MX2007005952A - Fluorescence polarization assay for determining histidine decarboxylase activity - Google Patents

Abstract

A fluorescence polarization assay for determining the HDC modulating activity of a candidate compound comprising the steps of:a) providing a reaction mixture comprising a HDC, histidine, a fluorescently labeled histamine probe, a candidate compound and an anti histidine antibody having selectivity for histamine at least 10 fold greater than histidine;b) incubating the reaction mixture;c) determining whether inhibition of HDC has occured in the presence of the test compound, wherein an increase in fluorescence signal is an indication that the test compound inhibits the activity of HDC.

Description

FLUORESCENCE POLARIZATION TEST TO DETERMINE THE HYSTERINE ACTIVITY DESCARBOXILASA BACKGROUND OF THE INVENTION 1. TECHNICAL FIELD The field of the invention relates to fluorescence polarization assays for detecting HDC activity, which can be used in the diagnosis of diseases and in the identification of HDC inhibitory agents. 2. BACKGROUND INFORMATION Histamine is a potent bioamine with multiple activities in various pathological and physiological states (Jutel, Watanabe T, Akdis, Blaser K, Akdis CA: Immune regulation by histamine, Curr Opin. Immunol 2002; 14: 735-7 0 ). In addition to its well-characterized effects on acute inflammation and allergic responses, histamine regulates several aspects of immune responses with antigen specificity (Schneider E, Rolli-Derkinderen M, Arock M, Dy M: Trends in histamine research: new functions during immune responses and hematopoiesis Trends Immunol 2002; 23: 255-263). Recent findings, such as the discovery of a new histamine (H4) receptor in immunocompetent cells and the demonstration of a role for H1 and H2 receptors in the polarization of helper T cells, have generated much interest in the immunoregulatory mechanisms induced by histamine (Schneider E, Rolli-Derkinderen M, Arock M, Dy M, Trends Immunol, May 2002; 23 (5),: 255-63). Histidine decarboxylase (HDC) is the rate-limiting enzyme in histamine biosynthesis (Watanabe T, Yamatodani A, Maeyama K, Wada H: Pharmacology of a-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase.) Trends Pharmaceutical Sci 1990; 11: 363-367). The mammalian HDC is a member of a large family of pyridoxal 5-phosphate (PLP) -dependent enzymes (Christen P, Menta P: From Cofactor to enzymes, The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes. 2001; 1: 436-447). HDC is expressed in most tissues, but the highest levels are found in the skin, the GI tract and the airways. HDC is a 74 Kd enzyme that becomes a shorter form, 54 Kd (Yatsunami K, Tsuchikawa M, Kamada M, Hori K, Higuchi T: Comparative studies of human recombinant 74- and 54- kDa L-histidine decarboxylase, J. Biol. Chem. 1995; 270: 30813-30817). The two forms are active in vitro, but are not found in the same subcellular compartments; finding the 74 Kd form predominantly in the endoplasmic reticulum (Tanaka S, Nemoto K, Yamamura E, Ohmura S, Ichikawa A: Degradation of the 74 kDa form of l-histidine decarboxylase via the ubiquitin-proteasome pathway in a rat basophilic / mast cell line (RBL-2H3). FEBS Letters 1997; 417: 203-207). The recent generation of HDC-deficient mice provided a good system to study the role of endogenous histamine in a wide range of normal and disease processes (Ohtsu H, Watanabe T: New functions of histamine found in histidine decarboxylase gene knockout mice. Biophys Res Commun 2003; 443-447). HDC_ "mice have a reduced number of mast cells and a reduced content of granular material, such as mast cell proteases (Ohtsu H, Tanaka S, Terui T, Hori Y, Makabe-Kobayashi Y, Pejler G, Tchougounova E, Hellman L , Gertsenstein, Hirasawa N, Sakurai E, Buzas E, Kovacs P, Csaba G, Kittel A, Okada M, Will M, Sea L, Numayama-Tsuruta K, Ishigaki-Suzuki S, Ohuchi K, Ichikawa A, Falus A, Watanabe T, Nagy A: Mice lacking histidine decarboxylase exhibit abnormal mast cells, FEBS 2001; 502: 53-56.) These mice show a reduced airway hyper-response (Kozma GT, Losonczy G, Keszei M, Komlosi Z, Buzas E, Pallinger E, Appel J, Szabo T, Magyar P, Falus A, Szalai C: Histamine deficiency in gene-targeted mice strongly reduces antigen-induced airway hyper-responsiveness, eosinophilia and allergen-specific IgE International Immunol., 2003; 15 : 963-973), reduced vascular permeability (Ohtsu et al., Plasma extravasation induced by dietary supplemented h istamine in histamine-free mice. Eur J Immunol. 2002; 32: 1698-708), reduced skin inflammation (Ghosh AK, Hirasawa N, Ohtsu H, Watanabe T, Ohuchi K: Defective angiogenesis in the inflammatory granulation tissue in histidine decarboxylase-deficient mice but not in mast cell-deficient mice. J. Exp. Med. 2002; 195: 973-982.) And an increase in bone density (Fitzpatrick LA, Buzas E, Gagne TJ, Nagy A, Horvath C, Ferencz V, Mester A, Kari B, Rúa M, Falus A, Barson and J. Targeted deletion of histidine decarboxylase gene in mice increases bone formation and protects against ovariectomy-induced bone loss Proc Nati Acad Sci US 2003, 100 (10): 6027-32). In this way, potent inhibitors of HDC activity could be useful in allergic, inflammatory, immunological, bone and cardiovascular disorders. It has also been shown that histamine is a positive regulator of proliferation in some types of cancers (Hegyesi H, Somlai B, Varga VL, Toth G, Kovacs P, Molnar EL, Laszlo V, Karpati S, Rivera E, Falus A, Darvas Z. Suppression of melanoma cell proliferation by histidine decarboxylase specific antisense oligonucleotides, J Invest Dermatol, 2001 Jul; 117 (1): 151-3). The biological role of histamine has been studied considerably with pharmacological strategies that use specific agonists or antagonists of the histamine receptor. Despite the important role of HDC in allergic and inflammatory responses, very few small molecule inhibitors of this enzyme are known. Most of these inhibitors were discovered by rational design strategies and are analogous of histidine. A well-characterized HDC inhibitor is the irreversible alpha-fluoromethyl histidine inhibitor (Watanabe T, Yamatodani A, Maeyama K, Wada H, Pharmacology of alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase.) Trends Pharmacol Sci. 1990 11: 363-7 ). The ability to identify new classes of HDC inhibitors is limited by the lack of trials that are suitable for HTS. The most commonly used test for measuring HDC activity is based on the o-phthalaldehyde (OPT) method (Roskoski R, Roskoski L: A rapid histidine decarboxylase assay, Analytical Biochem 1978, 87: 293-297). This assay is not selective for histamine with respect to histidine and involves a chromatographic separation of the enzyme product from the substrate. Another more sensitive HDC assay utilizes the conversion of [14C] -labeled histidine to histamine labeled with [14C]. Then thin layer chromatography is used to separate the substrate and the product. Potentially, histamine ELISA kits could be adapted to measure HDC activity. However, these assays require an acetylation step (acetylated histamine) to achieve any useful selectivity and selectivity. In addition, these procedures require many washing steps that make them less susceptible to HTS techniques.

THE INVENTION A fluorescence polarization assay for determining HDC modulation activity of a candidate compound comprises the steps of: a) providing a reaction mixture comprising HDC, histidine, a fluorescence-labeled histamine probe, a candidate compound and an anti-histamine antibody having a selectivity for histamine at least 10 times higher than for histidine; b) incubating the reaction mixture; c) determining whether HDC inhibition has occurred in the presence of the test compound, where an increase in the fluorescence polarization signal is an indication that the test compound inhibits the activity of HDC. In another embodiment of the invention, the anti-histamine antibody has a selectivity for histamine at least 100 times higher than for histidine. In another embodiment of the invention, the reaction mixture is incubated for more than 15 minutes and, more preferably, for a period of between 60 to 120 minutes and even more preferably for a period of between about 80 to 100 minutes. In another embodiment of the invention, a human HDC is used.

In another embodiment of the invention, HDC is a recombinant enzyme or, alternatively, is partially purified. In another embodiment of the invention, the histamine probe has an affinity greater than 1um for the anti-histamine antibody. In another embodiment of the invention, the anti-histamine antibody used is generated by immunizing mice with a histamine linked via a linker region to a support and wherein said linker is structurally homologous to the fluorescein probe. In another embodiment of the invention, the linker region is 1,4-benzoquinone and the support is albumin. In another embodiment of the invention, the fluorescence-labeled histamine probe is chosen from FITC, rhodamine, TAMRA or Cy5. In another embodiment of the invention, the histidine concentration is between 10 μ? at 5 mM and, more preferably, between 100 μ? and 1 mM. Another aspect of the invention provides a method for detecting HDC activity in a sample of a patient as a diagnostic tool for a disease, wherein said method comprises: a) contacting said sample with a reaction mixture comprising an HDC , histidine, a a fluorescence-labeled histamine probe and an anti-histamine antibody having a selectivity for histamine at least 10 times higher than for histidine; b) incubating the reaction mixture; c) determining whether there has been an increase in HDC activity in the patient sample compared to the level of HDC activity in the control sample, where a reduction in the fluorescence polarization signal with respect to the control sample is an indication that the patient's sample is at risk of suffering from the disease. In another embodiment of the invention, the anti-histamine antibody used is generated by immunizing mice with a histamine linked via a linker region to a support, wherein said linker is structurally homologous to the histamine-fluorescein probe. In another embodiment of the invention, the disease is chosen among cancer, asthma and mastocytoma, immunological disorders and gastrointestinal disorders.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A shows the structure of histamine. Figure IB shows the structure of the disodium salt of N- [3 ', 6' -dihydroxy-3-oxospiro [isobenzofuran-1 (3H), 9 '- [9H] xanthene-5 (or 6) -yl] - N '- [2- (lH-imidazol-4-yl) ethyl] -2, 4- dimethylthiourea (FITC-histamine), the probe molecule. Figure 2 shows the binding of FITC-histamine to the anti-histamine antibody. The assay was performed in duplicate in a 96-well plate as described in the Methods section, with FITC-histamine at 6 nM. The data was adjusted in SAS and a Kd of 3.9 nM was determined. Figure 3 shows the displacement of the binding of FITC-histamine to the anti-histamine antibody by histamine (·) or histidine (?). Determinations were made in triplicate in a 96-well plate as described in the Methods section, with 6 nM FITC-histamine, 50 nM anti-histamine antibody and the indicated concentration of competitor ligand. Figure 4A shows the evolution over time of HDC at various enzyme concentrations. Triplicate reactions were initiated in a 384-well plate by the addition of the indicated concentration of HDC, and polarization was determined by fluorescence at time points of 0 to 180 minutes of incubation. The concentrations of probe, substrate and antibody were those described in the conventional assay. Figure 4B. Titration of HDC to 90 minutes. Assays (384 wells) were performed in quadruplicate using the indicated concentration of HDC for 90 minutes at 37 ° C in the Allegro ™ system. The assay window is defined as the difference in mP between the white wells (without enzyme) and the reaction wells. Figure 5 shows the inhibition of HDC by methylester histidine (A), α-fluoromethylhist idine (B) and the dipeptide histidine-phenylalanine (C). The reactions were performed using the conventional conditions described in the Methods section and the indicated concentration of inhibitor. The IC50 values were obtained by adjusting the data through the use of XLFit4 (IDBS Software). The error bars show the mean ± S >; D of determinations in triplicate. Figure 6 shows the scatter plot of the white (·) and positive control (?) Values from a single-day screening test of 90 plates (384 wells).

DETAILED DESCRIPTION OF THE INVENTION Histamine probe labeled with fluorescent. The present invention provides a fluorescent probe. The preferred probe is FITC-Histamine (Thiourea, N- [3 ', 6' dihydroxy-3-oxospiro [isobenzofuran-1 (3H), 9 '- [9H] xanthene-5 (or 6-il) -N'- [2- (1H-imidazol-4-yl) ethyl] -2, -dimethyl-, disodium salt) obtainable from Molecular Probes (Eugene, OR). Other suitable probes include histamine labeled with rhodamine, TAMRA or Cy5.

Anti-histamine antibodies. The present invention provides an assay using anti-histamine antibodies. A suitable monoclonal antibody that can be used is the antibody against histamine D22.12, which can be obtained from Argene (Varilhes, France). Antibody D22.12 was generated by immunizing mice with albumin-coupled histaminyl-1-benzoquinone (Guesdon JL, Chevrier D, Mazie JC, David B, Avrameas S: onoclonal anti-histamine antibody.) Preparation, characterization and application to enzyme immunoassay of histamine, J. Immunol. ethods 1986; 87: 69-78), while all other antibodies that were tested were generated by immunization with histamine or acetylated histamine coupled to albumin. The high binding affinity of D22.12 for histamine-fluorescein could be due to a structural homology between the immunogen used to obtain D22.12 (histaminyl benzoquinone) and the histamine-fluorescein probe. Other anti-histamine antibodies suitable for use in the invention could be generated using immunogens with similar linkers that also have structural homology to the histamine probe. Different anti-histamine antibodies can be used depending on the fluorophore used as a probe.

Histidine can be obtained from Sigma Chemical Co.

(St. Louis, MO).

HDC Enzyme Source a) Recombinant - In the preferred embodiment of the invention, the human HDC enzyme is used. It is contemplated that the whole length protein or a truncated form such as the 53 Kd form is used, provided the truncated form retains the histidine decarboxylase activity. Preferably, the human HDC protein (SEQ ID NO: 1) or a fragment thereof is used. The HDC protein can also be fused to protein signals, such as glutathione S-transferase (GST), to facilitate purification. b) Purified - The method of the invention can be practiced using a purified HDC enzyme. As used in the text of this document, the term "partially purified" is understood to include an HDC enzyme that has been partially purified to a greater extent than the HDC enzyme as it would be found in the human cell. Purification procedures for HDC are known in the art and are taught by Watabe A, Fukui T, Ohmori E, Ichikawa A: Purification and properties of L-histidine decarboxylase from mouse stomach. Biochem. Pharmacol. 1992; 43: 587-593 whose contents are incorporated in this document.

Conventional assay for detecting inhibitors In the conventional assay, HDC, diluted in an HDC buffer containing a reducing agent such as DTT and the enzymatic cofactor PLP, is added to a sample plate. To the plate is added test compound in HDC Buffer plus an adequate amount of D SO or buffer alone. They combine marked histamine with fluorescence and histidine in FP Buffer and the mixture is transferred to the plate in 10 μ ?. Finally, 90 nM anti-histamine antibody is added in 20 μ? of FP Buffer. In this manner, the final concentrations in the assay are: HDC 25 at 50 nM, FITC-histidine 3 at 6 nM, histidine 300 at 600 uM, anti-histamine antibody 25 at 50 nM, and DMSO at 1 to 5%. The plate is then incubated at 37 ° C for at least 15 minutes. The fluorescence polarization signal is read on an instrument suitable for fluorescence polarization reading such as LJL Analyst (Molecular Devices, Sunnyvale, CA) with excitation at 485 nm, emission at 530 nm, a dichroic fluorescein mirror at 505 nm and the G factor set to 1. A reduction in the fluorescence signal is an indication that the test compound inhibits the activity of HDC.

Modifications of the conventional method for detecting candidate inhibitors- Optimization of parameters The present invention provides an HDC assay for determining the modulating activity of HDC of a candidate compound and a diagnostic method for determining HDC levels in patient samples. It is contemplated that one skilled in the art may practice the invention in a manner in which various parameters may be altered to adapt the assay for its own use. Some of the parameters which can be altered include: anti-histamine antibody concentration, histidine substrate concentration, probe concentration, source and concentration of HDC enzyme, concentration of test compound, order of component additions, volume of the individual components, the total reaction volume, the addition of a pre-incubation step of HDC with the test compound before the reaction, the duration of the reaction, the temperature of the reaction, the type of test plate , a cooling or heating step, the addition of a step to stop the enzymatic reaction (for example: acid, base, salt, known HDC inhibitors or the like), the type of instrument and related parameters used to read the bias signal by fluorescence.

Selectivity of the fluorescent probe for its target receptor molecule. In one embodiment of the invention, the selectivity of the anti-histamine antibody for histamine to histidine is greater than 100X. It would be expected that a suitable candidate inhibitor would have an IC50 of < 10 μp ?.

Conventional assay for diagnosing diseases Methods for extracting and preparing tissues or serum from normal or standard samples are known in the art. sick to measure HDC activity. For example: (1) 1- Sieja K, Stanosz S, von Mach-Szczypinski J, Olewniczak S, Stanosz M. Concentration of histamine in serum and tissues of the primary ductal breast cancers in women. Breast. 2005 Jun; 14 (3): 236-41; (2) E. Masini, V. Fabbroni, L. Giannini, A. Vannaccil, L. Messerini, F. Perna, C. Cortesini and F. Cianchi Histamine and histidine decarboxylase up-regulation in colorectal cancer: correlation with tumor stage Inflamm. beef. 54, Supple ent 1 (2005) S80-S81; (3) 3- Fogel WA, Dudkowska M, Wagner W, Grzelakowska-Sztabert B, Manteuffel-Cymborowska M. Ornithine and histidine decarboxylase: activities in hypertrophic and hyperplastic mouse kidney. Inflamm Res. 2005 Apr; 54 Suppl 1: S62-3. These methods could be applied to prepare samples for testing in the fluorescent HDC assay described herein. The method of the present allows HDC activity to be assayed with high performance in samples of blood, plasma or normal tissue or target diseased.

EXAMPLES Reagents FITC-Histamine was obtained (Thiourea, N- [3 ', 6' dihydroxy-3-oxospiro [isobenzofuran-1 (3H), 9 '- [H] xanthene-5 (or 6-il) -N'- [2- (1H-imidazol-4-yl) ethyl] -2, -dimethyl-, disodium salt) in Molecular Probes (Eugene, OR). The monoclonal antibody of histamine 22.12 was obtained in Argene (Varilhes, France). L-Histidine, histamine, potassium phosphate (1M mono- and dibasic solutions), polyethylene glycol of molecular weight 400, ethylene glycol tetraacetic acid (EGTA), dithiothreitol, pyridoxal-5-phosphate and sodium chloride were from Sigma Chemical Co. (St. Louis, MO). 3- [(3-Colamidopropyl) dimethylammonium] -1-propanesulfonate (CHAPS) was obtained from Pierce Chemical Co. (Rockford, IL). The dimethylsulfoxide was from Baker Chemical Corp. The 384-well plates of black opaque polystyrene were obtained from Corning-Costar. The known HDC inhibitors, histidine-methyl ester and His-Phe, were from Sigma Chemical Co. and alpha-fluoromethylhistidine was obtained from the Boehringer Ingelheim Pharmaceuticals compound library.

Buffers The HDC buffer comprises 200 mM potassium phosphate (pH 6.8), 2% PEG-400, 0.2 mM EGTA, 0.03% CHAPS. The FP buffer is 16.6 mM Tris-HCl (pH 7.5) and 50 mM NaCl.

HPLC Determination of Histamine Content HPLC separations were performed on an Agilent 1090M equipped with a diode array detector. A Delta-Pak HPI C4 300A column, 2.0 x 150 MI (Waters) was used. The mobile phase consisted of 20 ml of Low UV Reagent PIC® B-8 (Waters) in 1000 ml of 10 mM triethylamine phosphate, pH 3.0.

All separations were carried out at room temperature (22 ° C) with a flow rate of 0.2 ml / min and were controlled at a wavelength of 215 nm. Histamine concentrations were calculated using standard curves for the concentration versus area response. The standard curves were generated using duplicate injections of histamine in control buffer at concentrations of 0 to 200 or 600 μ? . Six equally spaced concentrations were used. The standard curves were calculated for the concentration response in μ? versus area using linear regression on a TI-68 calculator. The correlation coefficients were greater than 0.999.

Cloning of human HDC The cDNA corresponding to the truncated form of 53 Kd of the full-length human HDC (accession number: NM_002112) was amplified by PCR using total RNA from the human mast cell line HMC-1 (Maeda K, Taniguchi H , Ohno I, Ohtsu H, Yamauchi K, Sakurai E, Tanno Y, Butterfield JH, Watanabe T, Shirato K: Induction of L-histidine decarboxylase in a human mast cell line, HMC-1, Exp Hematol., 1998; 26: 325 -31.). The primers used were: 5 '-atgatggagcctgaggagtacagag and 3'-acactactgactcaggatgagagt. The HDC cDNA (1.5 Kb) was cloned into the pcDNA4.1 vector. The clone pcDNA .1-HDC was used as a template to obtain a PCR product that contained the first 1431 bases (amino acid residues 1-477) of HDC and which incorporated a 5 'thrombin cleavage site and adjacent to the first base. This PCR product was cloned into pDEST 20 using Gateway cloning technology (Invitrogen Life Technologies) following the manufacturer's protocols. The purified DNA sequence of the final expression clone was confirmed and then used to transform E. coli DHlOBac cells for transposition in the bacmid. Recombinant bacmid DNA was purified from individual colonies and transposition was verified by PCR analysis.

Expression in baculovirus and purification of GST-HDC A volume of 20 L of SF900II-SFM (Invitrogen Cat. No. 10902-088) was sterile filtered in 750 L MBR stirred tank bioreactors. The MBR bioreactor it was equipped with pH, dissolved oxygen and temperature probes and the established control points were: pH 6.2, OD 50%, Temp 27 ° C, RPM 110. Four 1 L shaking flasks of Sf9 cells were cultured to a cell density comprised between 2.5xl06 and 3xl06 cells / mL and were used to inoculate the bioreactor, resulting in 24 L of a medium with an approximate cell density of 4 xlO5 at 5xl05 cells / mL. Samples were taken daily from the inoculated bioreactor to determine the cell density, viability and diameter of the cells using a Cedex cell counter (Innovatious).

A nutrient (glucose, glutamine) and residue (ammonia) analysis was also performed daily using a Bioprofile 100 analyzer (Nova Biomedical). Twenty-four hours after the initial inoculation of the cells, the bioreactor was infected with GST-HDC baculovirus to achieve an MOI (multiplicity of infection) of 0.1. A 1.5 mL sample of cell supernatant was collected (centrifuged, decanted and frozen) before infection and every 24 hours until the assay was collected for analysis by SDS-PAGE and WESTERN. Twenty-four hours after infection, 25 mg of Leupeptin were dissolved in an SF900II-SFM medium, sterilized by filtration and injected into the bioreactor. The collections were made 48 hours after infection. The infected cells were centrifuged in a 12 L centrifuge (Sorvall BP12) @ 3000 rpm, 4 ° C for 10 min by centrifugation. The pellets were combined in a centrifuge bottle and subjected to a final centrifugation at 3500 rpm for 10 minutes, at 4 ° C. The sediments were weighed and frozen at -80 ° C until use. The final sediment yield was 300 grams. For protein purification, all buffers were prepared in distilled and deionized water and all procedures were performed at 4 ° C. The cell pellet was mixed with lysis buffer (20 mM Hepes, pH 7.5, 150 mM KCl, 10% glycerol, 2 mM DTT, 1 mM EGTA, cocktail tablets of Roche protease inhibitor, and 0.01 mM PLP) at a ratio of 5 ml / g of cell pellet. The cells were homogenized on ice using PolyTron PT 2100 (Kinematica AG, Switzerland), and then sonicated 3 times for 5 minutes with a Branson 450 sonicator (Converter, USA) at a 50% duty cycle. The cell lysate was centrifuged at 18,600 g for 30 min followed by 225,071 g for 60 min. The clarified lysate was loaded directly onto a 50 mL Glutathione Sepharose 4B column (Amersham, Sweden) using an AKTA Prime chromatography system (Amersham, Sweden). After loading, the column was washed with 4 column volumes (CV) of wash buffer (20 mM Hepes, pH 7.5, 150 mM KC1, 10% Glycerol, 2 mM DTT, 0.5 mM EDTA, 0.5 mM PMSF and 0.01 mM PLP), and then eluted with 10 CV of elution buffer (20 mM reduced GTH, 2 mM DTT and wash buffer, pH 8.0). The HDC product eluting from the column was pooled according to a visual inspection of an SDS-PAGE gel. The conventional yield was 33 mL of HDC at 1.8 mg / mL. The pooled product was dialyzed into 5 L of buffer 1 (20 mM Hepes, pH 7.5, 0.1 mM EGTA, 0.2 mM PMSF, 0.25 mM DTT, 10% Glycerol, and 0.01 mM PLP) for 4 hours, and then dialyzed to pH 5. L of buffer 2 (0.1 M Phosphate, pH 7.5, 2% PEG-400, 0.1 mM EGTA, 2 mM PMSF, 10% Glycerol and 0.02 mM PLP) for 17 hours. Aliquots of the final product were collected, immediately frozen in liquid nitrogen and stored at -80 ° C.

Histidine Decarboxylase Assay In the conventional assay, HDC, diluted to 90 nM in HDC Buffer plus 0.9 mM DTT and 99 μ M PLP, was added to a black opaque 384-well plate in 20 μ?,. Test compound in HDC Buffer plus 6% DMSO or Buffer alone in an amount of 10 μ ?? was added to the assay plate. FITC-histidine 36 nM and 3.6 mM histidine were combined in FP Buffer and the mixture was transferred to the plate in an amount of 10 μ? . Finally, 90 nM anti-histidine antibody was added in 20 μ ?, of FP Buffer. In this way, the final concentrations in the assay were: 30 nM HDC, 6 nM FITC-histidine, 600 μ histidine, 30 nM anti-histamine antibody, 1% DMSO. The plate was incubated at 37 ° C for 90 minutes. The fluorescence polarization signal was read on an LJL Analyst (Molecular Devices, Sunnyvale, CA) with excitation at 485 nm, emission at 530 nm, a dichroic fluorescein mirror at 505 nm and the G factor set at 1. A version of the 96-well plate assay, used in the development of the assay, as described above with volumes twice as large as those indicated for the 384-well plates.

High Performance Selection The assay was automated in a Zymark Allegro ™ robotic system (Caliper-Zymark, Hopkinton, MA), using a Multidrop to add enzyme, Sciclone to add substrate / probe and Test compound, and a Multidrop to add the antibody. The plates were incubated at 37 ° C in a humidified medium and the fluorescence polarization was read in an LJL Analyst integrated in the Allegro system using the parameters described above. The compounds were selected at a concentration of 5 μg / mL. The POC values were calculated with respect to a blank containing complete reaction minus HDC and a 100% control containing HDC buffer with 1% DMSO instead of compound.

RESULTS The product structure of the HDC enzyme (histamine) and the histamine probe (FITC-histamine) are shown in FIGURE 1. The interaction of several commercial anti-histamine antibodies with the FITC-histamine probe is first examined. (data not revealed). One of these antibodies (D22.12) showed a strong binding to the probe measured by fluorescence polarization and was retained for further characterization. In the first place, the affinity of the probe for the antibody and the specificity of the interaction were determined, these two characteristics being crucial for constructing a solid assay in a competitive manner. FIGURE 2 shows a probe and antibody binding curve determined by anisotropy measurement. The concentration of the probe was maintained at 6 nM and the antibody concentration varied by approximately 3 logarithmic units. A dissociation constant of 3.9 nM was determined after the adjustment of the data. The specificity of the antibody for histamine with respect to histidine was demonstrated as shown in FIGURE 3. The concentrations of probe and antibody were kept constant at 6 and 50 nM, respectively, while the concentrations of histamine and histidine varied as sample. Histidine could not compete with FITC-histamine for binding to the antibody in the range of 5 logarithmic units examined. However, histamine freely competed with the probe for antibody binding, producing an IC50 value of 135 μ ?. In this way, the strong and specific binding of FITC-histamine to the monoclonal antibody histamine and the ability to compete for that binding with the product of the enzymatic reaction suggests that it is possible to create a competitive competitive FP assay for HDC. FIGURE 4A shows a time course of the enzymatic reaction at various concentrations of HDC. Histidine, at 600 uM, had a value of Km approximately twice greater than the indicated Km value of 200-400 uM (atabe et al 1992). At concentrations of HDC > 100 nM, the reaction is linear only for about 30 minutes. In order to balance the size of the assay window with the requirements of the enzyme for a large-scale selection and linearity of the reaction, it was decided to use HDC 25-50 nM and a time of 90 minute incubation in the conventional assay. This was further refined in FIGURE 4B, which shows a HDC titration to a 90 minute incubation performed in the Allegro robotic system. In this experiment an enzyme concentration of 30 nM was reached. In this manner, final assay conditions of 30 nM HDC, 30 nM antibody and 6 nM FITC-histamine were set in a total volume of 60 μ? for 90 minutes at 37 ° C. The concentration of PLP was set at 33 μ? to maintain the saturation of the enzyme. FIGURE 5 shows the behavior of 3 known HDC inhibitors: histidine methyl ester, α-fluoromethyl histidine and the dipeptide histidine-phenylalanine in the conventional assay. IC50 values of 7.7 and M, 1.4 uM and 228.1 uM were determined for the 3 compounds, respectively. These values are in agreement with those obtained using the HPLC assay. The described assay was then used to select compounds from a library at a final concentration of 5 ng / mL. 384-well plates were prepared to contain 352 wells of compound, 16 wells of control (without compound) and 16 wells white (without enzyme) per plate. The compounds, in pure DMSO, were diluted in buffer to give a final concentration of DMSO of 1% in the assay. It was shown that this concentration of DMSO had no effect on enzyme activity or stability (data not shown). He The test was further automated in the Allegro robotic system, allowing yields of approximately 100 plates per day. FIGURE 6 shows a scatter chart of the blank and control wells for a single screening test of 90 plates, with a mean value of Z 'of 0.6 and a test window of 80-100 mP. In the trial, more than 600,000 compounds were selected with a confirmed success rate of 0.05% using 60% of the control as a criterion for correct answers. Confirmed hits were then tested on a 10-point dose response curve to assess power.

DISCUSSION An important parameter that contributes to the performance of an FP assay is the affinity of the fluorescent probe for its target molecule or receptor. As a rule, the Kd for the binding of the probe to its receptor is inversely proportional to the bound fraction. In this way, a high affinity binding allows an optimal stoichiometry of fluorescent ligand / receptor and a solid FP signal. Multiple anti-histamine antibodies were selected to find one that had an adequate affinity for the histamine-fluorescein probe of the present invention. Only one of these antibodies, D22.12, had sufficient affinity for the development of an FP assay. Antibody D22.12 was generated by immunizing mice with coupled 2-histaminyl-l, 4-benzoquinone to albumin (Guesdon et al., 1986) while all other antibodies that were tested were generated by immunization with histamine or acetylated histamine coupled to albumin. The high binding affinity of D22.12 for histamine-fluorescein could be due to a structural homology between the immunogen used to obtain D22.12 (histaminil benzoquinone) and the histamine-fluorescein probe. The Km of HDC (54 Kd form) by its histidine substrate is 275 μ? (data not revealed) . Thus, an important requirement for the development of an HDC assay is histamine selectivity with respect to histidine. The FP assay of the present invention shows a more than 100 fold greater selectivity for histamine compared to histidine. However, as shown in Figure 3, the histidine concentration must be maintained below 2 mM due to a non-specific increase in the FP signal. This limitation should not be a problem in most applications to measure HDC activity since the Km of the enzyme is much less than the maximum amount of histidine tolerated by the assay. Decarboxylases are a large family of enzymes that play important physiological roles (Christen et al, 2001). For example, DOPA decarboxylase is responsible for the synthesis of the key neurotransmitters dopamine and serotonin through the decarboxylation of L-3,4- dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan respectively. Current methods used to measure transmitters such as serotonin and dopamine are analogous to histamine detection techniques. In this way, the assay described herein for HDC could be applied to related enzymes such as dopa decarboxylase and allow the development of new inhibitors with better pharmacological characteristics.

Claims (19)

1. CLAIMS 1. Fluorescence polarization assay for determining HDC modulation activity of a candidate compound characterized in that it comprises the steps of: a) providing a reaction mixture comprising a HDC, histidine, a fluorescence-labeled histamine probe, a candidate compound and an anti-histamine antibody having a histamine selectivity at least 10 times higher than by histidine; b) incubating the reaction mixture; c) determining whether HDC inhibition has occurred in the presence of the test compound, where an increase in the fluorescence polarization signal is an indication that the test compound inhibits the activity of HDC.
2. 2. Test according to claim 1, characterized in that the anti-histamine antibody has a selectivity for histamine at least 100 times higher than for histidine.
3. 3. Fluorescence polarization assay according to claim 1, characterized in that the reaction mixture is incubated for more than 15 minutes.
4. 4. Fluorescence polarization assay according to claim 3, characterized in that the reaction mixture is incubated for a period of between 60 and 120 minutes.
5. 5. Fluorescence polarization assay of according to claim 4, characterized in that the reaction mixture is incubated for a period of about 80 to 100 minutes.
6. 6. Method according to claim 1, characterized in that the HDC is the polypeptide of SEQ. FROM IDENT. DO NOT. : 1. Method according to claim 1, characterized in that the HDC is a recombinant enzyme. 8. Method according to claim 1, characterized in that the HDC is partially purified. 9. Method according to claim 1, characterized in that the histamine probe has an affinity greater than 1 μp? for the anti-histamine antibody. Method according to claim 1, characterized in that the anti-histamine antibody used is generated by immunizing mice with a histamine linked by a linker region to a support and wherein said linker is structurally homologous to the fluorescein probe. 11. Method according to claim 10, characterized in that the linker region is 1, -benzoquinone coupled to a support. Method according to claim 10, characterized in that said support is albumin. Method according to claim 1, characterized in that the histamine probe labeled with Fluorescence is chosen from FITC, rhodamine, tetramethylrhodamine and Cy5. 14. Method according to claim 1, characterized in that the histidine concentration is between 10 μ? and 5 mM. 15. Method according to claim 10, characterized in that the histidine concentration is between 100 μ? and 1 mM. 16. Method for detecting HDC activity in the sample of a patient as a diagnostic tool for a disease, characterized in that said method comprises: a) contacting said sample with a reaction mixture comprising histidine, a histamine probe labeled with fluorescence and an anti-histamine antibody having a selectivity for histamine at least 10 times higher than for histidine; b) incubating the reaction mixture; c) determining whether there has been an increase in HDC activity in the patient sample compared to the level of HDC activity in the control sample, where a reduction in fluorescence polarization with respect to the control sample is an indication of that the patient is at risk of suffering from the disease. Method according to claim 16, characterized in that the anti-histamine antibody used is generated by immunizing mice with a histamine bound by a linker region to a support, wherein said linker is structurally homologous to the histamine-fluorescein probe. Method according to claim 16, characterized in that the disease is selected from cancer, asthma, mast cell carcinoma, immunological disorders, bone disorders and gastrointestinal disorders. 19. Method according to claim 18, characterized in that the disease is cancer.