Homogeneous time-resolved G protein-coupled receptor–ligand binding assay based on fluorescence cross-correlation spectroscopy

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor–ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.     

Specific allowance for antibody bivalence in the determination of dissociation constants by kinetic exclusion assay

Theory that takes rigorous account of antibody bivalence in the characterization of immunospecific reactions by kinetic exclusion assay is presented. In addition to reinforcing the basic correctness of quantitative expressions currently being used for the determination of dissociation constants (K_d) by this method, the current study highlights a requirement for conformity of the system with critical assumptions/approximations therein. Published results for the interaction between the extracellular domain of human insulin-like growth factor (hIGFR) and anti-hIGFR are used to illustrate aspects of the theoretical predictions for a system to which those assumptions/approximations may well apply; and those for a cadmium–ethylenediaminetetraacetic acid (Cd–EDTA) antibody interaction to emphasize the consequences of adopting the same analytical procedure in a situation where one of those assumptions does not apply. The major weakness of current protocols for the characterization of antigen–antibody interactions by kinetic exclusion assay is an absence of any check on the likely magnitude of the probability of antibody capture by the affinity beads—a parameter that needs to be 5% or lower for validity of the quantitative expression on which the analysis is based.     

Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes

Genotyping in closed tube is commonly performed using polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide probes using fluorescence resonance energy transfer (FRET). Here we introduce a homogeneous human leukocyte antigen (HLA)–DQA1∗05 end-point PCR assay based on switchable lanthanide luminescence probe technology and a simple dried blood sample preparation. The switchable probe technology is based on two non-luminescent oligonucleotide probes: one carrying a non-luminescent lanthanide chelate and the other carrying a light-absorbing antenna ligand. Hybridization of the probes in adjacent positions to the target DNA leads to the formation of a highly luminescent lanthanide chelate complex by self-assembly of the reporter molecules. Performance of the HLA–DQA1∗05 assay was evaluated by testing blood samples collected on sample collection cards and was prepared by lysing the punched samples (3-mm discs) using alkaline reaction conditions and high temperature. Testing of 147 blood samples yielded 100% correlation to the heterogeneous DELFIA technology-based reference assay. Genotyping requires carefully designed probe sequences able to discriminate matched and mismatched target sequences by hybridization. Furthermore, definite genotype discrimination was achieved because inherently non-luminescent switchable probes together with time-resolved measurement mode led to very low background signal level and, therefore, very high signal differences averaging 54-fold between DQA1∗05 and other alleles.     

Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases

Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI–MS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis–Menten constant (K_m), and catalytic turnover rate (V_max/[E]_t) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (V_max/[E]_t) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5 s⁻¹, respectively) suggested that CN1 is approximately 6-fold more efficient. The turnover rate of Dug1p for Cys-Gly dipeptide at pH 8.0 was found to be slightly lower (73.8 s⁻¹). In addition, we determined kinetic parameters of CN2 at pH 9.2 and found that the turnover rate was increased by 4-fold with no significant change in the K_m. Kinetic parameters obtained by the ESI–MS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP–HPLC)-based assay. Furthermore, we used tandem MS (MS/MS) analyses to characterize carnosine and measured its levels in CHO cell lines in a time-dependent manner. The ESI–MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo.     

Real-time monitoring of disintegration activity of catalytic core domain of HIV-1 integrase using molecular beacon

HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN–CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN–CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN–CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity.     

A spectrophotometric assay for measuring acetyl–coenzyme A carboxylase

Acetyl–coenzyme A (CoA) carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacteria and eukaryota. This enzyme is the target of drug design for treatment of human metabolic diseases and of herbicides acting specifically on the eukaryotic form of the enzyme in grasses. Acetyl–CoA carboxylase activity screening in drug and herbicide design depends mostly on a time-consuming enzyme assay that is based on the incorporation of radiolabeled bicarbonate into the product malonyl–CoA. Here we describe a new simple, continuous, and quick photometric assay avoiding radioactive substrate. It couples the carboxylation of acetyl–CoA to the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of malonyl–CoA, which is catalyzed by recombinant malonyl–CoA reductase of Chloroflexus aurantiacus. This assay can be adapted for high-throughput screening.     

A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway and is a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40-μl assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)–thiosemicarbazide (TSC) or DAMO–antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives, including dimethyl sulfoxide, reducing agents, detergents, and bovine serum albumin. DAMO–TSC (Z′-factors 0.7–0.8) was determined to be superior to DAMO–antipyrine (Z′-factors 0.5–0.6) with significantly less variability within replicates. An HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO–TSC. This robust method has no heating requirement, which was the main obstacle to applying previous assays to HTS. More important, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large-scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.     

A homogeneous, high-throughput fluorescence resonance energy transfer-based DNA polymerase assay

A homogeneous, fluorescence resonance energy transfer (FRET)-based DNA polymerase assay that is suitable for high-throughput screening for inhibitors, and can also be used for steady-state kinetic investigations, is described. The activity, kinetic mechanism, and processivity of the isolated alpha subunit of DNA polymerase III, the product of the dnaE gene, from the gram-negative pathogen Haemophilus influenzae were investigated using the FRET assay.     

A high-throughput matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry-based assay of chitinase activity

A high-throughput matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF MS) assay is described for determination of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates and is readily achievable on a microliter scale (2 μl of total volume containing 2 μg of substrate and 1 ng of protein). The speed and sensitivity of the assay make it potentially well suited for the high-throughput screening of chitinase inhibitors. The mass spectrum is acquired in approximately 2 min, as opposed to typically 30–40 min for a single run with a high-performance liquid chromatography (HPLC)-based assay. By using the multiple-place MALDI MS targets, we estimate that 100 assays could be run in approximately 2–3 h without needing to remove the target from the instrument. In addition, because the substrate and product chitomers are visualized simultaneously in the TOF spectrum, this gives immediate information about the cleavage site and mechanism of the enzyme under study. The assay was used to monitor the purification and transgenic expression of plant class IV chitinases. By performing the assay with chitomer substrates and C-glycoside chitomer analogs, the enzyme mechanism of the class IV chitinases is described for the first time.     

Microplate-based kinetic method for assay of mitochondrial NADH-- and succinate--cytochrome c reductase activities

This article describes a microplate-based kinetic assay for mitochondrial NADH– and succinate–cytochrome c reductase activities in rat brain mitochondria. The assay reported here is based on the conventional spectrophotometric method and involves substrate-driven reduction of exogenous cytochrome c. Conditions regarding linearity with respect to time and protein concentration have been standardized. Furthermore, the methods were tested for inhibition of respective activities by specific inhibitors. The microplate format described here can be employed for rapid and simultaneous measurements of mitochondrial NADH– and succinate–cytochrome c reductase activities in a large number of samples.     

Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase

Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC₅₀ values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.     

A continuous spectrophotometric assay for monitoring adenosine 5′-monophosphate production

A number of biologically important enzymes release adenosine 5′-monophosphate (AMP) as a product, including aminoacyl–tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5′-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5′-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD⁺) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses.     

A fluorescence resonance energy transfer-based method for histone methyltransferases

A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye and a quencher, respectively. When lysine-9 residues in the peptides were methylated, they were protected from cleavage by endoproteinase–EndoLysC, whereas unmethylated peptides were cleaved, resulting in an increase in fluorescent intensity.     

Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry

Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein–protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein–peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a predefined raster of matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium–tin oxide. Furthermore, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI–TOF instruments and has general utility for the label-free analysis of microarray assays.     

Scintillation proximity assay as a high-throughput method to identify slowly dissociating nonpeptide ligand binding to the GnRH receptor

Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K(on) and K(off) are time and labor intensive, prompting the development of a screening assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K(i)) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K(i) values over time (Ki(30 min)/Ki(10 h) > 6). They used scintillation proximity assay technology to perform these binding experiments because this homogeneous assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K(i) value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K(i) ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.     

Scintillation proximity assay (SPA) as a new approach to determine a ligand’s kinetic profile. A case in point for the adenosine A<Subscript>1</Subscript> receptor

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a “mix and measure” format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A₁ receptor (hA₁R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k _on and k _off) of unlabeled hA₁R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.     

A simple and fast kinetic assay for phytases using phytic acid-protein complex as substrate

Phytase (EC 3.1.3.–) hydrolyzes phytate (IP₆) present in cereals and grains to release inorganic phosphate (P_i), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the P_i liberated from IP₆. This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP₆–lysozyme as a substrate. The assay is based on the principle that IP₆ forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP₆ in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released P_i from IP₆. This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl₂, and phosphate, showed a concentration-dependent interference.     

Capillary electrophoresis-based assay of phosphofructokinase-1

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg–ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg–ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg–ATP and Mg–ADP. The separation was enhanced by the addition of Mg²⁺ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC₅₀ value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.     

Assay methods for small ubiquitin-like modifier (SUMO)–SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system

SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation. We applied a version of this assay to the detection of in vitro SUMO–SIM interactions using a bacterial expression system. These novel assays enable screening of inhibitors of SUMO-dependent protein–protein interactions, either in vivo or in vitro, in a high-throughput manner.