small molecule library (~21,000 compounds) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1,494 primary hits. We have optimised the HTRF® Transcreener® ADP assay for high-throughput screening to identify PMCA4 inhibitors. The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non-hormonal contraception or anti-malaria therapy.

Design of a Coupled Bioluminescent Assay for a Recombinant Pyruvate Kinase from a Thermophilic Geobacillus

A simple and rapid method using coupled bioluminescent assay was developed to determine level of ADP. ADP is involved in many biological reactions and ADP assay can be used for assaying some reactions universally by monitoring ADP formation or depletion. ADP analysis involves incubation of ADP or extracts containing ADP with pyruvate kinase (PK) and PEP. The ATP formed by this reaction is determined by measuring the intensity of the initial light flash produced when luciferin-luciferase preparation injected into the reaction mixture. In regard to the main role of the PK in this assay, the gene of PK from a Geobacillus species has been cloned in expression vector pET28a (+), sequenced and overexpressed in Escherichia coli. Recombinant protein was purified using Ni-NTA column and then the purified PK was used in a coupled bioluminescent assay for ADP measurement. Kinetic properties of PK are determined according to a bioluminescent assay using firefly luciferase.

A kinetic assay of mitochondrial ADP–ATP exchange rate in permeabilized cells

We previously described a method to measure ADP–ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg 2+] reported by an Mg 2+-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg 2+. In the current article, we describe a modification of this method suited for following ADP–ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF 3 − and sodium orthovanadate (Na 3VO 4) to medium containing digitonin-permeabilized cells inhibits all ADP–ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP–ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F 1F o–ATPase due to its sensitivity to BeF 3 − and Na 3VO 4. With this assay, ADP–ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein.

A Biosensor for Fluorescent Determination of ADP with High Time Resolution

Nearly every cellular process requires the presence of ATP. This is reflected in the vast number of enzymes like kinases or ATP hydrolases, both of which cleave the terminal phosphate from ATP, thereby releasing ADP. Despite the fact that ATP hydrolysis is one of the most fundamental reactions in biological systems, there are only a few methods available for direct measurements of enzymatic-driven ATP conversion. Here we describe the development of a reagentless biosensor for ADP, the common product of all ATPases and kinases, which allows the real-time detection of ADP, produced enzymatically. The biosensor is derived from a bacterial actin homologue, ParM, as protein framework. A single fluorophore (a diethylaminocoumarin), attached to ParM at the edge of the nucleotide binding site, couples ADP binding to a >3.5-fold increase in fluorescence intensity. The labeled ParM variant has high affinity for ADP (0.46 μm) and a fast signal response, controlled by the rate of ADP binding to the sensor (0.65 μm−1s−1). Amino acids in the active site were mutated to reduce ATP affinity and achieve a >400-fold discrimination against triphosphate binding. A further mutation ensured that the final sensor did not form filaments and, as a consequence, has extremely low ATPase activity. The broad applicability of N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC)-ParM as a sensitive probe for ADP is demonstrated in real-time kinetic assays on two different ATPases and a protein kinase.

Evaluating the utility of the HTRF ® Transcreener ™ ADP assay technology: A comparison with the standard HTRF assay technology

The HTRF (homogeneous time-resolved fluorescence) Transcreener ADP assay is a new kinase assay technology marketed by Cis-Bio International (Bagnols-Cèze, France). It measures kinase activity by detecting the formation of ADP using a monoclonal antibody and HTRF detection principles. In this article, we compare this technology with a standard HTRF kinase assay using EGFR [L858R/T790M] mutant enzyme as a case study. We demonstrate that the HTRF Transcreener ADP assay generated similar kinetic constants and inhibitor potency compared with the standard HTRF assay. However, the smaller dynamic window and lower Z′ factor of the HTRF Transcreener ADP assay make this format less preferable for high-throughput screening. Based on the assay principle, the HTRF Transcreener ADP assay can detect both kinase and ATPase activities simultaneously. The ability to probe ATPase activity opens up new avenues for assaying kinases with intrinsic ATPase activity without the need to identify substrates, and this can speed up the drug discovery process. However, caution must be exercised because any contaminating ATPase activity will result in an invalid assay. The inability to tolerate high concentrations of ATP in the assay will also limit the application of this technology, especially in compound mechanistic studies such as ATP competition. Overall, the HTRF Transcreener ADP assay provides a new alternative tool to complement existing assay technologies for drug discovery.

Development and Validation of a High-Density Fluorescence Polarization-Based Assay for the Trypanosoma RNA Triphosphatase TbCet1

RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high throughput screening.

Characterization and Optimization of a Red-Shifted Fluorescence Polarization ADP Detection Assay

ATP depletion and ADP formation are generic detection methods used for the identification of kinase and other ATP-utilizing enzyme inhibitors in high-throughput screening campaigns. However, the most widely used nucleotide detection approaches require high ATP consumption rates or involve the use of coupling enzymes, which can complicate the selection of lead compounds. As an alternative, we have developed the Transcreener (BellBrook Labs, Madison, WI) platform, which relies on the direct immunodetection of nucleotides. Here we describe the development of antibodies with >100-fold selectivity for ADP versus ATP, which enable robust detection of initial velocity rates (Z' > 0.7 at 10% substrate consumption) at ATP concentrations ranging from 0.1 microM to 1,000 microM in a competitive fluorescence polarization (FP) immunoassay. Competitive binding experiments indicate similar affinities for other nucleotide diphosphates, including 2' -deoxy ADP, GDP, and UDP. The antibody-tracer complex and the red-shifted, ratiometric FP signal are stable for at least 24 h at room temperature, providing suitable conditions for high-throughput screening. A method for calculating a kinase ATP Km with this FP immunoassay is also presented. The Transcreener ADP assay provides a simple, generic assay platform for inhibitor screening and selectivity profiling that can be used for any ADP-generating enzyme.

Development of an Eg5 assay using the HTRF® Transcreener® ADP kit

Based on Cisbio Bioassays' patented HTRF® technology, HTRF Transcreener® ADP assay is a universal method for identifying and characterizing the phosphotransferase activity of ATPases and kinases. ADP is directly detected by a specific monoclonal antibody labeled with europium cryptate and correlates closely with the amount of phosphorylated substrate in a kinase assay. Here we describe a typical four-step assay development with the Eg5 ATPase using the HTRF Transcreener ADP assay.

Discovery of Acetyl-Coenzyme A Carboxylase 2 Inhibitors: Comparison of a Fluorescence Intensity-Based Phosphate Assay and a Fluorescence Polarization-Based ADP Assay for High-Throughput Screening

Acetyl-coenzyme A carboxylase (ACC) enzymes exist as two isoforms, ACC1 and ACC2, which play critical roles in fatty acid biosynthesis and oxidation. Though each isoform differs in tissue and subcellular localization, both catalyze the biotin- and ATP-dependent carboxylation of acetyl-coenzyme A to generate malonyl-coenzyme A, a key metabolite in the control of fatty acid synthesis and oxidation. The cytosolic ACC1 is expressed primarily in liver and adipose tissue, and uses malonyl-coenzyme A as a key building block in fatty acid biosynthesis. The mitochondrial ACC2 is primarily expressed in heart and skeletal muscle, where it is involved in the regulation of fatty acid oxidation. Inhibitors of ACC enzymes may therefore be useful therapeutics for diabetes, obesity, and metabolic syndrome. Two assay formats for these ATP-utilizing enzymes amenable to high-throughput screening are compared: a fluorescence intensity-based assay to detect inorganic phosphate and a fluorescence polarization-based assay to detect ADP. Acetyl-coenzyme A carboxylase inhibitors were identified by these high-throughput screening methods and were confirmed in a radiometric high performance liquid chromatography assay of malonyl-coenzyme A production.

Bioluminescent Assay of Bacterial Intracellular AMP, ADP, and ATP with the Use of a Coimmobilized Three-Enzyme Reagent (Adenylate Kinase, Pyruvate Kinase, and Firefly Luciferase)

A three-enzyme coimmobilized system (firefly luciferase, pyruvate kinase, and adenylate kinase) was constructed for the bioluminescent assay of ATP, ADP, and AMP in bacterial cell extracts. Data for the reproducibility and sensitivity of the proposed method are presented. Detection limits were 1.5 pmol of ADP and 15 pmol of AMP in the sample. With this system, changes in adenine nucleotide concentrations in bacterial cells were measured during the actions exerted by external chemical and physical sources, such as additives to nutrient media and low-power He-Ne laser irradiation.

Bioluminometric Assay of ADP and ATP at High ATP/ADP Ratios: Assay of ADP After Enzymatic Removal of ATP

Bioluminometric Assay of ADP and ATP at High ATP/ADP Ratios: Assay of ADP After Enzymatic Removal of ATP