A generic, homogenous method for measuring kinase and inhibitor activity via adenosine 5'-diphosphate accumulation

The authors describe an assay to measure the generation of adenosine 5'-diphosphate (ADP) resulting from phosphorylation of a substrate by a kinase. ADP accumulation is detected by conversion to a fluorescent signal via a coupled enzyme system. The technology has potential applications for the assessment of inhibitor potency and mode of action as well as kinetic analysis of enzyme activity. The assay has a wide dynamic range (0.25-75 microM) and has been validated with several kinases including the highly active cyclic adenosine monophosphate-dependent protein kinase (PKAalpha), casein kinase 1 (CK1), and the weakly active kinase Jun N-terminal kinase 2 (Jnk2alpha2). Kinase activity can be measured either in an end point or continuous mode. Assay performance in end point mode was compared with an adenosine 5'-triphosphate (ATP) depletion assay and in continuous mode with a pyruvate kinase/lactate dehydrogenase coupled assay. The ability to characterize kinase kinetics was demonstrated by deriving ATP/substrate affinity (Michaelis-Menten constant; K(m)) values for PKAalpha, CK1, and Jnk2alpha2. The assay readily measured activity with kinase reactions using protein substrates, indicating the suitability for use with large macromolecules. A wide range of inhibitor activities could be determined even in the presence of high ATP concentrations, making the assay highly suitable to characterize the mode of action of the inhibitor in question. Collectively, this assay provides a homogenous, generic method for a number of applications in kinase drug discovery.     

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.     

Characterization of a set of HIV-1 protease inhibitors using binding kinetics data from a biosensor-based screen

The interaction between 290 structurally diverse human immunodeficiency virus type 1 (HIV-1) protease inhibitors and the immobilized enzyme was analyzed with an optical biosensor. Although only a single concentration of inhibitor was used, information about the kinetics of the interaction could be obtained by extracting binding signals at discrete time points. The statistical correlation between the biosensor binding data, inhibition of enzyme activity (K(i)), and viral replication (EC(50)) revealed that the association and dissociation rates for the interaction could be resolved and that they were characteristic for the compounds. The most potent inhibitors, with respect to K(i) and EC(50) values, including the clinically used drugs, all exhibited fast association and slow dissociation rates. Selective or partially selective binders for HIV-1 protease could be distinguished from compounds that showed a general protein-binding tendency by using three reference target proteins. This biosensor-based direct binding assay revealed a capacity to efficiently provide high-resolution information on the interaction kinetics and specificity of the interaction of a set of compounds with several targets simultaneously.     

Determining appropriate substrate conversion for enzymatic assays in high-throughput screening

It is generally accepted that the conversion of substrate should be kept at less than 10% of the total substrate used when studying enzyme kinetics. However, 10% or less substrate conversion often will not produce sufficient signal changes required for robust high-throughput screening (HTS). To increase the signal-to-background ratio, HTS is often performed at higher than 10% substrate conversion. Because the consequences of high substrate conversion are poorly understood, the screening results are sometimes questioned by enzymologists. The quality of an assay is judged by the ability to detect an inhibitor under HTS conditions, which depends on the robustness of the primary detection signal (Z factor) and the sensitivity to an inhibitor. The assay sensitivity to an inhibitor is reflected in the observed IC(50) value or percent inhibition at a fixed compound concentration when single-point data are collected. The major concern for an enzymatic assay under high substrate conversion is that the sensitivity of the screen may be compromised. Here we derive the relationship between the IC(50) value for a given inhibitor and the percentage of substrate conversion using a first-order kinetic model under conditions that obey Henri-Michaelis-Menten kinetics. The derived theory was further verified experimentally with a cAMP-dependent protein kinase. This model provides guidance for assay developers to choose an appropriate substrate conversion in designing an enzymatic assay, balancing the needs for robust signal and sensitivity to inhibitors.     

Importance of albumin binding in the assay for carnitine palmitoyltransferase.

Alterations in the long-chain acyl-CoA binding to albumin in the carnitine palmitoyltransferase (CPT) assay appreciably affect the reaction at commonly used substrate concentrations. Since in the CPT assay the latter are typically well below saturation or Vmax. values, the measured enzyme activity depends on both the absolute quantity of albumin in the CPT assay and any biochemical modification of its binding. The present study verifies the striking dependence of the K0.5 for palmitoyl-CoA on albumin and the misleading 'activation' of the enzyme by compounds that also avidly bind to albumin. In assessing the intracellular physiological relevance of any modifier of CPT, the effects of protein binding in the assay assume particular importance. Indeed, any compound that alters CPT activity may do so, not directly, but as an assay artifact changing the free or unbound substrate concentrations.     

Determination of ligand-MurB interactions by isothermal denaturation: application as a secondary assay to complement high throughput screening

We used a temperature-jump isothermal denaturation procedure with various methods of detection to evaluate the quality of putative inhibitors of MurB discovered by high-throughput screening. Three optical methods of detection-ultraviolet hyperchromicity of absorbance, fluorescence of bound dyes, and circular dichroism-as well as differential scanning calorimetry were used to dissect the effects of two chemical compounds and a natural substrate on the enzyme. The kinetics of the denaturation process and binding of the compounds detected by quenching of flavin fluorescence were used to quantitate the dose dependencies of the ligand effects. We found that the first step in the denaturation of MurB is the rapid loss of flavin from the active site and that the two chemical inhibitors appeared to destabilize the interaction of the cofactor with the enzyme but stabilize the global unfolding. The kinetics of the denaturation process as well as the loss of flavin fluorescence on binding established that both compounds had nanomolar affinities for the enzyme. We showed that coupling of the various detection methods with isothermal denaturation yields a powerful regimen to provide analytical data for assessing inhibitor specificity for a protein target.     

Screening of herbal constituents for aromatase inhibitory activity

Random Forest screening of the phytochemical constituents of 240 herbs used in traditional Chinese medicine identified a number of compounds as potential inhibitors of the human aromatase enzyme (CYP19). Molecular modelling/docking studies indicated that three of these compounds (myricetin, liquiritigenin and gossypetin) would be likely to form stable complexes with the enzyme. The results of the virtual screening studies were subsequently confirmed experimentally, by in vitro (fluorimetric) assay of the compounds' inhibitory activity. The IC-50s for the flavones, myricetin and gossypetin were determined as 10 and 11 microM, respectively, whilst the flavanone, liquiritigenin, gave an IC-50 of 0.34 microM--showing about a 10-fold increase in potency, therefore, over the first generation aromatase inhibitor, aminoglutethimide.     

Properties of Na+, K+-ATPase of liver plasma membranes in the hamster

This study was designed to establish the properties of liver plasma membranes (LPM) Na+,K+-ATPase in the hamster and to determine whether a similar assay may be used to measure enzyme activity in the hamster and in the rat. Maximal Na+,K+-ATPase activity was obtained when the assay medium contained 5 mM Mg APT2- with or without 1 mM free Mg2+, 120 mM Na+, 12,5 mM K+. The incubation must be performed at 37 degrees C, pH 7.4. In the absence of free Mg2+, the saturation curve with respect to the substrate Mg ATP2- resulted in biphasic complex kinetics with a maximal activity at a substrate concentration of 5 mM. In the presence of 1 mM free Mg2+ activation of Na+,K+-ATPase and modification of the kinetics were observed: the biphasic curve tended to disappear and to become of the Michaelis-Menten type. The apparent Km for Mg APT2- was 0.36 mM and the Vmax 34.5 mumol.h-1.mg protein-1. In the presence of 10 mM free Mg2+ a decrease in the Vmax was observed without any effect on the apparent Km for Mg APT2-. It is concluded that the same incubation medium may be used to assay LPM N+,K+-ATPase from hamster and rat and that the addition of 1 mM free Mg2+ to the incubation medium is recommended to obtain Michaelis-Menten kinetics in order to eliminate complex kinetics due to the absence of free Mg2+.     

Molecular determinants of sarco/endoplasmic reticulum calcium ATPase inhibition by hydroquinone-based compounds

The ion transport activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA) is specifically and potently inhibited by the small molecule 2,5-di-tert-butylhydroquinone (BHQ). In this study, we investigated the relative importance of the nature and position of BHQ's four substituents for enzyme inhibition by employing a combination of experimental and computational techniques. The inhibitory potencies of 21 commercially available or synthesized BHQ derivatives were determined in ATPase activity assays, and 11 compounds were found to be active. Maximum inhibitory potency was observed in compounds with two para hydroxyl groups, whereas BHQ analogues with only one hydroxyl group were still active, albeit with a reduced potency. The results also demonstrated that two alkyl groups were an absolute requirement for activity, with the most potent compounds having 2,5-substituents with four or five carbon atoms at each position. Using the program GOLD in conjunction with the ChemScore scoring function, the structures of the BHQ analogues were docked into the crystal structure of SERCA mimicking the enzyme's E(2) conformation. Analysis of the docking results indicated that inhibitor binding to SERCA was primarily mediated by a hydrogen bond between a hydroxyl group and Asp-59 and by hydrophobic interactions involving the bulky inhibitor alkyl groups. Attempts to dock BHQ into crystal structures corresponding to the E(1) conformation of the enzyme failed, because the conformational changes accompanying the E(2)/E(1) transition severely restricted the size of the binding site, suggesting that BHQ stabilizes the enzyme in its E(2) form. The potential role of Glu309 in enzyme inhibition is discussed in the context of the computational results. The docking scores correlated reasonably well with the measured inhibitory potencies and allowed the distinction between active and inactive compounds, which is a key requirement for future virtual screening of large compound databases for novel SERCA inhibitors.     

Fluorescence assay of polyamide-DNA interactions

Polyamides (PAs) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition. This capability provides a viable route to their development as therapeutics. Presented here is a simple and convenient fluorescence assay for PA-DNA binding. PAs are titrated into a sample of a hairpin DNA featuring a TAMRA dye attached to an internal dU near the PA binding site. In a study of 6 PAs, PA binding leads to a steady reproducible decrease in fluorescence intensity that can be used to generate binding isotherms. The assay works equally well with both short (6- to 8-ring) and long (14-ring) PAs, and K(d) values ranging from approximately 1 nM to at least 140 nM were readily obtained using a simple monochromator or filter configuration. Competition assays provide a means to assessing possible dye interference, which can be negligible. The assay can also be used to determine PA extinction coefficients and to measure binding kinetics; thus, it is an accessible and versatile tool for the study of PA properties and PA-DNA interactions.     

Equilibrium competition binding assay: inhibition mechanism from a single dose response

Inhibition of a receptor by a small-molecule compound in many cases is achieved via a competitive, uncompetitive or non-competitive mechanism. The receptor-inhibitor interaction is often probed through the displacement of a ligand in an equilibrium competition binding experiment. The previous solutions to receptor inhibition mechanisms were borrowed from steady-state enzyme inhibition mechanisms. The inhibition mechanism is determined by a visual inspection or a global fit of ligand dose response curves at a series of inhibitor concentrations. However these solutions only apply to situations when both the ligand and the inhibitor are not significantly depleted by the receptor. In most published equilibrium receptor binding studies, only the relative potency of the inhibitor is calculated. Ranking inhibitors tested under differing experimental conditions is often not possible. In the current paper, we offer exact mathematical solutions to uncompetitive and non-competitive inhibition, and demonstrate that in most cases both the inhibition mechanism and absolute potency of an inhibitor can be simultaneously determined from a single dose response of the inhibitor at a fixed concentration of the ligand. Therefore, an equilibrium competition assay provides a quick and facile method to determine the inhibition mechanism of a large number of inhibitors. The theory herein described is applicable to equilibrium competition binding experiments such as radioligand assays and fluorescence polarization assays.     

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure–activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC₅₀ values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC₅₀ values with steep dose–response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay’s utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein–ligand interactions.     

Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver

1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0 degrees C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of P(i)) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis-Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis-Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn(2+) was slightly more, and Ca(2+) somewhat less, stimulatory than Mg(2+). The Mg(2+)-dependent fraction showed Michaelis-Menten kinetics with respect to the added Mg(2+). 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.     

Transient response of encapsulated enzymes in hollow-fiber reactor

A mathematical model for the transient response of encapsulated enzymes is developed showing the effects of the outer boundary layer, the encapsulating membrane, the partition coefficient, and diffusion with reaction within the encapsulating medium. The model incorporates both first-order kinetics and Michaelis-Menten kinetics for the reaction rate. Using typical hollow-fiber or microcapsule parameters, the model shows that (a) the partition coefficient affects the overall rate only when the rate-limiting step is diffusion through the membrane, (b) the transient overall effectiveness factor rises sharply with time and approaches an asymptotic value for most situations, and (c) the first-order approximation to Michaelis-Menten kinetics is not valid when the initial outside bulk concentration is higher than the Michaelis constant and the overall rate is reaction limited. The model is compared with experimental data using uricase in a hollow-fiber enzyme reactor configuration. Batch assay and CSTUER (continuous-stirred ultrafiltration enzyme reactor) studies were conducted on the free enzyme to provide some of the parameters used in the model. The CSTUER data fit the case of substrate inhibition kinetics with the apparent Michaelis constant approaching zero. The hollow-fiber reactor was conducted with uricase dissolved in both a buffer solution and a concentrated hemoglobin solution. Diffusivities of the solute were measured in both solutions as was the osmotic pressure of the hemoglobin solution. While experimental data for uricase in buffer solution could easily be matched by the model, that in the concentrated hemoglobin solution could not.     

High-throughput scintillation proximity assay for stearoyl-CoA desaturase-1

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([(3)H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compounds was screened at 11 μM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [(3)H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.     

Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection.

We developed a method to determine dihydrofolate reductase (DHFR) activity at pH 7.4 (37 degrees C) by monitoring its product, tetrahydrofolate (H(4)folate), using HPLC with electrochemical detection. After the assay mixture was deproteinized by 0.5 M perchloric acid, the H(4)folate concentration was measured. Using sodium ascorbate at 20 mM, H(4)folate was stable in our assay system. The enzyme activity was also stable. The detection limit of this method was less than 1 nM of H(4)folate in the enzyme assay system, which was 1/100 lower than those for the NADPH-spectrophotometric assay, which is commonly used for analysis of DHFR activity. This value of 1 nM allowed us to control the conversion from dihydrofolate (H(2)folate) to H(4)folate less than 10% of initial substrate concentrations during assay, when we used a concentration around K(m) values reported for DHFR from various sources. The rate of reduction showed a linearity at concentrations around the K(m). The reduction rate must be evaluated exactly around the K(m), in order to obtain an accurate profile of Michaelis-Menten kinetics. This assay method has a sensitivity high enough to determine the reduction rate at H(2)folate concentrations around K(m). In addition, the assay procedure is very simple. Therefore, our method may be useful for studying DHFR.     

Non-isotopic dual parameter competition assay suitable for high-throughput screening of histone deacetylases

Histone deacetylases reside among the most important and novel target classes in oncology. Selective lead structures are intensively developed to improve efficacy and reduce adverse effects. The common assays used so far to identify new lead structures suffer from many false positive hits due to auto-fluorescence of compounds or triggering undesired signal transduction pathways. These drawbacks are eliminated by the dual parameter competition assay reported in this study. The assay involves a new fluorescent inhibitor probe that shows an increase in both, fluorescence anisotropy and fluorescence lifetime upon binding to the enzyme. The assay is well suited for high-throughput screening.     

Bacterial complementation as a means to test enzyme–ligand interactions

A bacterial complementation assay has been developed for the rapid screening of a large number of compounds to identify those that inhibit an enzyme target for structure-based inhibitor design. The target enzyme is the hypoxanthine phosphoribosyltransferase (HPRT). This enzyme has been proposed as a potential target for inhibitors that may be developed into drugs for the treatment of diseases caused by several parasites. The screening assay utilizes genetically deficient bacteria complemented by active, recombinant enzyme grown in selective medium in microtiter plates. By comparing absorbance measurements of bacteria grown in the presence and absence of test compounds, the effect of the compounds on bacterial growth can be rapidly assayed. IC₅₀ values for inhibition of bacterial growth are a reflection of the ability of the compounds to bind and/or inhibit the recombinant enzyme. We have tested this bacterial complementation screening assay using recombinant HPRT from the parasites Plasmodium falciparum and Trypanosoma cruzi, as well as the human enzyme. The results of these studies demonstrate that a screening assay using bacterial complement selection can be used to identify compounds that target enzymes and can become an important part of structure-based drug design efforts. Received: 4 December 1997 / Received revision: 17 March 1998 / Accepted: 26 March 1998     

Imidazole derivatives as a novel class of hybrid compounds with inhibitory histamine N-methyltransferase potencies and histamine hH3 receptor affinities

In this study, a novel series of imidazole-containing compounds with dual properties, that is, inhibitory potency at the enzyme histamine N(tau)-methyltransferase (HMT) and antagonist potency at histamine H(3) receptors was designed and synthesized. Pharmacologically, these new hybrid drugs were evaluated in functional assays for their inhibitory potencies at rat kidney HMT and for their antagonist activities on synaptosomes of rat cerebral cortex. For selected compounds, binding affinities at recombinant human histamine H(3) receptors were determined. The first compounds (1-10) of the series proved to be H(3) receptor ligands of high potency at rat synaptosomes or of high binding affinity at human H(3) receptors, respectively, but of only moderate activity as inhibitors of rat kidney HMT. In contrast, aminoquinoline- or tetrahydroacridine-containing derivatives 11-17 also displayed HMT inhibitory potency in the nanomolar concentration range. Preliminary data from molecular modeling investigations showed that the imidazole derivative 15 and the HMT inhibitor quinacrine possess identical binding areas. The most interesting compound (14) is simultaneously a highly potent H(3) receptor ligand (K(i)=4.1nM) and a highly potent HMT inhibitor (IC(50)=24nM), which makes this derivative a valuable pharmacological tool for further development.