Process for glucuronidation screening

Abstract

A fluorescence polarization process used to identify activity of conjugative enzymes involved in xenobiotic transformations, such as glucuronosyltransferases is provided.

Description

FIELD

[0002] The field of this invention relates to a process for screening for enzymes activity. More particularly the process is a method that can be used to identify activity of glucuronosyltransferases.

BACKGROUND

[0003] Drug metabolism problems such as production of toxic metabolites and unfavorable pharmacokinetics cause almost half of all drug candidate failures during clinical trials. Although glucuronidation is one of the most important routes of biotransformation, the broad and overlapping substrate specificity of the hepatic UDP-glucuronosyltransferases (UGTs) that catalyze glucuronidation remains poorly understood. The two main reasons for this situation are the lack of isolated individual UGT isozymes and the lack of assay methods suitable for detecting glucuronidation of diverse chemicals.

[0004] The UDP-glucuronosyltransferases are a family of enzymes that catalyze the glucuronidation of endogenous and xenobiotic chemicals (Equation 1), generating products that are more hydrophilic and thus more readily excreted in bile or urine.

[0005] Equation 1:

Uridine diphosphate-glucuronic acid (UDPGA)+aglycone→UDP+glucuronide

[0006] The UGTs play a key role in several important metabolic functions, including:

[0007] elimination of drugs such as non-steroidal anti-inflammatories, opioids, antihistamines, antipsychotics and antidepressants,

[0008] detoxification of environmental contaminants such as benzo(a)pyrenes,

[0009] regulation of hormone levels for androgens, estrogens, progestins, and retinoids,

[0010] elimination of the heme degradation product bilirubin.

[0011] Although glucuronidation generally is classified as Phase II metabolism—the phase occurring after P450 dependent oxidative metabolism—many compounds do not require prior oxidation because they already possess functional groups that can be glucuronidated. Examples of first pass metabolism catalyzed by UGTs include the UGT2B7-dependent glucuronidation of morphine and the glucuronidation of 5-lipoxygenase inhibitors (anti-inflammatories). In the latter case glucuronidation was demonstrated to be the rate-limiting step for in vivo plasma clearance.

[0012] Notably, glucuronidation does not always cause decreased biological activity and/or detoxification. Glucuronides of some drugs are toxic, and have been linked with adverse drug reactions including immune hypersensitivity. Glucuronidation can modulate the potency of some drugs: the 6-glucuronide of morphine is a more potent analgesic than the parent compound, whereas the 3-glucuronide is a morphine antagonist. In addition, steroid glucuronidation can produce more active or toxic metabolites under pathophysiological conditions or during steroid therapies.

[0013] UGTs are 50-60 kDa integral membrane proteins with the major portion of the protein, including the catalytic domain, located in the lumen of the endoplasmic reticulum and a C-terminal anchoring region spanning the ER membrane. Two UGT families—UGT1 and UGT2—have been identified in humans. Although the members of these families are less than 50% identical in primary amino acid sequence, they exhibit significant overlap in substrate specificity.

[0014] The members of the UGT1 family that are expressed in human liver, where the majority of xenobiotic metabolism takes place, include UGT 1A1, 1A3, 1A4, 1A6, and 1A9. Although the UGT2 family has not been studied as extensively, it is known that UGT2B4, 2B7, 2B10, 2B15 and 2B17 are expressed in the liver. Mutations in UGTs are known to have deleterious effects, including hyperbilirubinemia which occurs with a frequency of 5-12% and can lead to neurotoxicity and in severe cases, death. As is the case for other drug metabolizing enzymes such as P450s, interindividual differences in UGT expression levels have been observed and linked to differences in drug responses. For instance, low expression of UGT1A1, as in patients with Gilbert's syndrome, has been associated with the toxicity of Irinotecan, a promising anticancer agent. In addition, UGT upregulation in tumor tissues has been identified as a possible cause of anticancer drug resistance.

[0015] Specificity for Aglycones. UGT substrates are known as aglycones. The products of the reaction are called glucuronides. All of the known UGTs exhibit broad substrate specificity, with a single isozyme catalyzing glucuronidation of a broad range of structurally unrelated compounds. Not surprisingly there also is a great deal of overlap in the specificities of UGT isozymes. The sites of glucuronidation generally are nucleophilic nitrogen, sulfur or oxygen atoms in functional groups such as aliphatic alcohols, phenols, carboxylic acids, primary through tertiary amines, and free sulfyhydryls. The aglycone binding site is believed to be in the N-terminal portion of the UGT polypeptide, the region of the protein that shows the greatest variability in sequence among isozymes. However, efforts to define the aglycone binding site by correlating N-terminal amino acid sequences of UGT isozymes with their substrate specificities have been unsuccessful.

[0016] Despite their broad substrate specificities, UGTs can be highly regio- and stereo-selective. It has been suggested that substrates bind loosely to a very “open” substrate binding pocket—as with some P450s—and rotate until reactive functional groups are suitably oriented to the bound UDPGA and the amino acids involved in catalysis. Although several studies on the substrate specificities of individual recombinant UGTs have been performed, most have been limited to a relatively small number of compounds within one or two structural classes.

[0017] HTS assay methods described herein can be used to rapidly screen large numbers of diverse chemicals thus allowing a systematic effort to fully define the “chemical space” recognized by each of the key hepatic UGTs. Moreover, these HTS assay methods will fulfill the immediate needs of the pharmaceutical industry by providing a means to screen large numbers of diverse compounds for glucuronidation with a panel of the key human UGT isozymes. The information obtained with these HTS assays can be used in the following ways:

[0018] After isozyme identification, more detailed kinetic studies with the appropriate UGT isozyme can be used to predict in vivo clearance rates, reducing the number of compounds that fail in clinical studies due to poor pharmacokinetics.

[0019] Knowledge of metabolism by a specific UGT alerts the drug discovery team to potential pharmacogenetic problems, since genetic differences in UGT levels are recognized as an important factor in varying responses to therapeutics.

[0020] Identification of the UGT responsible for the metabolism of a drug will aid in judicious selection of the in vitro assays or animal models used for preclinical assessment of possible drug-drug interactions and toxicology testing, thereby reducing inappropriate or unnecessary use of animals for experiments.

[0021] Metabolism data can be used as a component of rational drug design. A better understanding of the structure-activity relationships that define substrate specificity for the various UGT isozymes would provide a basis for structural modifications of primary compounds to change their metabolism profile. This approach was used successfully for development of ABT-761, a 5-lipoxygenase inhibitor.

[0022] The testing of glucuronidated compounds can lead to the discovery of valuable prodrugs that are inactive until metabolized in the body into an active form.

[0023] To confirm the need for improved technology to probe the specificity of UGT isozymes, it is useful to review the methods currently employed for in vitro drug metabolism studies, and the reasons why they are not adequate for immediate drug discovery needs.

[0024] Sources of UGTs. The important drug metabolizing UGT isozymes are located in the endoplasmic reticulum of liver cells. Natural sources of UGT for in vitro assays include liver slices, cultured cells, and cell fractions such as human liver microsomes. The major drawbacks of these unpurified systems are that they contain a mixture of multiple UGT isozymes and other drug metabolizing enzymes. As a result, they are of limited use in obtaining meaningful data on a specific UGT isozyme—particularly in an HTS format. Heterologous expression systems such as mammalian and BaV-infected insect cells have made it possible to produce large amounts of microsomal membranes highly enriched in a single UGT isozyme.

[0025] Assay methods. UGTs generally are assayed by isolation and quantification of the radioactively labeled metabolites produced from the parent compound in reactions containing radiolabeled UDPGA. In most cases, this involves chromatographic techniques such as thin layer chromatography (TLC) or high pressure liquid chromatography (HPLC), and in some cases phase separations. There are two major drawbacks to these assays methods. First, the need to isolate the reaction products makes the methods too cumbersome and time consuming for use in any type of high volume assay format. Second, different glucuronidated metabolites have different chromatographic properties, raising an obvious technical barrier to screening diverse compounds for metabolism by a panel of isolated UGT isozymes. For some substrates, products and reactants can be differentiated on the basis of altered absorbance or fluorescence after glucuronidation. However, these methods are limited to a few UGT isozymes.

SUMMARY

[0026] The present invention provides a universal HTS activity assay that enables screening for glucuronidation of large numbers of diverse chemicals by any isolated recombinant UGT using a single detection method. The method is based upon glucuronides, the products of UGT reactions, inhibiting the formation of a fluorescent product by a bacterial β-glucuronidase. The method is non-radioactive, homogenous and can be used for identification of novel UGT substrates and inhibitors in a high throughput screening (HTS) format. UGT assay methods provided herein are based on inhibition of a fluorescent β-glucuronidase reporter reaction. This approach provides the following advantages over existing methods:

[0027] Universal Assay Method. The assay method is useful for all UGT isozymes and for all aglycone substrates, thus making it ideal for screening large numbers of diverse compounds.

[0028] Nonradioactive. The assay does not employ radioisotopes, thus eliminating the hazards and regulatory and handling costs associated with such agents.

[0029] Homogeneous Assay Method. The assay is homogenous, eliminating separation steps and possibly allowing continuous monitoring of reaction rate, in turn allowing more flexibility for kinetic analyses.

[0030] The novel HTS assay method will allow investigators to survey the full range of potential substrate specificity for the key hepatic UGT isozymes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1, panel A, depicts the kinetics of the cleavage of various concentrations of 4-methylumbelliferyl-β-D-glucoronide by β-glucuronidase, as measured by fluorescence intensity. Panel B depicts the velocity of cleavage of various concentrations of 4-methylumbelliferyl-β-D-glucoronide by β-glucuronidase.

[0032] FIG. 2 depicts the effect of varying concentrations of β-D-glucuronide on the ability of β-glucuronidase to cleave various substrates.

[0033] FIG. 3, panel A, depicts the results of an experiment wherein a microsomal preparation from either recombinant insect cells engineered to express the UDP glycosyl transferase UGT1A6 (“Baculosomes UGT1A6”) or non-recombinant insect cells (“Control Baculosomes”) is incubated with α-naphthol for various periods of time and any resulting α-naphthyl glucuronide is extracted and added to a mixture of 4-methylumbelliferyl-β-D-glucoronide and β-glucuronidase. The effect of such an extract on the ability of β-glucuronidase to cleave 4-methylumbelliferyl-β-D-glucoronide as compared to reaction lacking such extract is shown (“% of activity”). Panel B depicts the results of an experiment wherein a microsomal preparation from either recombinant insect cells engineered to express the UDP glycosyl transferase UGT1A6 (“Baculosomes UGT1A6”) or non-recombinant insect cells (“Control Baculosomes”) is incubated with or without α-naphthol for various periods of time and then directly added (without extraction) to a mixture of 4-methylumbelliferyl-β-D-glucoronide and β-glucuronidase. The effect of the baculosomes on the ability of β-glucuronidase to cleave 4-methylumbelliferyl-β-D-glucoronide as compared to reaction without baculosomes is shown (“% of activity”).

[0034] Assay Principle: Cleavage of 4-methylumbelliferyl β-D-glucuronide by bacterial β-glucuronidase generates the highly fluorescent compound, 4-methylumbelliferone. Diverse β-D-glucuronides act as competitors of β-glucuronidase, thus providing the basis of a coupled assay method for detection of glucuronide production by recombinant UGT isozymes. Using human recombinant human UGT 1A6 as an example, the present invention demonstrates the feasibility of using this coupled assay method for fluorescence detection of UGT activity with several structurally diverse substrates. The 4-methylumbelliferyl β-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of β-D glucuronides generated using recombinant glycosyl transferase preparations.

[0035] Methods

[0036] β-D-glucuronidase (Part G-7396), α-naphthyl β-D-glucuronide, β-trifluoromethylumbelliferyl β-D-glucuronide, β-estradiol 3-(β-D-glucuronide), p-acetominophenyl β-D-glucuronide, 5β-androstane-3α, 17α-diol-11-one-17-carboxilic acid-3-(β-D-glucuronide), UDPGA and 4-methylumbelliferone β-D-glucuronide were obtained from Sigma, St. Louis Mo. Tetrahydrocortisone 3-β-D-glucuronide was obtained from Molecular Probes, Eugene, Oreg. Recombinant control and UGT1A6 membrane preparations were generated and are commercially available from PanVera Corporation, Madison Wis. All other reagents were analytical grade or better and purchased from a variety of commercial sources.

[0037] Results and Discussion

[0038] An assay that can be used to demonstrate the presence of β-D-glucuronides that might be generated from different classes of UGT enzymes is provided. An assay based on β-glucuronidase activity provides a high throughput screening method to identify structurally different β-D-glucuronides. Cleavage of 4-methylumbelliferyl β-D-glucuronide yielded the highly fluorescent compound 4-methylumbelliferone. Under linear conditions of protein concentration and incubation time, the apparent Km value for cleavage of 4-methylumbelliferyl-β-D-glucuronide was approximately 56 μM (FIG. 1, panels A and B).

[0039] In order to show the feasibility of developing a high throughput inhibition assay, we examined the potential of a variety of structurally dissimilar, commercially available β-D-glucuronides to act as inhibitors for the cleavage of 4-methylumbelliferyl-β-D-glucuronide. These β-D-glucuronides represent compounds that might potentially be formed by UGT activity e.g. phenol and steroid glucuronides. FIG. 2 shows that all of the β-glucuronides tested inhibited the production of 4-methylumbelliferone by β-glucuronidase. The potential to inhibit 4-methylumbelliferyl-β-D-glucuronide cleavage appeared to be dependent on the chemical nature of the substituted aglycone, with β-estradiol-3-(62-D-glucuronide) and α-naphthyl β-D-glucuronide showing the strongest and weakest inhibition, respectively. Uridine 5′-diphosphoglucuronic acid (UDPGA), an essential cofactor for UGTs was not an effective competitor over the range tested, probably due to the α configuration of sugar bond to UDP (Parkinson, 1996).

[0040] Two approaches were taken to test the feasibility of coupling the UGT-dependent production of glucuronides to inhibition of fluorescent product formation by β-D-glucuronidase. The first involved extracting the UGT reaction products and adding them to the β-D-glucuronidase fluorescence assay reagents. The second involved adding the β-D-glucuronidase reaction components directly to the quenched UGT reaction. In the first approach, α-naphthyl β-D-glucuronide was generated in with a reaction involving recombinant human UGT1A6 and α-naphthol. Following extraction, 4-methylumbelliferyl-β-D-glucuronide assay reagents were added to the extracted, dried reaction residues. In a separate experiment it was shown that aglycones as well as glucuronides are extracted with ethyl acetate and hence transferred to the β-glucuronidase reporter assay. FIG. 3, panel A, shows that residues from the UGT1A6 assay, but not the control microsome assay, inhibited 4-methylumbelliferyl-β-D-glucuronide cleavage. Residues from the 0 time point extractions do not appear to inhibit the 4-methylumbelliferone cleavage assay which indicated that the aglycone α-naphthol does not interfere non-specifically with this assay under these conditions. Inhibition of 4-methylumbelliferyl-β-D-glucuronide cleavage was dependent on the incubation time of the UGT1A6 assay, presumably reflecting increased α-naphthyl glucuronide accumulation. (Accumulation of β-naphthyl glucuronide in the UGT1A6 assay was independently verified in a parallel experiment using [14C] UDPGA, data not shown.)

[0041] In the second approach, 4-methylumbelliferyl-β-D-glucuronide assay reagents were added directly to a β-naphthyl glucuronidation assay that was performed in a 96 well plate. FIG. 3, panel B, shows that 4-methylumbelliferyl β-D-glucuronide cleavage was αpercent slower when added to the UGT1A6 assay compared to the control. The homogeneous format circumvented the need for extraction, but required longer incubation periods with the recombinant enzyme. These results demonstrate that: i) the 4-methylumbelliferyl-β-D-glucuronide assay can be used to detect β-D-glucuronides generated in biological preparations, ii) the choice of α-naphthol β-D-glucuronide, which is a weak competitive inhibitor (FIG. 2) adds credence to this assay. More potent inhibitors such as estradiol β-D-glucuronide (FIG. 2) are likely to be more effective, iii) the assay is amenable to a high throughput format, as a two step procedure, and iv) heterologous expression systems are useful for the generation of high concentrations of β-D-glucuronides, such as α-naphthyl β-D-glucuronide, which was a weak competitor in the β-D-glucuronide cleavage assay (FIG. 2).

[0042] In summary, we have demonstrated the feasibility of using a fluorescent assay that can be adapted to a high throughput format to measure the presence of β-D-glucuronides. The assay described here uses relatively inexpensive reagents and does not require chromatographic resolution (TLC or HPLC) or radioactivity. The assay can be performed in a homogeneous format and could be easily automated. The assay will not eliminate the usefulness of other analytical procedures needed to determine the kinetic parameters of glucuronidation, but shows potential as a rapid preliminary screening method.

[0043] In addition, similar principles and procedures described here for UDP-glycosyltransferases could be applied to other important classes of phase II conjugating enzymes, e.g. sulfotransferases, N-acetyltransferases, glutathione S-transferases. For example, sulfation catalyzed by members of the sulfotransferase enzyme family is a major metabolic pathway which modulates the biological activity of numerous endogenous and xenobiotic chemicals. Sulfate conjugates from sulfation assays would be expected to compete in a similarly designed assay that used a sulfatase to generate a fluorescent product. The following procedure can be used to demonstrate the presence of sulfated compounds generated from multiple sources of sulfotransferases including recombinant enzymes, tissue homogenates and biological fluids. Aryl-sulfatase enzyme, which is capable of cleaving a wide variety of sulfated compounds, can be used to design a high-throughput fluorescent assay where the products of sulfotransferase conjugation would act as competitive substrates for aryl-sulfatase cleavage of the reporter fluorescent or chromogenic substrate. The sulfatases belong to a highly conserved gene family. Considerable sequence similarity exists among sulfatases of both prokaryotic and eukaryotic origins. Sulfatases of prokaryotic origin are preferable to use because of their wider substrate specificity (for example, sulfatase type IV-VIII; H1-H5 from Sigma). Possible substrates for assaying aryl-sulfatase activity include the fluorogenic ELF-97sulfatase substrate (ELF-97 sulfate, E-6579, Molecular Probes) and the chromogenic indolyl substrates (B-8406, B-8410, Molecular Probes). ELF-97 sulfate is expected to yield a photostable yellow-green fluorescent precipitate, whereas the indolyl sulfates (B-8406, B-8410) produce dark blue and magenta precipitates, respectively. The measurement of the cleavage of ELF-97 sulfate (Molecular Probes, OR) by sulfatase to the highly fluorescent compound will be possible using excitation wavelength of 345 nm and emission wavelength of 530 nm. Competitive inhibition of ELF-97 sulfatase cleavage will be used to detect the presence of sulfated metabolites of the test compound in the sulfotransferase reaction assay mixture.

[0044] Production of purified, full length UGT2B7 from BaV infected insect cells. To establish a model for ultimate development of QSAR methods for all key UGTs, UGT2B7 is purified from BaV-infected insect cells for incorporation into the HTS assay method. Although there are no published reports on purification of UGTs from BaV-infected insect cells, we will employ methods used successfully to purify various UGT isozymes from native sources and to maintain their stability with lipids. We and other investigators already have demonstrated that the properties of the BaV-expressed UGTs are similar to those of the enzymes expressed in mammalian cells. Use of the BaV expression system as a source for the purified enzyme will allow cost effective production of sufficient protein for use of the proposed HTS assay in pharmaceutical drug discovery programs and for the planned QSAR studies.

[0045] Production of a soluble aglycone binding domain for structural studies. Structural studies on UGT/aglycone complexes will aid in the development and validation of QSAR models. NMR analysis combined with molecular docking simulations is used to determine the bioactive conformations of substrates and to identify molecular interactions involved in binding and subsequent glucuronidation. Experiments with UGTs containing mutations in proposed active site residues are used to test the structural models. These structural studies will provide a powerful approach that is synergistic with HTS screening for identification of the key molecular recognition factors that affect UGTs catalysis. To circumvent the difficulties inherent in structural studies with membrane proteins, an affinity-tagged soluble UGT2B7 aglycone binding domain in E. coli will be produced and purified. This approach provides the following advantages: a) elimination of the need for lipids to maintain structural integrity due to removal of the C-terminal portion of the protein that interacts with the membrane, b) high level expression of a soluble domain in the optimal size range for NMR studies, and c) affinity tagging of binding domains, thus simplifying purification.

[0046] The HTS assay is used to identify and determine the kinetic parameters for diverse UGT substrates used in the QSAR study. QSAR is a rational design tool that has been used for more than 30 years by medicinal chemists to improve the potency and metabolism properties of drugs, and to assess chemical toxicity. The concept is based on the ability to quantitatively relate changes in the bioactivity of small molecules to changes in their physical properties. In a typical QSAR study, a defined set of chemicals—the training set—is tested for interaction with a protein of interest, usually receptor binding or enzymatic conversion. Correlations are then sought between the physicochemical properties of the chemicals and their bioactivity. A primary goal of QSAR is to predict the potency of chemicals outside of the training set. In a medicinal chemistry setting, QSAR analysis typically is performed using a high affinity receptor and a training set of 10-50 compounds in a single congeneric series—a group of chemicals sharing a common molecular core with relatively minor substituent or structural variations on that core. Such an approach is not applicable to the UGTs because a single isozyme may have very broad substrate specificity, encompassing several structural classes.

[0047] To effectively address the structural diversity of UGT substrates, UGT QSAR that will focus on the catalytic mechanism (i.e., how the physicochemical properties of UGT substrates affect their binding to UGT and subsequent nucleophilic attack on the UDPGA molecules) will be used. The key molecular recognition factors that determine aglycone binding and reactivity at the active site with the assumption that two structures can present similar recognition factors within dissimilar molecular frameworks will be identified. Establishing the QSAR based on the fundamental interactions that trigger catalysis should enable prediction of glucuronidation for molecules with diverse molecular frameworks. The assay methods developed are used to generate quantitative kinetic data (Vmax and Km) using in vitro assays with purified recombinant enzyme, thereby allowing a mechanism based QSAR analysis of UGT catalysis and eliminating many of the extrinsic factors that might interfere with the analysis using less refined systems.

[0048] The success of QSAR in these applications partially depends upon how effectively the structures in the training set probe the “chemical space” defined by the enzyme active site and the mechanism of its bioactivity. Although the high throughput assay methods developed will allow a “shotgun approach” by screening hundreds of thousands of compounds, such a collection of purified compounds would cost several million dollars. Instead, a smaller focused library of several thousand compounds that are likely to be glucuronidated will be designed. This is achieved using database searching tools with known glucuronidation reactions and chemical logic as inputs. The focused library then is screened using the HTS method described herein to identify those compounds metabolized by UGT2B7. This subset, an estimated 500-1500 compounds, will comprise the QSAR training set. Thus the HTS assay methods provided herein are used in both a qualitative screening mode to develop the UGT2B7 QSAR training set, and in an analytical mode for probing the molecular determinants of UGT catalysis.

[0049] Additional UGT isozymes are incorporated into the HTS activity assay. The invention also provides a second assay based on detection of UDP, a reaction product common to all UGT isozymes. When used in HTS format, this assay can be utilized with all of the key hepatic UGT isozymes. Four of the UGT1 family isozymes have been produced as microsomal membrane preparations from BaV-infected insect cells. These UGTs and additional members of the UGT2 family are purified and incorporated into the universal UDP-based HTS assay, thereby providing the capability to screen compound libraries for UGT isozyme identification and pharmacokinetics with the full spectrum of hepatic glucuronidation activity.

[0050] The soluble aglycone binding domain provides structural information on bound aglycones. Structural analyses of bound aglycones and their interactions with active site amino acid residues enhance the QSAR by providing an independent approach for identifying the key molecular recognition factors that determine aglycone binding and reactivity. Three-dimensional models of the bound aglycones aid in the application of powerful 3D-QSAR approaches, such as comparative molecular field analysis. Mapping of aglycone-UGT interactions aid in the application and validation of fragment based QSAR approaches such as Holographic QSAR (Tripos, St. Louis, Mo.) or Multicase (Multicase, Inc. Beachwood, Ohio). In the end, it is important to note that QSAR is based on statistical correlations. The structural studies will provide a means of assessing whether these correlations are consistent with empirically determined steric and spatial constraints for substrate binding and catalysis.

[0051] Heterologous expression and characterization of full length UGT 2B7. It has recently demonstrated that stably expressed human UGT2B7 catalyzed the glucuronidation of opioids such as morphine and buprenorphine with high efficiency (Dr. Thomas Tephly, University of Iowa College). In addition, UGT2B7 has been shown to catalyze the glucuronidation of NSAIDS and catechol estrogens. Dr. Tephly3 s studies with UGT 2B7expressed in mammalian cells have clearly established the isozyme3 s importance in the metabolism of drugs and endogenous steroids, and will provide important benchmark data for evaluating the enzyme purified from BaV-infected insect cells during studies.

[0052] Clinical studies have shown that morphine-6-O-glucuronide is 2-3 times more effective as an analgesic than the parent compound and that it binds with high affinity to opioid receptors. In contrast, morphine-3-O-glucuronide does not bind to the opioid receptors and is devoid of analgesic effects. Furthermore, morphine-3-O-glucuronide has been shown to counteract the analgesic activity of morphine and morphine-6-O-glucuronide.

[0053] As a first step towards understanding the metabolism of morphine a UGT2B7, cDNA was isolated from a human liver library based on homology with the full length rat liver cDNA for UGT2B1, which has activity toward opioids. Stable UGT2B7 cell lines were established from embryonic human kidney cells (HK293 cells) and microsomal membrane fractions from these cells were used to assess the properties of the recombinant enzyme. Glucuronidation activity towards opioids (5 mM morphine (pH 8.4), 5 mM codeine (pH 7.7), 2 mM nalorphine (pH 8.4), 2 mM naloxone (pH 7.7), 2 mM naltrexone (pH 7.0) and 0.5 mM buprenorphine (pH 7.0)) was demonstrated using the method described by Puig and Tephly. These studies also indicated that UGT2B7 promotes the glucuronidation of the 3-OH and 6-OH of codeine with an efficiency ratio similar to the ratio of glucuronides found in human urine, or 7 to 1; 3-OH to 6-OH. In addition, codeine, the 3-methoxy derivative of morphine, was efficiently converted to the 6-O-glucuronide by UGT2B7.

[0054] Besides its important role in opioid and NSAID metabolism, UGT2B7 is polymorphic, having been cloned and expressed previously with a tyrosine (UGT2B7Y(268)) or a histidine (UGT2B7H(268) at amino acid 268. Questions have been raised concerning the substrate specificity or even the relative reactivity of the two enzyme forms with certain substrates. UGT2B7Y(268) was reported to be active toward menthol and androsterone glucuronide formation by one laboratory, while another has reported that UGT2B7H(268) is inactive with these substrates. In addition, these investigators proposed that individual differences in the expression levels of the two UGT2B7 forms accounts for the variability in the ratio of (S)- to (R)-glucuronides of oxazepam detected in urine and plasma samples.

TABLE 1
Kinetics of glucuronidation with various opioid substrates using membrane
preparations of HK293 cells stably expressing either UGT2B7Y(268) or UGT2B7H(268)
using two different passages of cultured cells.
	UGT2B7Y	UGT2B7H
		Vmax	Efficiency		Vmax	Efficiency
	Km	pmol/min/mg	Vmax/Km	Km	pmol/min/mg	Vmax/Km
Substrate	(μM)	protein	(× 100)	(μM)	protein	(× 100)
morphine, 3-glu	458, 490	5050, 5900	1100, 1200	633, 331	4779, 3054	754, 922
morphine, 6-glu	432, 670	485, 749	112, 111	311, 236	413, 498	132, 211
nalorphine	77, 121	2870, 3650	3700, 3000	141, 171	3990, 4670	2850, 2700
naloxone	50, 40	3750, 2550	7500, 6300	41, 60	3810, 4530	9300, 7550
naltrexone	140, 180	840, 1130	600, 600	63, 200	1000, 1120	1600, 560
hydromorphone	410, 500	1450, 1770	350, 350	390, 360	1500, 1600	380, 450
oxymorphone	1360, 900	5640, 6040	400, 650	540, 670	7510, 8570	1385, 1285
codeine	230, 360	190, 40	83, 11	791, 550	97, 110	12, 20
buprenorphine	3, 1	580, 900	19400	22 ± 6	400 ± 40	1840
S-propanolol	184, 54	70, 140	38, 259	72 ± 19	148 ± 30	205
R-propanolol	180	96	53	101, 72	194, 86	192
androsterone	7.4, 6.1	372, 962	5027, 15874	7.2, 12.2	584, 909	8100, 7443

[0055] To confirm the roles of the two forms of UGT2B7 in the metabolism of drugs and endobiotics, studies on the glucuronidation of opioid compounds, agonists, partial agonists and opioid antagonists using stably expressed UGT2B7Y and H and optimized experimental conditions were performed (Dr. Telphy) (Table 1). This work also demonstrated the role of UGT2B7 in glucuronidation of certain androgenic steroids and xenobiotics including propranolol, and additional compounds not shown in Table I.

[0056] Development of HTS Assay Methods for UGTs.

TABLE 2
Detection of glucuronidation by coupling to the inhibition of a fluorescent β-
glucuronidase reporter reaction.
1. UGI reaction:             1
                             2
2. β-glucuronidase reaction: 3
                             glucuronic acid + 4-methylumbelliferone* (360 nm Ex, 440 nm Em)

[0057] The assay outlined in the above box is described in detail earlier in the application. Because this assay was developed for limited qualitative analysis only, it does not meet all of the requirements for the UGT QSAR proposed. However, it demonstrates the feasibility of using a simple non-radioactive assay in a high-throughput format to detect UGT activity. The assay principle is based on the ability of glucuronides produced in UGT reactions to competitively inhibit a fluorescence based β-glucuronidase reporter reaction.

[0058] After identifying a suitable β-glucuronidase and determining the optimal concentrations of various assay components, the fluorescent assay was tested for its ability to detect β-glucuronides generated by a recombinant UGT enzyme. Products from a UGT1A6 glucuronidation assay inhibited the methylumbelliferyl-β-D-glucuronide cleavage assay and the extent of inhibition was dependent on the incubation period for the UGT1A6 reaction. (The accumulation of β-naphthyl glucuronide in the UGT1A6 reaction was independently verified in a parallel experiment using UDP[14C]-GA). To establish the feasibility of performing the assay in an HTS format, the assay was conducted in a 96 well plate in a homogenous format, i.e., without extracting the glucuronide produced in the UGT reaction prior to starting the β-glucuronidase reaction. Inhibition of the β-glucuronidase reaction was dependent on the presence of recombinant UGT1A6 and on the aglycone substrate β-naphthol. The ability of several diverse glucuronides to inhibit the β-glucuronidase reaction was demonstrated in separate experiments.

[0059] HTS Activity Assay For UGT2B7. To overcome the limitations of existing UGT assay methods, an assay that measures UDP, which is produced via lysis of UDP-glucuronic acid in stoichiometric amounts with the glucuronidated products according to the following reaction is provided.

UDP-glucuronic acid+aglycone→UDP+glucuronide  Equation 1

[0060] Unique advantages of UGT assays based on UDP detection include: a) reliance on the detection of a single product regardless of the UGT isozyme or the aglycone substrate being tested, thus greatly simplifying incorporation into an HTS format, b) direct quantification of reaction rate, thus allowing measurement of the kinetic parameters required for a mechanistic QSAR approach and providing a distinct advantage over assay methods based on competitive inhibition (such as the β-glucuronidase linked method described above), c) ability to use any of several absorbance based methods to detect nucleotides and allow signal detection in a multiwell format with no post reaction separation steps.

[0061] A UGT source for the HTS assays has been selected for both economic and technical reasons. Purified enzyme is required to eliminate interference by extrinsic factors with the QSAR analysis, and a recombinant source is required to provide a consistent source of highly enriched enzyme. While both transfected mammalian cells and BaV-infected insect cells have been used successfully for expression of UGTs, the BaV system offers greater economy and efficiency through lower media costs and easier scale up. The expression levels for UGTs in BaV-infected insect cells generally exceed those in transfected mammalian cells, and the enzymes retain their native kinetic properties. Methods for purification and lipid stabilization have been developed previously for isolation of the enzymes from native sources and are applied to BaV-expressed UGT2B7. Dr. Tephly3 s determinations of kinetic parameters for glucuronidation of opioids, steroids, and other compounds using mammalian cell membranes (Table I) will serve as a benchmark for evaluating the substrate specificity and catalytic efficiency of BaV-expressed UGT2B7 during expression and purification.

[0062] Purification and characterization of human UGT2B7 from baculovirus-infected insect cells. The cDNA for UGT2B7 has been subcloned into the BaV transfer vector pBlueBac (Invitrogen, San Diego, Calif.) and cotransfected with baculovirus DNA into Sf-9 cells. Recombinant plaques were isolated and used to propagate high titer stocks of recombinant virus. The expression methods that are employed are similar to those used successfully for other UGT isozymes, including 1A1, 1A6, 1A7, and 1A10. Parameters to be optimized for expression of UGT2B7 are media conditions, multiplicity of infection (MOI), cell type, and harvest time post infection. The initial approach, at the 100 ml scale, is to infect a serum free-adapted Sf-9 cell line at a high MOI (5-10) in Sf-900-II media (Gibco/BRL, Bethesda, Md.), and monitor expression over a period of five days post infection. After samples are removed, microsomal membrane fractions are prepared (100,000×g pellet) and assayed for the presence of UGT2B7 using androsterone glucuronidation activity (described below), Western blots, and SDS-PAGE. If low levels of expression are obtained in the initial experiments, the effect of media additives such as fetal calf serum and the use of different host cells (T. ni.) are tested. If these efforts yield reasonable expression levels (>1,000 pmol/min/mg), additional small scale runs will be performed to reduce the MOI and thus preserve the virus stock. The optimized shake flask methods then will be scaled up to 10-20 liter stirred bioreactors (B. Braun Biotech, Allentown, Pa.). Generally, this process is straightforward since oxygen availability and shear stress are fairly constant over this range with the culture methods used.

[0063] Microsomal membranes from large scale batches (10-20 liters) of UGT2B7 expressing insect cells will be prepared and tested for activity and native substrate specificity. Some investigators have reported activation of “latent” UGT activity by addition of detergents, consistent with their location in the lumen of the endoplasmic reticulum. In general, we have not found that adding detergents such as CHAPS significantly increases UGT activity with the BaV expressed enzymes. Nonetheless, the effect of detergents on UGT2B7 activity will be tested during the course of expression optimization.

[0064] Following expression optimization, large scale (20-40 liters) batches of BaV infected insect cells will be grown and used as a source for the purified enzyme. Although the UGTs present some purification challenges due to their association with the membrane of the endoplasmic reticulum, methods for detergent solubilization and chromatographic separation have been developed for a number of rat, rabbit and human UGT isozymes isolated from liver tissue.

[0065] Briefly, cells will be lysed by extrusion using a Manton Gaulin Press. The microsomal fraction will be isolated by centrifugation and the membrane bound enzymes solubilized by detergent treatment. Although most UGT purifications described in the literature report the use of Emulgen 911 for solubilization, we also will test Triton N-101 due to its lower expense, greater availability in a more highly purified form, and our successful use in replacing Emulgen 911 for all of the P450 isozymes so far tested. The detergent-solubilized UGT will be purified using an empirically optimized combination of column chromatography steps. Those to be assessed include affinity chromatography (UDP-hexanolamine-Sepharose and omega-(beta-carboxyprionyl-amino)octyl-Sepharose), anion and cation exchange, hydroxylapatite, and chromatofocusing. Most of these resins have been used to purify various UGT isozymes from native sources.

[0066] The enzyme activity will be monitored during the course of the purification using the standard radioassay for androsterone glucuronidation. Purity will be assessed using SDS-PAGE. As with the P450s, the addition of lipid—most frequently phosphatidylcholine—is required to restore activity and maintain stability of purified UGTs. Optimal lipid composition and concentration for each of these purposes will be determined empirically for UGT2B7. Two-dimensional gel electrophoresis also will be used to determine whether multiple glycosylated forms of the UGT are being produced in insect cells. Some UGTs from native sources have been shown to be glycosylated, though no functional significance has been identified for this modification. Nevertheless, elimination of any type of structural heterogeneity is desirable for the intended QSAR analysis. Moreover, we have found that post-translational modifications sometimes can be optimized empirically by altering insect cell culture conditions.

[0067] A panel of aglycone substrates consisting primarily of opioids and steroids similar to those shown in Table I will be used to assess the substrate specificity of the BaV expressed, purified UGT2B7. Glucuronidation activity will be measured using standard methodology: quantification of radioactive glucuronide produced in reactions containing UDP-[14C]-GlcA. Briefly, 100 μl incubations containing aglycone (100 μM-5 mM), purified UGT2B7, 100 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM saccharolactone, 0.1 mg/ml phosphatidylcholine, and 80 μM UDP-[14C]-GA will be incubated at 37° C. for an appropriate period (0.5-2 hours depending upon the substrate). Different post reaction separation methods will be used depending upon the aglycone used. For opioids, the reactions will be quenched with 100 μl pre-chilled ethanol and vortexed. Precipitated protein will be removed by centrifugation. TLC will be used to resolve the glucuronidated products as described, which then will be quantified by scintillation counting following visualization by autoradiography. Reactions containing steroid substrates will be quenched with 100 μl of 0.7M glycine-HCl/1% (v/v) Triton X-100, and the glucuronide product will be extracted with water-saturated ethyl acetate. After vortexing and centrifugation, 200 μl of the organic (upper) phase will be transferred to scintillation cocktail, and radioactivity determined by scintillation counting.

[0068] The specificity of the purified UGT2B7 will be assessed by determining overall catalytic efficiency with each aglycone substrate, as reflected in a ratio of the two fundamental Michaelis kinetic parameters, Vmax/Km. Reaction rates will be determined over a range of substrate concentrations and nonlinear regression will be used to fit the UGT rate data to velocity versus substrate curves (Graph Pad, Prism). Vmax/Km values will be determined and compared with those determined with the enzyme isolated from HK293 cells (Table 1). In addition, the ability of UGT2B7 to glucuronidate morphine at both the 3-OH and 6-OH will be used to assess whether the purified enzyme retains native regioselectivity. In both human liver microsomes and HK293 microsomes expressing UGT2B7, the ratio of approximately 7:1 is observed for the Vmax for the formation of morphine-3-glucuronide vs. morphine-6-glucuronide. This ratio will be determined for the purified enzyme using HPLC-based methods for separation and quantification of the two glucuronides.

[0069] To ensure that the proposed HTS assays fully address the UGT QSAR study requirements and current market needs, the following feasibility goals must be met for production of the purified protein:

[0070] Production of 20 mg batches of UGT2B7 with 90% purity as assessed by Coomassie-blue stained SDS-PAGE.

[0071] Adequate stability (maintenance of 80% of activity) at room temperature for 2 hrs after a single freeze-thaw.

[0072] Substrate specificity similar to the enzyme expressed in HK293 cells as assessed by rank ordering of test substrates based on Vmax/Km values (Table I).

[0073] Development of an HTS UGT activity assay based on UDP detection. The basis of the proposed UGT assay is measurement of UDP formation indirectly by coupling to the apyrase catalyzed production of inorganic phosphate. Phosphate will be detected colorimetrically at concentrations as low as 100 nM (10 pmol) in microtitre plates (100 μl volume) following formation of a complex with molybdate and malachite green. This will be sufficiently sensitive for initial velocity kinetics with the UGTs because very few known aglycone substrates have Km values lower than 10 μM. Thus, detectable levels of product can be formed from subsaturating concentrations of substrate without depleting the pool of substrate to a level that would significantly affect reaction rate.

TABLE 3
Apyrase coupled assay for the colorimetric measurement of UGT activity.
1. UGT reaction:          UDPGA + Aglycone → Glucuronide + UDP
2. Apyrase reaction:      UDP → UMP + Pi
3. Colorimetric reaction: Pi + molybdenum/malachite green → A630 @
                          1 mM = 100

[0074] The protocol for the phosphate-linked assay method is as follows: 1) UGT reaction components, including purified UGT2B7, lipids, buffer, effectors, and UDPGA and apyrase will be precombined and dispensed into microtitre plates; 2) the reactions will be initiated by the addition of aglycone substrates from concentrated stock solutions in DMSO and incubated at predetermined temperatures and time periods; 3) the reactions will be quenched by the addition of the highly acidic molybdate/malachite green color reagent; 4) after a suitable time period (10-30 minutes) for color development, absorbance will be determined using a multiwell plate reader. Standard curves for phosphate are determined and used to convert the absorbance values to nanomoles of glucuronide produced assuming a 1:1 stoichiometry with UDP formation (Equation 1). The simplicity of this method makes it highly useful for the HTS approaches required to adequately survey the structural diversity of UGT substrates.

[0075] UGT reactions are optimized for enzyme amount, buffer and metal composition, lipid composition and concentration, and UDPGA concentration. Apyrase reactions are optimized primarily for the amount of enzyme required to rapidly hydrolyze the minimum amounts of UDP expected in the assay (approx 10 pmol), and tested for inhibition by a number of aglycones from various structural classes. The colorimetric phosphate detection method is optimized primarily for composition of the color reagent (acidity, amounts of molybdenum and malachite green, addition of detergent or organic solvents to increase solubility malachite green) and tested for interference from UGT reaction components and aglycones.

[0076] For the QSAR studies, the assay is capable of quantitatively measuring UGT turnover of structurally diverse aglycones, including both very good and very poor substrates.

[0077] The assay has sufficient sensitivity for kinetic analysis with substrates that are glucuronidated very slowly (low Vmax) or at low concentrations (low Km). The assay is used for qualitative screening of compounds to detect glucuronidation, and for detailed kinetic analyses of those aglycones identified as substrates. Since an excess of enzyme can be used, it should be possible to detect even very poor UGT substrates in the screening mode. Based on the activities obtained with crude cell fractions, and the turnovers for other purified UGTs, purified UGT2B7 is expected to have a Vmax of at least 100 nmol/min/mg with good substrates such as morphine. Thus, using 1 μg of UGT2B7 per assay, and assuming subsaturating substrate concentration and a low Vmax such that the enzyme is operating at a rate of only 1.0 nmol/min/mg, the amount of product generated in 100 minutes is 100 pmol, ten-fold higher than the detection limit of the assay.

[0078] However, there are some special considerations that must be addressed to use the assay in the analytical mode at very low substrate concentrations. As a practical guideline for initial velocity kinetics, it is undesirable to consume more than 10% of the aglycone substrate during the course of the reaction. At the same time, sufficient product is needed to allow detection using the apyrase coupled reaction. Determination of UGT kinetic

Claims

1. A process for detecting metabolite formation, comprising: identifying conjugative enzymes involved in xenobiotic transformations.