WO2013112358A1

WO2013112358A1 - Luciferase-linked methods for detecting adenosine and uses thereof

Info

Publication number: WO2013112358A1
Application number: PCT/US2013/022015
Authority: WO
Inventors: Emmanuel S. BURGOS; Vern L. Schramm
Original assignee: Albert Einstein College Of Medicine Of Yeshiva University
Priority date: 2012-01-23
Filing date: 2013-01-18
Publication date: 2013-08-01

Abstract

Luciferase-linked methods for detecting adenosine and for screening for inhibitors of enzymes that produce adenosine are disclosed. The invention also provides methods for detecting the presence of Sadenosyl-L-homocysteine hydrolase (SAHH) comprising: (a) hydrolysis of S-adenosyl-Lhomocysteine (SAH) catalyzed by S-adenosyl-L-homocysteine hydrolase (SAHH) to adenosine and L-homocysteine; (b) converting adenosine to adenosine monophosphate (AMP) by adenosine kinase in the presence of a phosphate donor; (c) converting AMP to adenosine 5 '-triphosphate (ATP) by pyruvate phosphate dikinase (PPDK) in the presence of phosphoenolpyruvate (PEP) and inorganic pyrophosphate; and (d) reacting A TP with luciferin and Oz in the presence of luciferase to produce AMP and a luminescent signal, wherein the luminescent signal indicates the presence of S-adenosyl-L-homocysteine hydrolase (SAHH).

Description

LUCIFERASE-LINKED METHODS FOR DETECTING ADENOSINE

AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application priority of U.S. Provisional Patent Application No. 61/589,552, filed January 23, 2012, the content of which is herein incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers GM41916 and CA135405 awarded by the National Institutes of Health, U.S. Department of Health and Human Services. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates to luciferase-linked methods for detecting adenosine and for screening for inhibitors of enzymes that produce adenosine.

BACKGROUND OF THE INVENTION

[0004] Throughout this application various publications are referred to by superscripts. Citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.

[0005] S-Adenosyl-L-homocysteine hydrolase (SAHH) is the sole enzyme responsible for the catabolism of S-adenosyl-L-homocysteine (SAH) in mammals; it catalyzes the hydrolysis of SAH to adenosine (ADO) and L-homocysteine (Hey).¹ Although the reaction is reversible in vitro, the occurrence of adenosine kinase (AK) and adenosine deaminase (ADA) in cells shifts the chemical equilibrium away from SAH synthesis.² SAH is both a product of biomethylation reactions and also a strong inhibitor. The selective inhibition of SAHH may promote indirect inhibition of the S- adenosylmethionine mediated transmethylations.³'⁴ Mechanistic and structural studies have led to an improved understanding of the enzyme,^5"9 the design of immunosuppressive and anti-inflammatory agents,^10"12 the development of new therapeutics with anti-cancer,^13"15 and cholesterol-lowering effects.^16"18 More recently, SAHH inhibition has been a focus of anti-parasitic studies.^19"21 [0006] Neplanocin A (ΓΝΗ) and its structural analogues display nanomolar affinity toward SAHH in vitro (Fig. 1). However, in vivo, adenosine analogues are often substrates for ADA and AK enzymes, and the formation of neplanocin D and phosphorylated neplanocin A (Fig. 1) explains the high cytotoxicity of these drugs independent of their SAH inhibition.^22"24 Although new molecules with different scaffolds are known (e.g. ilimaquinone, D-eritadenine; Fig. 1), there is a need for more specific and powerful SAHH inhibitors.^25"28 Rapid and sensitive assays may permit the identification of such compounds by evaluating hits from computational docking, screening chemical libraries or fragment-based design.

[0007] Current methods to monitor SAHH activity include 1) the detection of ADO formed during the hydrolysis reaction via UV absorbance (coupling with ADA; 8265 = 7760 M^"1 cm^"1),¹ 2) the use of radiolabeled substrates combined with the isolation/separation of their corresponding products by resins or HPLC,²⁹'³⁰ and 3) the detection of Hey with Ellman's reagent (5-thio-2-nitrobenzoic acid, DTNB; 8412 = 13700 M^"1 cm^"1) or more recently the use of fluorosurfactant-capped gold nanoparticules.³¹'³²

[0008] Adenosine is also a critical metabolite in human metabolism through its interaction with adenosine receptors.⁴⁴ There are four know adenosine receptor subtypes (Al, A2A, A2B and A3). Adenosine receptors have been identified as therapeutic targets in human conditions as diverse as hypertension, brain and heart ischemia, sleep disorders, inflammatory disorders and cancer. Adenosine is also an inhibitory neurotransmitter. Detection of adenosine in biological samples is therefore important in research and diagnosis of disorders related to its receptor function.

[0009] The present invention addresses the need for an improved method for detecting adenosine, which is applicable to any reaction in which adenosine is produced such as, for example, an assay for S-adenosyl-L-homocysteine hydrolase (SAHH), as well as a need for an improved method for screening for inhibitors of enzymes that form adenosine.

SUMMARY OF THE INVENTION

[0010] The present invention provides methods of detecting the presence of adenosine or an enzyme that forms adenosine as a product comprising: (a) converting adenosine to adenosine monophosphate (AMP) by adenosine kinase in the presence of a phosphate donor; (b) converting AMP to adenosine 5 '-triphosphate (ATP) by pyruvate phosphate dikinase (PPDK) in the presence of phosphoenolpyruvate (PEP) and inorganic pyrophosphate; and (c) reacting ATP with luciferin and 02 in the presence of luciferase to produce AMP and a luminescent signal, wherein the luminescent signal indicates the presence of adenosine or an enzyme that forms adenosine as a product.

[0011] The invention also provides methods for detecting the presence of S- adenosyl-L-homocysteine hydrolase (SAHH) comprising: (a) hydrolysis of S-adenosyl-L- homocysteine (SAH) catalyzed by S-adenosyl-L-homocysteine hydrolase (SAHH) to adenosine and L-homocysteine; (b) converting adenosine to adenosine monophosphate (AMP) by adenosine kinase in the presence of a phosphate donor; (c) converting AMP to adenosine 5 '-triphosphate (ATP) by pyruvate phosphate dikinase (PPDK) in the presence of phosphoenolpyruvate (PEP) and inorganic pyrophosphate; and (d) reacting ATP with luciferin and (¾ in the presence of luciferase to produce AMP and a luminescent signal, wherein the luminescent signal indicates the presence of S-adenosyl-L-homocysteine hydrolase (SAHH).

[0012] The invention further provides methods of determining whether or not a compound is an inhibitor of S-adenosyl-L-homocysteine hydrolase (SAHH) or other enzymes that form adenosine as a product.

BRIEF DESCRIPTION OF THE FIGURES

[0013] Figure 1. SAHH substrate and inhibitors. Neplanocin A (ΓΝΗ) is a 'suicide' inhibitor with a tight affinity for the hydrolase enzyme. However, in vivo, this drug loses its inhibitory power upon phosphorylation or deamination by adenosine kinase (AK) or adenosine deaminase (ADA). In the later case, neplanocin D is formed. New classes of inhibitors include D-eritadenine, initially isolated from shiitake mushrooms (Lentinula edodes), exhibits a low nanomolar K{ toward HsSAHH while (-)-ilimaquinone (Smenospongia sp.) or 5'-deoxy-5'-amino-P-D-adenosine (ΓΝΗ-1) display low micromolar affinity with the same enzyme. Note the resemblances with the «S-adenosyl-L- homocysteine substrate (SAH).

[0014] Figure 2. The coupled luciferase-based assay for detection of ADO released during hydrolysis of SAH as catalyzed by SAHH. Abbreviations are provided in the text.

[0015] Figure 3A-3C. Adenosine detection and analysis of SAHH activity using the coupled assay. (A) ADO calibration. Using PPDK (100 U L^"1) and ^i AK (6 U L^"1), ADO is rapidly converted to light. This assay displays a broad dynamic range for ADO (1-80 pmol). (B) Detection limits for the luciferase-coupled assay. The procedure allows ultra-sensitive detection of SAHH activity (10^~7 unit per well). (C) Application of the luciferase assay to the determination of human SAHH kinetic parameters. The enzyme displays a K_m of 22 ± 2 μΜ and a k_cat of 0.075 ± 0.006 s^"1.

[0016] Figure 4A-4B. Ki evaluation for D-eritadenine. (A) Light output profile monitored for 60 minutes at 80, 40, 20, 10, 5 and 2.5 nM of inhibitor. (B) D-eritadenine displays slow-onset inhibition with a K* of 1.28 ± 0.05 nM (solid trace).

[0017] Figure 5A-5B. Evaluation and validation of the SAHH coupled assay for high throughput screening. (A) Luminescence recording for samples (s; 20 μΜ SAH and buffer B₂ supplied with 3.2 nM hydrolase) and controls (c; same as s but without HsSAHH); 16 repeats for each 'samples' and 'controls' sets were run. The assay displays an extremely low background. (B) Determination of the screening window coefficient Z'. Representation of the mean for s and c (plain lines) and their corresponding data variability band (dashed lines); Z' = 0.92.

[0018] Figure 6. Effect of DMSO on the reaction catalyzed by SAHH and on the luciferase assay components.

[0019] Figure 7A-7C. Ki evaluation for 5'-deoxy-5'-amino- ?-D-adenosine (ΓΝΗ- 1). A) Light output profile monitored for 20 minutes at 20, 10, 4, 2 and 0.8 μΜ of ΓΝΗ-1. B) ΓΝΗ-1 displays a slight slow-onset inhibition (dashed trace) with a Ki* of 2.17 ± 0.08 μΜ. C) Assay control for molecules displaying micromolar Kis. At the end of the inhibition experiment with ΓΝΗ-1 (20 μΜ), ADO was added to a 50 μΜ final concentration and luminescence was monitored; the coupling enzymes AK/PPDK/FLUC are not inhibited by high concentrations of ΓΝΗ-1 (diamonds signs).

[0020] Figure 8A-8B. The phopshorylation of neplanocin A by AgAK and its implications for screening with the luciferase-based assay. A) HPLC analysis of species encountered during the assay. ADO and GTP (Trace a) are converted to AMP and GDP (Trace b) upon addition of AgAK; starting materiel SAH, substrate of SAHH (Trace c) and neplanocin A (Trace d); according to the production of GDP, neplanocin A (ΓΝΗ) is a substrate of AgAK and is phosphorylated into ΓΝΗ-Ρ (Trace e). B) Under conditions where AgAK is omitted (left panel), ΓΝΗ (6 μΜ) fully inhibits the production of ADO by SAHH (100 nM; solid trace, round signs) in the presence of SAH (20 μΜ). However, ΓΝΗ is fully phosphorylated when the kinase is added to the enzyme mixture (30 mU mL^" ¹ final concentration) and there was no inhibition observed (right panel). The AMP production for the control (no inhibitor; dashed line, square signs) and the measure experiments (with inhibitor; solid line, round signs) are nearly identical.

[0021] Figure 9. Effect of DMSO on the chemiluminescence output. A new ADO calibration was established in the presence of DMSO (1.75%, v/v) using the typical experimental conditions; the linear response display the same sensitivity as in the calibration experiment where DMSO was omitted (12792 RLU pmol^"1 vs. 12570 RLU pmol^"1). Thus, the coupling enzymes AK/PPDK/FLUC are not affected by this organic solvent.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The invention provides a method for detecting the presence of adenosine or an enzyme that forms adenosine as a product comprising:

(a) converting adenosine to adenosine monophosphate (AMP) by adenosine kinase in the presence of a phosphate donor;

(b) converting AMP to adenosine 5 '-triphosphate (ATP) by pyruvate phosphate dikinase (PPDK) in the presence of phosphoenolpyruvate (PEP) and inorganic pyrophosphate; and

(c) reacting ATP with luciferin and O2 in the presence of luciferase to produce AMP and a luminescent signal,

wherein the luminescent signal indicates the presence of adenosine or an enzyme that forms adenosine as a product.

[0023] The enzyme that forms adenosine as a product can be, for example, S- adenosyl-L-homocysteine hydrolase (SAHH), nucleotidase or alkaline phosphatase. Adenosine can be formed, for example, by the hydrolysis of S-adenosyl-L-homocysteine (SAH) catalyzed by S-adenosyl-L-homocysteine hydrolase (SAHH) to adenosine and L- homocysteine.

[0024] The invention provides, for example, a method for detecting the presence of S-adenosyl-L-homocysteine hydrolase (SAHH) comprising:

(a) hydrolysis of S-adenosyl-L-homocysteine (SAH) catalyzed by S-adenosyl-L- homocysteine hydrolase (SAHH) to adenosine and L-homocysteine;

(b) converting adenosine to adenosine monophosphate (AMP) by adenosine kinase in the presence of a phosphate donor;

(c) converting AMP to adenosine 5 '-triphosphate (ATP) by pyruvate phosphate dikinase (PPDK) in the presence of phosphoenolpyruvate (PEP) and inorganic pyrophosphate; and

(d) reacting ATP with luciferin and O2 in the presence of luciferase to produce AMP and a luminescent signal,

wherein the luminescent signal indicates the presence of S-adenosyl-L-homocysteine hydrolase (SAHH). [0025] In the methods disclosed herein, the AMP that is produced in the presence of luciferase can be use as a source of AMP for conversion to ATP by pyruvate phosphate dikinase (PPDK).

[0026] The adenosine kinase can be from any source and is preferably from Anopheles gambiae.

[0027] The phosphate donor can be, for example, guanosine triphosphate (GTP), cytosine triphosphate (CTP) or uracil triphosphate (UTP), and is preferably GTP.

[0028] Luciferase is a common bioluminescent enzyme. Organisms from which luciferase can be obtained include, but are not limited to fireflies, bacteria (e.g. Vibrio fischeri), jellyfish (e.g. Aequorea victoria), beetles, and squid. In the preferred embodiment of the present invention, the luciferase is firefly luciferase.

[0029] In the methods disclosed herein, adenosine can be detected at a concentration of 1 picomole or less.

[0030] In the methods disclosed herein, preferably the luminescent signal has an intensity that is proportional to the amount of adenosine or to the amount of an enzyme that forms adenosine as a product, such as, for example, the amount of S-adenosyl-L- homocysteine hydrolase (SAHH).

[0031] In the methods disclosed herein, adenosine or an enzyme that forms adenosine can be in a biological sample, such as, for example, blood, tissue or cells. The biological sample can be from a subject having a disease or condition, such as, for example, hypertension, brain or heart ischemia, a sleep disorder, an elevated cholesterol level, an inflammatory disorder or cancer.

[0032] The methods disclosed herein do not require the formation of adenine.

[0033] The invention also provides methods of determining whether or not a compound is an inhibitor of an enzyme that forms adenosine as a product, comprising carrying out any of the methods disclosed herein in the presence and in the absence of the compound, wherein a decrease in the luminescent signal in the presence of the compound is indicative that the compound is an inhibitor of an enzyme that forms adenosine as a product, and wherein a lack of decrease in the luminescent signal in the presence of the compound is indicative that the compound is not an inhibitor of an enzyme that forms adenosine as a product.

[0034] The enzyme that forms adenosine as a product can be, for example, S- adenosyl-L-homocysteine hydrolase (SAHH), nucleotidase or alkaline phosphatase. The compound can be, for example, an inhibitor of S-adenosyl-L-homocysteine hydrolase (SAHH). [0035] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXPERIMENTAL DETAILS

Introduction

[0036] A firefly luciferase-based assay is presented that can detect picomole levels of ADO, which can be generated, for example, during the SAH hydrolysis catalyzed by SAHH. The product ADO is converted to adenosine monophosphate (AMP) by the highly efficient AK from Anopheles gambiae (K_m ^ADO = 230 nM; k_cat = 2.7 x 10⁶ M^" ¹ s^"1) in the presence of the phosphate donor guanosine triphosphate (GTP).³³ AMP is converted to ATP by pyruvate phosphate dikinase and firefly luciferase (PPDK and FLUC, respectively) to give a sustained light output (λ = 570 nm). This is distinct from, but shares some common elements with the assays previously reported for the detection of ricin and methyltransferases activity.³⁴'³⁵ This assay, illustrated in Figure 2, is a dramatic improvement compared to former methods. This procedure is compatible with a 96 well plate screening format, is 4000-fold more sensitive than current methodologies and permits the detection of ultra low SAHH activity (10^~7 unit) without the use of radioactive substrates. SAH hydrolysis can be monitored continuously over an extended period of time (60 min), hence the assay is appropriate to reveal the unprecedented kinetic resolution of slow-onset inhibition with SAHH inhibitors. This assay is also superior to the use of thio-detecting chromophores as DTNB rapidly reacts with cysteine residues from SAHH and inactivates the hydrolase.³⁶ Procedures measuring Hey with DTNB cannot detect low concentrations of SAHH or monitor the enzymatic reaction for more than five minutes.³¹

Materials and Methods

[0037] Reagents. Common reagents (Sigma-Aldrich) were used without further purification; GTP and SAH were purified by HPLC (Luna²-Cis; phenomenex), desalted, concentrated and stored at -80 °C.³⁷ Reducing agent Tris(hydroxypropyl)-phosphine (THP) was from Novagen. The SAHH inhibitors, D-eritadenine was from Santa Cruz Biotechnology (No. sc-207632) and neplanocin A was from Cayman Chemicals (No. 10584). ATP detection was achieved with "ATPLite™ lstep" (Perkin-Elmer); molecular biology grade water was used for all assays (Fisher Scientific; No. BP2819-1). Enzymatic activities were determined by HPLC and one unit (1 U) is defined as the amount of enzyme which converts 1 μιηοΐ of substrate to product per min at 25 C.

[0038] 5'-deoxy-5'-amino-fi-D-adenosine (INH-1). This compound was synthesized and purified following a reported procedure; ^XH NMR and mass spectroscopy data were in agreement with that reported in the literature.³⁸

[0039] Enzymes. The full length human S-adenosyl-L-homocysteine hydrolase

(HsSAHH) was from Abeam (No. ab99326). The N-terminal 6x His-Tag adenosine kinase from Anopheles gambiae (AgAK) was expressed and purified as reported previously.³³ The Clostridium symbiosum pyruvate phosphate dikinase (CsPPDK) was expressed according to a published protocol (generous gift provided by Dr. Debra Dunaway-Mariano; University of New Mexico).³⁹

[0040] Coupled assay buffer. The formation of ADO as a product of the SAHH reaction (2-40 mU L^"1) was monitored using the coupling enzymes AgAK (6 U L^"1) and CsPPDK (100 U L^"1). A 4x concentrated buffer Bi (200 mM TRIS-acetate pH 7.7, 1 mM PEP, 1 mM PPi and 4 mM ammonium chloride; treated with charcoal and filtered sterilized) containing AK and PPDK was supplemented with MgCl₂ (10 mM), GTP (0.5 mM), THP (1 mM), ATPlite (following supplier protocol) and appropriate concentrations of SAHH. Each experiment uses 25 μϊ_^ of this final buffer B2, 2* concentrated and diluted to a final volume of 50 μί.

[0041] Enzymatic assays. Kinetic constants were measured at 25 °C by monitoring luminescence at 570 nm using a SpectraMax L instrument configured with two photo- multipliers (Molecular Devices) in 96 well half-area flat bottom plates (Corning; No. 3992). Briefly, 25 of buffer B₂ (containing 6.4 nM of HsSAHH; i.e. 4 mU L^"1) was mixed with an equal volume of SAH standard solutions (10 to 40 μΜ). Initial rates were plotted against substrate concentrations and fitted to the Michaelis-Menten equation to yield corresponding K_m and k_cat values. Typical inhibition assays consisted of several samples containing SAH (40 μΜ) and various inhibitor concentrations with one control sample (40 μΜ SAH, without inhibitor). To initiate the reaction, 25 μϊ_^ of buffer B2 was added and luminescence was recorded for 20-60 min. The slow-onset inhibition phase was analyzed using the equation:

m

K ^* where V_s' and V/ are the steady state rates with and without inhibitor, respectively; K_m is the Michaelis constant for SAH; [SAH] and [I] are the concentrations of SAH and inhibitor, respectively. If the concentration of inhibitor is less than ten times the concentration of enzyme (e.g. D-eritadenine), the following equation is used to correct for inhibitor depletion by the enzyme:

with [I]* the effective inhibitor concentration, V and V₀° the initial rates with and without inhibitor, respectively; and [Ε]τ is total SAHH concentration.

[0042] Z' factor. The screening window coefficient was determined as previously described using SAHH at 10^~7 unit and 20 μΜ substrate.⁴⁰ Each experiment consisted of 16 sample replicates (s; enzyme with SAH) and 16 control replicates (c; with SAH but without SAHH). Statistical analysis of the initial rates using the following equation yields the corresponding factor Z':

, _{= 1} 3 + 3σ

k - where / is the mean value of the initial rates for samples s and a_s is the standard deviation of the initial rates for samples s (3 a_s corresponds to a 99.73% confidence interval).

[0043] Phosphorylation of neplanocin A by AgAK. Analyses were performed by HPLC on a reverse phase C₁₈ column (Luna², 150x4.6 mm, 5 μιη, 100 A; phenomenex) using a protocol previously described;⁴⁵ ADO, AMP, GDP, GTP, SAH, ΓΝΗ and ΓΝΗ-Ρ could be easily separated and the analyses demonstrate that ΓΝΗ is a substrate of AgAK according to the formation of GDP.

[0044] SAHH inhibition by neplanocin A (INH). In the absence of AgAK, two experiments were performed in parallel. The SAHH enzyme mixture (100 mM TRIS- acetate pH 7.7, 0.5 mM PEP, 0.5 mM PPi, 2 mM ammonium chloride, 10 mM MgCl₂, 0.5 mM GTP, 1 mM THP and 250 mU L^"1 of SAHH) was rapidly added to an equal volume of SAH (40 μΜ; with or without 12 μΜ ΓΝΗ). Samples were taken every ten minutes, quenched by ultrafiltration (Biomax UltraFree 0.5 mL centrifugal filter, 10 KD NMWL; Millipore) and analyzed by HPLC to follow ADO production. In presence of the same kinase, two similar experiments were carried; the SAHH mixture (see above; supplied with 60 U L^"1 of AgAK) was added to an equal volume of SAH (40 μΜ; with or without 12 μΜ ΓΝΗ). Every ten minutes, samples were processed as described above to quantify the formation of AMP.

Results and Discussion

[0045] The quantitation of ATP by luciferase is a sensitive analytical method, which can be generalized to detect any metabolite that can be converted to AMP. Phosphoenol pyruvate dikinase is used to convert AMP to ATP. The luciferase reaction generates light, oxyluciferin and AMP, and cycling of AMP to ATP sustains the luminescent signal (Fig. 2).

[0046] A highly efficient AK was used to extend the use of the FLUC system to the detection of ADO produced during the reaction catalyzed by SAHH. Under typical conditions, ADO is converted to ATP and chemiluminescence (Fig. 3A).The method displays a dynamic range of ADO detection as low as one and as high as 80 picomoles (Fig. 3 A). The rates of the coupling reactions do not limit the observed rates of the assay. Initial velocities for adenosine formation were measured with increasing SAHH concentrations at 20 μΜ substrate. A linear relationship between luminescence output and enzyme activity was observed (Fig. 3B). AgAK is highly efficient at phosphorylating ADO to AMP and SAH hydrolysis is readily quantitated even at elevated SAHH concentrations (Fig. 3B). Using this assay the kinetic profile was determined as a function of SAH concentration (5-20 μΜ, at 3.2 nM SAHH). The data fit to the Michaelis-Menten equation gives a k_cat of 0.075 ± 0.006 s^"1 and a K_m of 22 ± 2μΜ, consistent with reported values (Fig. 3C).⁷'³¹'³²

[0047] The inhibition constants (Ki) for three known inhibitors of SAHH, D- eritadenine, 5'-deoxy-5'-amino- ?-D-adenosine (ΓΝΗ-1) and neplonocin A were determined to demonstrate the applicability of this assay (ΓΝΗ; Fig. 1). These inhibitors include 'suicide' and 'reversible' inhibitors, to permit analysis of both constant rate and inactivation kinetic parameters.

[0048] With varied D-eritadenine concentrations (0-80 nM), 3.2 nM SAHH and 20 μΜ SAH, the inhibition profile for SAHH was established. A two-phase, slow-onset inhibition is observed. Inhibition with a linear rate of ADO formation was observed during the first fifteen minutes of the assay (Fig. 4A). Analysis of this portion of the steady-state kinetic data gave a of 11.3 ± 0.6 nM for D-eritadenine (Fig. 4B, dashed trace). This value is consistent with the literature values of 10-30 nM.⁵'¹⁸ At longer time periods, the light output from ADO production identified a second phase of inhibition associated with slow-onset inhibition (40-60 min; Fig. 4A). Slow-onset inhibition occurs when the initial [Εηζ·Ι] complex undergoes a slow conformational change that stabilizes its overall structure, leading to tighter inhibitor binding.⁴¹ For D-eritadenine, slow-onset increased inhibitor binding by 10-fold relative to the R value (K* = 1.28 ± 0.05 nM; Fig. 4B, solid trace).

[0049] A comparison of the X-ray structures for SAHH with bound D-eritadenine, ADO and SAH readily demonstrate that D-eritadenine causes the enzyme to adopt a more 'closed conformation' and interacts more tightly with the enzyme backbone than ADO or SAH.⁵ Specifically, hydrogen bonds between His352 and N6 and N7 of these inhibitors are significantly shorter when D-eritadenine is bound. The slow-onset inhibition observation provided here is in good agreement with the reported crystallographic analysis. However, there are no previous reports of slow-onset inhibition for this molecule.

[0050] The luciferase assay was also used to determine the inhibitory effect of INH-1. Although INH-1 also gives kinetic plots consistent with slow-onset inhibition (Fig. 7A), it is a relatively poor inhibitor of the SAH hydrolysis reaction (K* = 2.17 ± 0.08 μΜ; Fig. 7B).²⁵ Detailed evaluation of slow-onset inhibition for weak inhibitors requires high amounts of these molecules (e.g. up to 25 μΜ for INH-1) and raises a technical problem if the coupling enzymes are inhibited. With the luciferase assay, it is simple and important to perform controls to ensure the inhibitors solely target SAHH. One can easily identify 'false positives' from this assay. Addition of an ADO standard at the completion of the assay in the presence of inhibitor will give a light emission pattern equivalent to that without inhibitor to preclude inhibition of coupling enzymes. When this control is done in the presence of INH-1, no inhibition of the AK, PPDK, and FLUC coupling system is observed (Fig. 7C). Despite its relatively weak binding, the results here establish INH-1 as a slow-onset inhibitor. Although slow-onset inhibitors are most commonly associated with tight binding, SAHH contains protein domains known to reorganize on a slow time scale.⁴² The observed slow-onset kinetics are likely associated with these changes.

[0051] The utility of the luciferase assay was explored with an adenosine nucleoside analogue, neplanocin A (ΓΝΗ). Although this suicide inhibitor is expected to display strong affinity for SAHH (low nanomolar K), a 2 μΜ concentration of ΓΝΗ did not affect the hydrolase activity under the standard assay conditions.⁴³ It was anticipated that ΓΝΗ and related adenosine analogues might be phosphorylated by the AgAK using GTP as the phosphoryl donor during the assay since all coupling enzymes, including AgAK are present in large excess relative to SAHH. Upon addition of AgAK, ΓΝΗ and GTP were consumed and GDP is formed, according to HPLC analysis (Fig. 8A).

[0052] ΓΝΗ inhibition of SAHH was also monitored by ADO formation directly (HPLC analysis) during the SAHH-catalyzed reaction without coupling enzymes (Fig. 8B). Under these conditions, ΓΝΗ totally inhibits SAHH. When AgAK and GTP are added to the reaction mixture, inhibition by ΓΝΗ is weak (Fig. 8B). In vivo, cytotoxicity for ΓΝΗ and similar suicide inhibitors has been due to their reactivity with AK. ' ' The use of AgAK in the assay precludes its use for inhibitors acting as substrates for the AgAK-QT? pair. The luciferase assay for ADO quantitation provides a powerful tool in analytical chemistry for the identification of new SAHH inhibitors. Under the present experimental conditions, SAH decomposition is insignificant and the formation of ADO is monitored efficiently (Fig. 5A). Published assays for SAHH exhibit low sensitivity or require radioactive substrates. Since it was proposed that the luciferase assay may be suitable for the inhibitory evaluation of compounds, the suitability of the assay for high- throughput screening (HTS) was estimated. The screening window coefficient (Z' factor) for this coupled assay was 0.92 (Fig. 5B).This high value (maximum of 1.00 for a perfect assay) reflects the overall quality of this assay.⁴⁰

[0053] Since many compounds from commercial libraries are supplied in DMSO, a determination was made of the compatibility of DMSO with the assay. DMSO causes modest interference with chemiluminescence (inset, Fig. 6). However, the reduction of light output is not the result of interference with the coupling enzymes since the calibration curve is unchanged (Fig. 9). DMSO interferes with SAHH catalytic activity (Fig. 6). This solute effect of DMSO is independent of the assay procedure and is solely due to interactions between the solvent and SAHH. With appropriate controls, the screening of libraries supplied in DMSO is appropriate for the luciferase assay.

[0054] The luciferase-based quantitation of adenosine has implications beyond its use in enzymatic assays, as characterized here. Adenosine is also a critical metabolite in human metabolism through its interaction with adenosine receptors.⁴⁴ Adenosine receptors have been identified as therapeutic targets in human conditions as diverse as hypertension, brain and heart ischemia, sleep disorders, inflammatory disorders and cancer. Detection of adenosine in biological samples is therefore important in research and diagnosis of disorders related to its receptor function. A direct extension of the methods illustrated here, together with appropriate controls, can be readily applied to the detection of adenosine in samples of blood and other biological materials. [0055] In summary, a new assay was developed to monitor ADO. The assay has been exemplified and characterized with SAH hydrolysis catalyzed by SAHH. Detection of ADO is robust, continuous and can be achieved in the presence of a low quantity of enzyme. The present assay is expected to accommodate a broad range of inhibitors (e.g. low nanomolar to high micromolar Ki). Adenosine analogues phosphorylated by AgAK and GTP are precluded from the assay. Finally, the high Z' factor highlights the wide applications of this tool and the possible impact of the assay for identification of new inhibitors of SAHH and other enzymes that produce ADO using high-throughput screening. The development of SAHH inhibitors is of interest, for example, for new therapeutics with anti-cancer or cholesterol-lowering effects.

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Claims

What is claimed is:

1. A method for detecting the presence of adenosine or an enzyme that forms adenosine as a product comprising:

(c) reacting ATP with luciferin and (¾ in the presence of luciferase to produce AMP and a luminescent signal,

2. The method of Claim 1, wherein the enzyme that forms adenosine as a product is selected from the group consisting of S-adenosyl-L-homocysteine hydrolase (SAHH), nucleotidase and alkaline phosphatase.

3. The method of Claim 1, wherein adenosine is formed by the hydrolysis of «S-adenosyl- L-homocysteine (SAH) catalyzed by S-adenosyl-L-homocysteine hydrolase (SAHH) to adenosine and L-homocysteine.

4. The method of any of Claims 1-3, wherein AMP produced in step (c) is used as a source of AMP in step (b).

5. A method for detecting the presence of S-adenosyl-L-homocysteine hydrolase (SAHH) comprising:

(c) converting AMP to adenosine 5 '-triphosphate (ATP) by pyruvate phosphate dikinase (PPDK) in the presence of phosphoenolpyruvate (PEP) and inorganic pyrophosphate; and (d) reacting ATP with luciferin and (¾ in the presence of luciferase to produce AMP and a luminescent signal,

wherein the luminescent signal indicates the presence of S-adenosyl-L-homocysteine hydrolase (SAHH).

6. The method of Claim 5, wherein AMP produced in step (d) is used as a source of AMP in step (c).

7. The method of any of Claims 1-6, wherein the adenosine kinase is from Anopheles gambiae.

8. The method of any of Claims 1-7, wherein the phosphate donor is guanosine triphosphate (GTP), cytosine triphosphate (CTP) or uracil triphosphate (UTP).

9. The method of any of Claims 1-8, wherein the luciferase is firefly luciferase.

10. The method of any of Claims 1-9, wherein adenosine can be detected at a concentration of 1 picomole or less.

1 1. The method of any of Claims 1-10, wherein the luminescent signal has an intensity that is proportional to the amount of adenosine or to the amount of an enzyme that forms adenosine as a product.

12. The method of any of Claims 1-1 1, wherein adenosine or an enzyme that forms adenosine is in a biological sample.

13. The method of Claim 12, wherein the biological sample is blood, tissue or cells.

14. The method of Claim 12 or 13, wherein the biological sample is from a subject having hypertension, brain or heart ischemia, a sleep disorder, an elevated cholesterol level, an inflammatory disorder or cancer.

15. The method of any of Claims 1-14, wherein the method does not require the formation of adenine.

16. A method of determining whether or not a compound is an inhibitor of an enzyme that forms adenosine as a product, comprising carrying out the method of any of Claims 1-15 in the presence and in the absence of the compound, wherein a decrease in the luminescent signal in the presence of the compound is indicative that the compound is an inhibitor of an enzyme that forms adenosine as a product, and wherein a lack of decrease in the luminescent signal in the presence of the compound is indicative that the compound is not an inhibitor of an enzyme that forms adenosine as a product.

17. The method of Claim 16, wherein the compound is an inhibitor of «S-adenosyl-L- homocysteine hydrolase (SAHH).