Methods for assaying inhibitors of S-adenosylhomocysteine (SAH) hydrolase and S-adenosylmethionine (SAM)-dependent methyltransferase Number:7,384,760 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to methods for assaying inhibitors for S-adenosylhomocysteine (SAH) hydrolases and assaying inhibitors for S-adenosylmethionine (SAM)-dependent methyltransferases. The methods are amenable for use in high throughput formats. Kits for performing the methods are also provided.<H adenosylhomocysteine, adenosylmethionine, Methods, for, ass, 7384760.html, Patent, pto, 7384760

Title: Methods for assaying inhibitors of S-adenosylhomocysteine (SAH) hydrolase and S-adenosylmethionine (SAM)-dependent methyltransferase

Abstract: The present invention relates to methods for assaying inhibitors for S-adenosylhomocysteine (SAH) hydrolases and assaying inhibitors for S-adenosylmethionine (SAM)-dependent methyltransferases. The methods are amenable for use in high throughput formats. Kits for performing the methods are also provided.

Patent Number: 7,384,760 Issued on 06/10/2008 to Yuan, et al.

Inventors: Yuan; Chong-Sheng (San Diego, CA), Ye; Qi-Zhuang (San Diego, CA), Xie; Sheng-Xue (Lawrence, KS)
Assignee: General Atomics (San Diego, CA)

Appl. No.: 10/836,953

Filed: April 30, 2004

Current U.S. Class: 435/15 ; 435/18

Current International Class: C12Q 1/48 (20060101); C12Q 1/34 (20060101)

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Primary Examiner: Tate; Christopher R.
Assistant Examiner: Wood; Amanda P.
Attorney, Agent or Firm: Morrison & Foerster LLP

Claims

The claimed invention is:

1. A method for assaying for an inhibitor of a S-adenosylmethionine (SAM)-dependent methyltransferase, comprising: a) contacting a SAM-dependent methyltransferase with (i) a substrate of the methyltransferase, (ii) SAM, and (iii) in the presence or absence of a compound suspected of being an inhibitor of the methyltransferase, under a condition that a methyl group is transferred from SAM to the substrate and SAM is converted to SAH: b) contacting the resulting SAH with a SAH hydrolase and a tracer under a condition that allows hydrolysis of the SAH into adenosine (Ado) and homocysteine (Hcy) catalyzed by the SAH hydrolase; wherein the tracer is a labeled SAH or a labeled SAH analog and is not hydrolyzed by the SAH hydrolase; wherein the SAH hydrolase is wildtype or has one or more conservative amino acid substitutions that do not substantially alter its catalytic activity, and wherein i) the tracer generates a detectable signal after binding to the SAH hydrolase, or ii) the SAH hydrolase is immobilized on a suitable surface; c) detecting binding of the tracer to the SAH hydrolase; and d) comparing the amount of binding of the tracer to the SAH hydrolase in the presence of the compound to the amount of binding in the absence of the compound, whereby an increase in the amount of binding in the presence of the compound compared to the amount of binding in the absence of the compound indicates that the compound is an inhibitor of the SAM-dependent methyltransferase.

2. The method of claim 1, wherein the SAM-dependent methyl transferase is selected from the group consisting of a protein methyltransferase, a nucleic acid methyltransferase, a lipid methyltransferase, a polysaccharide methyltransferase and a small molecule methyltransferase.

3. The method of claim 1, wherein the substrate is selected from a group consisting of a protein, a nucleic acid, a lipid, and a small molecule, and wherein the SAM-dependent methyltransferase is selected from the group consisting of a protein methyltransferase, a nucleic acid methyltransferase, a lipid methyltransferase, and a small molecule methyltransferase.

4. The method of claim 1, wherein the label is a fluorescent.

5. The method of claim 4, wherein the binding of the tracer to SAH hydrolase is detected by detecting the fluorescent polarization of the tracer.

6. The method of claim 1, wherein a plurality of compounds suspected of being inhibitors of the SAM dependent methyltransferase are assayed simultaneously.

7. The method of claim 6, wherein the assay is conducted in a multi-well format.

8. The method of claim 6, wherein the SAH hydrolase is linked to a solid support.

9. The method of claim 8, wherein the SAH hydrolase is arranged in an array on the solid support.

Description

TECHNICAL FIELD

This invention relates generally to the field of assaying inhibitors of S-adenosylhomocysteine (SAH) hydrolase and assaying inhibitors of S-adenosylmethionine (SAM)-dependent methyltransferase.

BACKGROUND OF THE INVENTION

S-adenosylhomocysteine (SAH) hydrolase is a ubiquitous cellular enzyme catalyzing the hydrolysis of SAH to adenosine (Ado) and homocysteine (Hcy). SAH hydrolase has been an attractive therapeutic target for a number of medical indications including antiviral, anticancer, anti-inflammation, immunosuppression, and plasma Hcy-lowering for prevention or treatment of cardiovascular diseases due to its central role in regulation of biological methylation reactions. Yuan et al., Exp. Opin. Ther. Patents, 9: 1197-1206 (1999); Yuan et al., in Adv. Antiviral Drug Des. vol 2, pp. 41-88, De Clercq (ed)., JAI Press, Inc. London, UK (1996). Inhibition of SAH hydrolase results in inhibition of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. For example, inhibition of SAH hydrolase results inhibition of viral mRNA methylation, thus inhibiting viral replication (Scheme 1).

##STR00001##

Numerous inhibitors of SAH hydrolase have been identified from naturally occurring compounds and synthetic compounds, including irreversible and reversible inhibitors. See, e.g., Yuan et al., Exp. Opin. Ther. Patents, 9: 1197-1206 (1999); Wolfe and Borchardt, Journal of Medicinal Chemistry, 34:1521-1530 (1991); Votruba and Holy, Coll. Czech. Chem. Commun., 45:3039 (1980); Schanche et al., Molecular Plarmacology, 26:553-558 (1984); U.S. Ser. No. 10/410,879. It is an object of the invention to provide methods for screening inhibitors of SAH hydrolase.

S-adenosylmethionine (SAM)-dependent methyltransferase is an enzyme that catalyzes the transfer of a methyl group from SAM to a substrate and converts SAM to SAH. Methyltransferase, including SAM-dependent methyltransferase catalyzing abnormal methylation has been linked to pathological conditions (see, e.g., U.S. Pat. No. 5,876,996). For example, covalent modification of cellular substrates with methyl groups has been implicated in the pathology of cancer and other diseases (Gloria, et al., Cancer, 78:2300-2306 (1996)). Cytosine hypermethylation of eukaryotic DNA prevents transcriptional activation (Turker and Bestor, Mutat. Res., 386:119-130 (1997)). N.sup.6-methyladenosine is found at internal positions of mRNA in higher eukaryotes (Bokar, et al., J. Biol. Chem., 269:17697-17704 (1994)). Hypermethylated viral DNA is transcribed at higher rates than hypo- or hemimethylated DNA in infected cells (Willis, et al. Cell. Biophys., 15:97-111 (1989)).

In addition, many pathways of small molecule degradation, such as those of neurotransmitters, require methyltransferase activity (U.S. Pat. No. 5,876,996; and Kagan and Clarke, Arch. Biochem. Biophys., 310:417427 (1994)). Degradation of catecholamines (epinephrine ornorepinephrine) requires phenylethanolamine N-methyltransferase. Hydroxyindole methyltransferase converts N-acetyl-5-hydroxytryptamine to melatonin in the pineal gland.

In their roles as a rate-limiting step in methyltransferase reactions, SAM-dependent methyltransferases have been identified as targets for psychiatric, antiviral, anticancer and anti-inflammatory drug design (U.S. Pat. No. 5,876,996). For instances, sequence-specific methylation inhibits the activity of the Epstein-Barr virus LMP1 and BCR2 enhancer-promoter regions (Minarovits et al., Virology, 200:661-667 (1994)). 2'-5'-linked oligo(adenylic acid) nucleoside analogues synthesized by interferon-treated mouse L cells act as antiviral agents (Goswarmi, et al., J. Biol. Chem., 257:6867-6870 (1982)). Adenine analog inhibitors of AdoMet-MT decreased nucleic acid methylation and proliferation of leukemia L1210 cells (Kramer et al., Cancer Res., 50:3838-3842 (1990)). Therefore, another object of the invention is to provide methods for screening for inhibitors for SAM-dependent methyltransferases.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for assaying of an inhibitor of a S-adenosylhomocysteine (SAH) hydrolase, said method comprises: a) contacting a SAH hydrolase with (i) SAH, (ii) a tracer, wherein the tracer is a labeled SAH or a labeled SAH analog and is not hydrolyzed by the SAH hydrolase, and (iii) in the presence or absence of a compound suspected of being an inhibitor of the SAH hydrolase under a condition that allows hydrolysis of the SAH into adenosine (Ado) and homocysteine (Hcy) catalyzed by the SAH hydrolase in the absence of an inhibitor of the SAH hydrolase; and wherein i) the tracer generates a detectable signal after binding to the SAH hydrolase or ii) the SAH hydrolase is immobilized on a suitable surface; b) detecting binding of the tracer to the SAH hydrolase; and c) comparing the amount of binding of the tracer to the SAH hydrolase in the presence of the compound to the amount of binding in the absence of the compound, whereby an increase in the amount of binding in the presence of the compound compared to the amount of binding in the absence of the compound indicates that the compound is an inhibitor of the SAH hydrolase.

In some embodiments, the SAH hydrolase and a mutant SAH hydrolase are contacted with (i) SAH, (ii) the tracer, and (iii) the compound suspected of being an inhibitor of SAH hydrolase in step a), wherein the mutant SAH hydrolase has binding affinity for SAH and adenosine but has attenuated catalytic activity; wherein i) the tracer generates a detectable signal after binding to the SAH hydrolase and the mutant SAH hydrolase or ii) the SAH hydrolase and mutant SAH hydrolase are immobilized on a suitable surface; wherein the binding detected in step b) is binding of the tracer to the SAH hydrolase and the mutant SAH hydrolase.

In some embodiments, the label of the tracer is a florescence. In some embodiments, the binding of the tracer to the SAH hydrolase (in some embodiments, including mutant SAH hydrolase) is detected by detecting the fluorescent polarization of the tracer.

In some embodiments, the method is conducted using a single SAH hydrolase and a single compound suspected of being an inhibitor of the SAH hydrolase in one assay. In other embodiments, the method is conducted in a high throughput screening mode, i.e., a plurality of the SAH hydrolases and/or a plurality of the compounds suspected of being inhibitors of the SAH hydrolases are screened simultaneously. The methods can be conducted in a multi-well (e.g., 24-, 48-, 96-, or 384-well), chip or array format. The SAH hydrolase (in some embodiments, including mutant SAH hydrolase) may be linked to a solid support, and may be arranged in an array on the solid support.

The invention also provides a kit for assaying for an inhibitor of a SAH hydrolase, said kit comprises (i) SAH, (ii) a tracer, wherein the tracer is a labeled SAH or a labeled SAH analog and is not hydrolyzed by the SAH hydrolase, and (iii) a SAH hydrolase, wherein the tracer generates a detectable signal after binding to the SAH hydrolase or the SAH hydrolase is immobilized on a suitable surface. In some embodiments, said kit further comprises a mutant SAH hydrolase, wherein the mutant SAH hydrolase has binding affinity for SAH and adenosine but has attenuated catalytic activity; and wherein the tracer generates a detectable signal after binding to the SAH hydrolase and the mutant SAH hydrolase, or the SAH hydrolase and the mutant SAH hydrolase are immobilized on a suitable surface.

In another aspect, the invention provides a method for assaying for an inhibitor of a S-adenosylmethionine (SAM)-dependent methyltransferase, comprising: a) contacting a SAM-dependent methyltransferase with (i) a substrate of the methyltransferase, (ii) SAM, and (iii) in the presence or absence of a compound suspected of being an inhibitor of the methyltransferase, under a condition that a methyl group is transferred from SAM to the substrate and SAM is converted to SAH; b) contacting the resulting SAH with a SAH hydrolase and a tracer under a condition that allows hydrolysis of the SAH into adenosine (Ado) and homocysteine (Hcy) catalyzed by the SAH hydrolase; wherein the tracer is a labeled SAH or a labeled SAH analog and is not hydrolyzed by the SAH hydrolase; and wherein i) the tracer generates a detectable signal after binding to the SAH hydrolase, or ii) the SAH hydrolase is immobilized on a suitable surface; c) detecting binding of the tracer to the SAH hydrolase; and d) comparing the amount of binding of the tracer to the SAH hydrolase in the presence of the compound to the amount of binding in the absence of the compound, whereby an increase in the amount of binding in the presence of the compound compared to the amount of binding in the absence of the compound indicates that the compound is an inhibitor of the SAM-dependent methyltransferase.

In some embodiments, the SAM-dependent methyltransferase is selected from the group consisting of a protein methyltransferase, a nucleic acid methyltransferase, a lipid methyltransferase, a polysaccharide methyltransferase and a small molecule methyltransferase. In some embodiments, the substrate is selected from a group consisting of a protein, a nucleic acid, a lipid, and a small molecule, and wherein the SAM-dependent methyltransferase is selected from the group consisting of a protein methyltransferase, a nucleic acid methyltransferase, a lipid methyltransferase, and a small molecule methyltransferase.

In some embodiments, the resulting SAH is contacted with the SAH hydrolase, a mutant SAH hydrolase, and the tracer; wherein the mutant SAH hydrolase has binding affinity for SAH and adenosine but has attenuated catalytic activity; wherein i) the tracer generates a detectable signal after binding to the SAH hydrolase and the mutant SAH hydrolase, or ii) the SAH hydrolase and the mutant SAH hydrolase are immobilized on a suitable surface; and wherein the binding detected in step c) is binding of the tracer to the SAH hydrolase and the mutant SAH hydrolase.

In some embodiments, the label of the tracer is a florescence. In some embodiments, the binding of the tracer to the SAH hydrolase (in some embodiments, including mutant SAH hydrolase) is detected by detecting the fluorescent polarization of the tracer.

In some embodiments, the method is conducted using a single SAM-dependent methyltransferase and a single compound suspected of being an inhibitor of the SAM-dependent methyltransferase in one assay. In other embodiments, the method is conducted in a high throughput screening mode, i.e., a plurality of the SAM-dependent methyltransferases and/or a plurality of the compounds suspected of being inhibitors of the SAM-dependent methyltransferases are screened simultaneously. The methods can be conducted in a multi-well (e.g., 24-, 48-, 96-, or 384-well), chip or array format. The SAH hydrolase (in some embodiments, including mutant SAH hydrolase) may be linked to a solid support, and may be arranged in an array on the solid support.

The invention also provides a kit for assaying for an inhibitor for S-adenosylmethionine (SAM)-dependent methyltransferase, comprising a SAM-dependent methyltransferase, a SAH hydrolase, and a tracer; wherein the tracer is a labeled SAH or a labeled SAH analog and is not hydrolyzed by the SAH hydrolase; and wherein i) the tracer generates a detectable signal after binding to the SAH hydrolase, or ii) the SAH hydrolase is immobilized on a suitable surface. In some embodiments, the kit further comprises a mutant SAH hydrolase, wherein the mutant SAH hydrolase has binding affinity for SAH and adenosine but has attenuated catalytic activity; and wherein the tracer generates a detectable signal after binding to the SAH hydrolase and the mutant SAH hydrolase, or the SAH hydrolase and the mutant SAH hydrolase are immobilized on a suitable surface.

In the system assaying for inhibitors of SAH hydrolase, an increase of the tracer binding would be observed when an inhibitor of SAH hydrolase is present in the assay system. In the system for assaying inhibitors of SAM-dependent methyltransferase, an increase of the tracer binding would be observed when an inhibitor of SAM-dependent methyltransferase is present in the assay system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing fluorescence polarization of the assay described in Example 2 where a compound with good inhibitory activity against histone methyltransferase was present in the assay. FIG. 1B is a graph showing fluorescence polarization of the assay described in Example 2 where a compound without inhibitory activity against histone methyltransferase was present in the assay. The X axis corresponds to time (min); and the Y axis corresponds to fluorescence polarization.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

As used herein, "a" or "an" means "at least one" or "one or more."

As used herein, "SAH hydrolase" refers to an ubiquitous eukaryotic enzyme, which is also found in some prokaryotes, which catalyzes hydrolysis of SAH to adenosine (Ado) and Hcy. SAH hydrolase also catalyzes the formation of SAH from Ado and Hcy. The co-enzyme of SAH hydrolase is NAD.sup.+/NADH. SAH hydrolase may have several catalytic activities. In the hydrolytic direction, the first step involves oxidation of the 3'-hydroxyl group of SAH (3'-oxidative activity) by enzyme-bound NAD.sup.+ (E-NAD.sup.+), followed by .beta.-elimination of L-Hcy to give 3'-keto-4',5'-didehydro-5'-deoxy-Ado. Michael addition of water to the 5'-position to this tightly bound intermediate (5'-hydrolytic activity) affords 3'-keto-Ado, which is then reduced by enzyme-bound NADH (E-NADH) to Ado (3'-reduction activity). It is intended to encompass SAH hydrolase with conservative amino acid substitutions that do not substantially alter its activity.

As used herein, "mutant SAH hydrolase, wherein said mutant SAH hydrolase has binding affinity for SAH and adenosine but has attenuated catalytic activity" refers to a mutant form of SAH hydrolase that retains sufficient binding affinity for SAH and adenosine to be detected in the process or method, particularly assay, of interest. Typically this is at least about 10%, preferably at least about 50% binding affinity for SAH and adenosine, compared to its wildtype counterpart SAH hydrolase. Preferably, such mutant SAH hydrolase retains 60%, 70%, 80%, 90%, 100% binding affinity for SAH and adenosine compared to its wildtype counterpart for SAH and adenosine, or has a higher binding affinity than its wildtype counterpart for SAH and adenosine. Such mutant SAH hydrolase can be herein referred to as a "substrate trapping SAH and adenosine," i.e., a molecule that specifically binds to SAH and adenosine, but does not catalyze conversion therebetween.

As used herein, "attenuated catalytic activity" refers to a mutant SAH hydrolase that retains sufficiently reduced catalytic activity to be useful in the present method. The precise reduction in catalytic activity for use in the assays can be empirically determined for each assay. Typically, the enzyme will retain less than about 50% of one of its catalytic activities or less than 50% of its overall catalytic activities compared to its wildtype counterpart. Preferably, a mutant SAH hydrolase retains less than 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% of one of its catalytic activities or its overall catalytic activities compared to its wildtype counterpart. More preferably, a mutant SAH hydrolase lacks detectable level of one of its catalytic activities or its overall catalytic activities compared to its wildtype counterpart.

As used herein, "homocysteine (Hcy)" refers to a compound with the following molecular formula: HSCH.sub.2CH.sub.2CH(NH.sub.2)COOH. Biologically, Hcy is produced by demethylation of methionine and is an intermediate in the biosynthesis of cysteine from methionine. The term "Hcy" encompasses free Hcy (in the reduced form) and conjugated Hcy (in the oxidized form). Hcy can conjugate with proteins, peptides, itself or other thiols through disulfide bond.

As used herein, "S-adenosylmethionine (SAM)-dependent methyltransferase" refers to an enzyme that transfers a methyl group from SAM to a substrate and converts SAM to S-adenosylhomocysteine (SAH). SAM-dependent methyltransferase can transfer a methyl group from SAM to a carbon, an oxygen, a nitrogen or a sulfur atom of a substrate, and the SAM-dependent methyltransferase is thereby further classified as a C--, O--, N--, or S-methyltransferase, respectively. Any such SAM-dependent methyltransferase, including those with conservative amino acid substitutions that do not substantially alter its activity are contemplated herein.

As used herein, "substrate of a SAM-dependent methyltransferase" refers to a substance that receives the methyl group from SAM in a reaction catalyzed by the SAM-dependent methyltransferase. Examples of the substrates of the SAM-dependent methyltransferases include proteins, nucleic acids, lipids, polysaccharides and other small molecules. As used herein, "SAM" is not considered a "substrate of a SAM-dependent methyltransferase."

As used herein, "protein SAM-dependent methyltransferase" refers to an enzyme that transfers a methyl group from SAM to a protein substrate and converts SAM to SAH.

As used herein, "nucleic acid SAM-dependent methyltransferase" refers to an enzyme that transfers a methyl group from SAM to a nucleic acid substrate, such as a DNA or a RNA, and converts SAM to SAH.

As used herein, "lipid SAM-dependent methyltransferase" refers to an enzyme that transfers a methyl group from SAM to a lipid substrate and converts SAM to SAH.

As used herein, "polysaccharide SAM-dependent methyltransferase" refers to an enzyme that transfers a methyl group from SAM to a polysaccharide substrate and converts SAM to SAH.

As used herein, "small molecule SAM-dependent methyltransferase" refers to an enzyme that transfers a methyl group from SAM to a small molecule substrate and converts SAM to SAH.

In all instances the methyltransferases encompass variants and mutants thereof, particularly those with conservative amino acid substitutions (see, e.g., TABLE 1, below), that retain the methyltransferring activity. Such substitutions are preferably made in accordance with those set forth in TABLE 1 as follows:

TABLE-US-00001 TABLE 1 Original residue Conservative substitution Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe VaI (V) Ile; Leu

Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions.

As used herein, "small molecule" refers to a molecule that, without forming homo-aggregates or without attaching to a macromolecule or adjuvant, is incapable of generating an antibody that specifically binds to the small molecule. Preferably, the small molecule has a molecule weight that is about or less than 10,000 daltons. More preferably, the small molecule has a molecule weight that is about or less than 5,000 Dalton.

As used herein, "enzyme" refers to a protein specialized to catalyze or promote a specific metabolic reaction. Generally, enzymes are catalysts, but for purposes herein, such "enzymes" include those that would be modified during a reaction. Since the enzymes are modified to eliminate or substantially eliminate catalytic activity, they will not be so-modified during a reaction.

As used herein, "SAM-dependent homocysteine S-methyltransferase" refers to an enzyme that catalyzes formation of methionine and S-adenosyl-L-homocysteine (SAH) from homocysteine and S-adenosylmethionine (SAM). It is intended to encompass SAM-dependent homocysteine S-methyltransferase with conservative amino acid substitutions that do not substantially alter its activity.

As used herein, high-throughput screening (HTS) refers to processes that test a large number of compounds, such as compounds of diverse chemical structures against disease targets to identify "hits" (see, e.g., Broach, et al., High throughput screening for drug discovery, Nature, 384:14-16 (1996); Janzen, et al., High throughput screening as a discovery tool in the pharmaceutical industry, Lab Robotics Automation: 8261-265 (1996); Fernandes, P. B., Letter from the society president, J. Biomol. Screening, 2:1 (1997); Burbaum, et al., New technologies for high-throughput screening, Curr. Opin. Chem. Biol., 1:72-78 (1997)]. HTS operations are highly automated and computerized to handle sample preparation, assay procedures and the subsequent processing of large volumes of data.

B. Methods and Kits for Assaying Inhibitors of SAH Hydrolase

The invention provides methods for screening compounds that inhibit SAH hydrolase activity. These compounds can be potential drugs for treating various conditions and diseases.

In one aspect, the invention provides a method for assaying for an inhibitor of a S-adenosylhomocysteine (SAH) hydrolase, said method comprises: a) contacting a SAH hydrolase with (i) SAH, (ii) a tracer, wherein the tracer is a labeled SAH or a labeled SAH analog and is not hydrolyzed by the SAH hydrolase, and (iii) in the presence or absence of a compound suspected of being an inhibitor of the SAH hydrolase under a condition that allows hydrolysis of the SAH into adenosine (Ado) and homocysteine (Hcy) catalyzed by the SAH hydrolase in the absence of an inhibitor of the SAH hydrolase; and wherein i) the tracer generates a detectable signal after binding to the SAH hydrolase or ii) the SAH hydrolase is immobilized on a suitable surface; b) detecting binding of the tracer to the SAH hydrolase; and c) comparing the amount of binding of the tracer to the SAH hydrolase in the presence of the compound to the amount of binding in the absence of the compound, whereby an increase in the amount of binding in the presence of the compound compared to the amount of binding in the absence of the compound indicates that the compound is an inhibitor of the SAH hydrolase.

The assay may be conducted in the presence of a mutant SAH hydrolase. In these embodiments, the SAH hydrolase and a mutant SAH hydrolase are contacted with (i) SAH, (ii) the tracer, and (iii) the compound suspected of being an inhibitor of SAH hydrolase in step a), wherein the mutant SAH hydrolase has binding affinity for SAH and adenosine but has attenuated catalytic activity; wherein i) the tracer generates a detectable signal after binding to the SAH hydrolase and the mutant SAH hydrolase or ii) the SAH hydrolase and mutant SAH hydrolase are immobilized on a suitable surface; and the binding detected in step b) is binding of the tracer to the SAH hydrolase and the mutant SAH hydrolase.

1. SAH Hydrolase and Mutant SAH Hydrolase

The methods of the invention may be used to screen inhibitors of any SAH hydrolase. In some embodiments, the methods may be used to screen inhibitors of SAH hydrolase encoded by the following nucleic acid sequences having the GenBank Accession Nos.: AF129871 (Gossypium hirsutum); AQ003753 (Cryptosporidium parvum); AF105295 (Alexandrium fundyense); AA955402 (Rattus norvegicus); AA900229 (Rattus norvegicus); AA874914 (Rattus norvegicus); AA695679 (Drosophila melanogaster ovary); AA803942 (Drosophila melanogaster ovary; AI187655 (Manduca sexta male antennae); U40872 (Trichomonas vaginalis); AJ007835 (Xenopus Laevis); AF080546 (Anopheles gambiae); AI069796 (T. cruzi epimastigote); Z97059 (Arabidopsis thaliana); AF059581 (Arabidopsis thaliana); U82761 (Homo sapiens); AA754430 (Oryza sativa); D49804 (Nicotiana tabacum); D45204 (Nicotiana tabacum); X95636 (D. melanogaster); T18277 (endosperm Zea mays); R75259 (Mouse brain); Z26881 (C. roseus); X12523 (D. discoideum); X64391 (Streptomyces fradiae); W21772 (Maize Leaf); AH003443 (Rattus norvegicus); U14963 (Rattus norvegicus); U14962 (Rattus norvegicus); U14961 (Rattus norvegicus); U14960 (Rattus norvegicus); U14959 (Rattus norvegicus); U14937 (Rattus norvegicus); U14988 (Rattus norvegicus); U14987 (Rattus norvegicus); U14986 (Rattus norvegicus); U14985 (Rattus norvegicus); U14984 (Rattus norvegicus); U14983 (Rattus norvegicus); U14982 (Rattus norvegicus); U14981 (Rattus norvegicus); U14980 (Rattus norvegicus); U14979 (Rattus norvegicus); U14978 (Rattus norvegicus); U14977 (Rattus norvegicus); U14976 (Rattus norvegicus); U14975 (Rattus norvegicus); L32836 (Mus musculus); L35559 (Xenopus laevis); Z19779 (Human foetal Adrenals tissue); L23836 (Rhodobacter capsulatus); M15185 (Rat); L11872 (Triticum aestivum); M19937 (Slime mold (D. discoideum); M80630 (Rhodobacter capsulatus). In other embodiments, inhibitors of SAH hydrolase (human placental SAH hydrolase) encoded by the nucleotide sequences with the GenBank accession Nos. M61831-61832 are assayed using the methods of the invention. See also Coulter-Karis and Hershfield, Ann. Hum. Genet., 53(2):169-175 (1989)). In other embodiments, inhibitors of SAH hydrolase descried in U.S. Pat. No. 5,854,023 are assayed.

Any mutant SAH hydrolase that substantially retains its binding affinity or has enhanced binding affinity for SAH and adenosine (Ado), but has attenuated catalytic activity can be used in the methods of the invention. Mutant enzyme can be prepared using mutagenesis methods after obtaining nucleic acid encoding SAH hydrolase.

Nucleic acids encoding SAH hydrolase can be obtained by methods known in the art. Additional nucleic acid molecules encoding such enzymes are known and the molecules or sequences thereof are publicly available. If the molecules are available they can be used; alternatively the known sequences can be used to obtain clones from selected or desired sources. For example, the nucleic acid sequences of SAH hydrolases can be used in isolating nucleic acids encoding SAH hydrolases from natural sources. Alternatively, nucleic acids encoding SAH hydrolases can be obtained by chemical synthesis according to the known sequences.

Once nucleic acids encoding SAH hydrolases are obtained, these nucleic acids can be mutagenized and screened and/or selected for mutant SAH hydrolase having binding affinity for SAH and adenosine but having attenuated catalytic activity. Insertion, deletion, or point mutation(s) can be introduced into nucleic acids encoding SAH hydrolases according to methods known to those of skill in the art. Information regarding the structural-functional relationship of the SAH hydrolases can be used in the mutagenesis and selection of mutant SAH hydrolase having binding affinity for SAH and adenosine but having attenuated catalytic activity.

In one example, the mutant SAH hydrolase used in the method has a mutation in an amino acid residue that is directly involved in the SAH hydrolase's catalytic activity, its binding with NAD.sup.+, NADH, Hcy, SAH or adenosine. In another example, the mutant SAH hydrolase used in the method has a mutation in an amino acid residue that is adjacent to an amino acid residue that is directly involved in the SAH hydrolase's catalytic activity, its binding with NAD.sup.+, NADH, Hcy, SAH or adenosine. Information on the SAH hydrolase's catalytic domain, various binding domains including the NAD binding domain and conserved amino acid residues are generally known and can be used in the designing of a suitable mutant SAH hydrolase (See e.g., Turner et al., Nat. Struct. Biol., 5(5):369-76 (1998) entitled "Structure determination of selenomethionyl S-adenosylhomocysteine hydrolase using data at a single wavelength;" Yin et al., Biomedical Chemistry: Applying Chemical Principles to the Understanding and Treatment of Disease (Ed. Torrence), Chapter 2, Mechanism-based S-adenosylhomocysteine hydrolase inhibitors in the search for broad-spectrum antiviral agents), John Wiley & Sons, Inc. (2000); Hu et al., Biochemistry, 38(26):8323-33 (1999) entitled "Crystal structure of S-adenosylhomocysteine hydrolase from rat liver;" Creedon et al., J. Biol. Chem., 269(23):16364-70 (1994) entitled "Plasmodium falciparum S-adenosylhomocysteine hydrolase. cDNA identification, predicted protein sequence, and expression in Escherichia coli.;" and Henderson et al., Mol. Biochem. Parasitol., 53(1-2): 169-83 (1992) entitled "Cloning of the gene encoding Leishmania donovani S-adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy."

Once a mutant SAH hydrolase with desired properties, i.e., substantially retaining binding affinity for SAH and adenosine but having attenuated catalytic activity, is identified, such mutant SAH hydrolase can be produced by any methods known in the art including recombinant expression, chemical synthesis or a combination thereof. Preferably, the mutant SAH hydrolase is obtained by recombinant expression.

SAH hydrolase from mammalian sources are homotetramer of approximate molecular weight of 180-190 KD. The enzyme contains 4 molecules of tightly-bound NAD.sup.+ as a co-enzyme. The catalytic mechanism of the enzyme in the hydrolytic direction includes two consecutive reactions, i.e., the 3'-oxidation of the substrate to 3'-keto in concomitant with the reduction of the enzyme-bound NAD+ to NADH, and followed by the 5'-hydrolysis to release the reaction products Hcy and Ado (Refsum, et al., Clin. Chem., 31:624-628 (1985)). The C-terminal regions of all known SAH hydrolase are extremely conserved and contain essential amino acid residues to the enzyme catalysis. The crystal structure of human SAH hydrolase in complex with a substrate analog inhibitor w