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Title:  Human methionine synthase reductase: cloning, and methods for evaluating risk of, preventing, or treating neural tube defects, cardiovascular disease, cancer, and down's syndrome
United States Patent: 
7,063,944
Issued: 
June 20, 2006

Inventors:
 Gravel; Roy A. (Calgary, CA); Rozen; Rima (Montreal West, CA); Leclerc; Daniel (Montreal, CA); Wilson; Aaron (Montreal, CA); Rosenblatt; David (Montreal, CA)
Assignee:
  McGill University (Quebec, CA)
Appl. No.: 
487841
Filed: 
January 19, 2000


 

George Washington University's Healthcare MBA


Abstract

The invention features a novel gene encoding methionine synthase reductase. The invention also features a method for detecting an increased likelihood of hyperhomocysteinemia and, in turn, an increased or decreased likelihood of neural tube defects, cardiovascular disease, Down's Syndrome or cancer. The invention also features therapeutic methods for treating and/or reducing the risk of cardiovascular disease, Down's Syndrome, cancer, or neural tube defects. Also provided are the sequences of the human methionine synthase reductase gene and protein and compounds and kits for performing the methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Methionine synthase catalyzes the remethylation of homocysteine to methionine in a reaction in which methylcobalamin serves as an intermediate methyl carrier.

Over time, the cob(I)alamin cofactor of methionine synthase may become oxidized to cob(II)alamin, thus rendering the enzyme inactive. Regeneration of the functional enzyme occurs through the reductive methylation of the cob(II)alamin in a reaction in which S-adenosylmethionine is utilized as methyl donor (FIG. 1). The reductive activation system in the lower part of the scheme shown in FIG. 1 is the mechanism by which S-adenosylmethionine (Ado-Met) together with an electron reactivates the enzyme to the functional, methionine synthase-CH3-Co(III) state, resulting in the formation of S-adenosylhomocysteine (Ado-Hcy) as a reaction by-product.

Patients of the cblE complementation group of disorders of folate/cobalamin metabolism, who are defective in the reductive activation of methionine synthase, have megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. We have cloned a CDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. Using primers comprising sequences of consensus binding sites for FAD, FMN and NADPH, we performed RT-PCR and inverse PCR to clone a methionine synthase reductase cDNA. The cDNA hybridizes to an mRNA of 3.6 kb (as detected by Northern blot). The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted Mr of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase (see below). Methionine synthase reductase was localized to human chromosome 5 p 15.2-15.3 by fluorescence in situ hybridization (FISH).

A survey of the NCBI databases for homology to the human methionine synthase reductase using BLASTP or TBLASTN yielded the putative methionine synthase reductase of C. elegans (P value=9.times.10-92). Proteins of the FNR family were also found using the BLAST programs. The strongest homology was found with cytochrome P450 reductase (P values >3-10-68), followed by nitric oxide synthase (three isoforms, P values >4.times.10-52), and sulfite reductase (P values >6.times.10-39). Lower, but still significant homology was found with E. coli NADPH-ferredoxin(flavodoxin) reductase (P values >2.times.10-9) and flavodoxin (P values >3.times.10-2). Our finding suggests a convergent evolution of the two-gene flavodoxin/NADPH-ferredoxin(flavodoxin) reductase system to a single gene encoding a fused version of the two proteins in human cells. Alignment of the proteins provides for a large linker region bridging the two components.

The identity of our cloned cDNA sequence as that encoding methionine synthase reductase was confirmed by the identification of mutations in the corresponding gene in cblE patients having a functional deficiency of methionine synthase. Our key finding confirming the identification of the cDNA was a 4 bp frameshift mutation in two affected siblings. The occurrence of a functionally null mutation in a candidate gene provides compelling evidence that the mutation is causative of disease in the affected patients. Furthermore, a 3 bp deletion detected in a third patient is also highly likely to cause an enzyme defect, and the direct sequencing of PCR products suggested that the patient's second allele contains a mutation that renders the mRNA very unstable or poorly transcribed. In all, seven of ten tested cblE cell lines showed evidence of mutation although the sequence changes have yet to be determined in the remaining four.

The two mutations we have identified associated with cblE disease are located in the vicinity of the NADPH binding domain by comparison with proteins of the FNR family. The 4 bp deletion yields a truncated protein that is expected to be deficient in NADPH binding and possibly in FAD binding, since the C-terminus of the enzyme may be involved in both. The 3 bp deletion results in the deletion of Leu576, which is located between two sequences that may be involved in NADPH binding. Leu576 is well conserved among reductases that are similar to the methionine synthase reductase (FIG. 6C). This supports the idea that deletion of the Leu576 codon (1726deITTG) results in an enzymatic defect, although confirmation will require expression of the mutant protein. This residue is also conserved in the NADPH-ferredoxin (flavodoxin) reductase enzymes of several organisms, although the homology with this portion of the protein is low or absent in some cases. It is possible that the deletion affects the relationship between the two NADPH-binding sequences that are in its vicinity.

The cloning of human methionine synthase reductase cDNA enables the determination of the enzymatic mechanism involved in the reductive activation of methionine synthase. Furthermore, it is now possible to identify additional mutations in patients with severe deficiency of the enzyme activity, and to determine whether there exist common amino acid polymorphisms which lead to mildly elevated homocysteine levels. Such elevations may be a risk factor in cardiovascular disease, neural tube defects, and cancer.

Mutations in the human methionine synthase reductase gene that result in altered homocysteine and/or folate levels may be risk factors for the diseases listed above. The methods of the invention therefore provide diagnostic assays for such risk factors, as well as methods of treating or preventing cardiovascular disease, neural defects, cancer, megaloblastic anemia, and hypomethioninemia. In addition, the invention provides methods for screening assays for the isolation of potential therapeutic compounds that modulate methionine synthase reductase activity.

The assays described herein can be used to test for compounds that modulate methionine synthase activity and hence may have therapeutic value in the prevention of neural tube defects, prevention and/or treatment of cancer, cardiovascular disease, homocysteinemia, and megaloblastic anemia.

Test Compounds

In general, novel drugs for prevention of neural tube defects, or prevention and/or treatment of cancer, cardiovascular disease, and megaloblastic anemia are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their therapeutic activities for homocysteinemia, megaloblastic anemia, cardiovascular disease, cancer, and neural tube defects should be employed whenever possible.

When a crude extract is found to modulate methionine synthase reductase biological activity, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that modulates methionine synthase reductase biological activity. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using mammalian models of homocysteinemia, megaloblastic anemia, cardiovascular disease, cancer, and neural tube defects.

Methionine synthase reductase assays for the detection of compounds that modulate methionine synthase reductase activity and expression

Potentially useful therapeutic compounds that modulate (e.g. increase or decrease) methionine synthase reductase activity or expression may be isolated by various screens that are well-known to those skilled in the art. Such compounds may modulate methionine synthase reductase expression at the pre- or post-transcriptional level, or at the pre- or post-translational level.

A. Screens for compounds that modulate methionine synthase reductase enzymatic activity

Screens for potentially useful therapeutic compounds that modulate methionine synthase reductase activity may be readily performed. For example, the effect of a test compound on methionine synthase reductase activity may be determined by measuring formation of .sup.14CH.sub.3-cob(III)alamin, which results from the transfer of .sup.14CH.sub.3 from S-adenosylmethionine to methionine synthase-cob(II)alamin. A test compound that increases the enzymatic activity of a methionine synthase reductase would result in increased levels of methionine synthase-.sup.14CH.sub.3-cob(III)alamin, and a compound that decreases the enzymatic activity of a methionine synthase reductase would result in decreased levels of methionine synthase-.sup.14CH.sub.3-cob(III)alamin.

The effect of a test compound on methionine synthase reductase activity also may be determined by measuring the resulting activity of methionine synthase. The amount of reaction product (i.e., methionine) formation reflects the relative activity of methionine synthase, which in turn reflects the relative activity of methionine synthase reductase, which in turn indicates the effect of the test compound on methionine synthase reductase activity. For example, a sample containing methionine synthase and homocysteine may contain a mutant, inactive methionine synthase reductase which does not reduce oxidized methionine synthase, and hence, no methionine is formed. However, a test compound that increases the enzymatic activity of the mutant methionine synthase reductase will result in increased levels of methionine formation, relative to control samples not containing the test compound. Analogously, a compound that decreases methionine synthase reductase activity will result in the formation of decreased levels of methionine formation in reactions containing active methionine synthase reductase. That a test compound directly modulates methionine synthase reductase enzymatic activity, as opposed to methionine synthase enzymatic activity, can be confirmed by including control reactions that lack methionine synthase reductase. Such control reactions should not show altered levels of methionine production if the test compound directly modulates methionine synthase reductase activity.

Examples of methionine synthase activity assays, in vitro and in whole cells, are well-known to those skilled in the art (see, for example, Gulati et al., 1997, J. Biol. Chem. 272:19171-19175; see also Rosenblatt et al., 1984, J. Clin. Invest. 74:2149-2156).

B. ELISA for the detection of compounds that modulate methionine synthase reductase expression

Enzyme-linked immunosorbant assays (ELISAs) are easily incorporated into high-throughput screens designed to test large numbers of compounds for their ability to modulate levels of a given protein. When used in the methods of the invention, changes in a given protein level of a sample, relative to a control, reflect changes in the methionine synthase reductase expression status of the cells within the sample. Protocols for ELISA may be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1997. Lysates from cells treated with potential modulators of methionine synthase reductase expression are prepared (see, for example, Ausubel et al., supra), and are loaded onto the wells of microtiter plates coated with "capture" antibodies specific for methionine synthase reductase. Unbound antigen is washed out, and a methionine synthase reductase-specific antibody, coupled to an agent to allow for detection, is added. Agents allowing detection include alkaline phosphatase (which can be detected following addition of colorimetric substrates such as p-nitrophenolphosphate), horseradish peroxidase (which can be detected by chemiluminescent substrates such as ECL, commercially available from Amersham) or fluorescent compounds, such as FITC (which can be detected by fluorescence polarization or time-resolved fluorescence). The amount of antibody binding, and hence the level of a methionine synthase reductase polypeptide within a lysate sample, is easily quantitated on a microtiter plate reader.

As a baseline control for methionine synthase reductase expression, a sample that is not exposed to test compound is included. Housekeeping proteins are used as internal standards for absolute protein levels. A positive assay result, for example, identification of a compound that increases or decreases methionine synthase reductase expression, is indicated by an increase or decrease in methionine synthase reductase polypeptide within a sample, relative to the methionine synthase reductase level observed in cells which are not treated with a test compound.

C. Reporter gene assavs for compounds that modulate methionine synthase reductase expression

Assays employing the detection of reporter gene products are extremely sensitive and readily amenable to automation, hence making them ideal for the design of high-throughput screens. Assays for reporter genes may employ, for example, colorimetric, chemiluminescent, or fluorometric detection of reporter gene products. Many varieties of plasmid and viral vectors containing reporter gene cassettes are easily obtained. Such vectors contain cassettes encoding reporter genes such as lacZ/.beta.-galactosidase, green fluorescent protein, and luciferase, among others. Cloned DNA fragments encoding transcriptional control regions of interest (e.g. that of the mammalian methionine synthase reductase gene) are easily inserted, by DNA subcloning, into such reporter vectors, thereby placing a vector-encoded reporter gene under the transcriptional control of any gene promoter of interest. The transcriptional activity of a promoter operatively linked to a reporter gene can then be directly observed and quantitated as a function of reporter gene activity in a reporter gene assay.

Cells are transiently- or stably-transfected with methionine synthase reductase control region/reporter gene constructs by methods that are well known to those skilled in the art. Transgenic mice containing methionine synthase reductase control region/reporter gene constructs are used for late-stage screens in vivo. Cells containing methionine synthase reductase/reporter gene constructs are exposed to compounds to be tested for their potential ability to modulate methionine synthase reductase expression. At appropriate timepoints, cells are lysed and subjected to the appropriate reporter assays, for example, a colorimetric or chemiluminescent enzymatic assay for lacZ/.beta.-galactosidase activity, or fluorescent detection of GFP. Changes in reporter gene activity of samples treated with test compounds, relative to reporter gene activity of appropriate control samples, indicate the presence of a compound that modulates methionine synthase reductase expression.

D. Quantitative PCR of methionine synthase reductase MRNA as an assay for compounds that modulate methionine synthase reductase expression

The polymerase chain reaction (PCR), when coupled to a preceding reverse transcription step (rtPCR), is a commonly used method for detecting vanishingly small quantities of a target mRNA. When performed within the linear range, with an appropriate internal control target (employing, for example, a housekeeping gene such as actin), such quantitative PCR provides an extremely precise and sensitive means of detecting slight modulations in mRNA levels. Moreover, this assay is easily performed in a 96-well format, and hence is easily incorporated into a high-throughput screening assay. Cells are treated with test compounds for the appropriate time course, lysed, the mRNA is reverse-transcribed, and the PCR is performed according to commonly used methods, (such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1997), using oligonucleotide primers that specifically hybridize with methionine synthase reductase nucleic acid. Changes in product levels of samples exposed to test compounds, relative to control samples, indicate test compounds that modulate methionine synthase reductase expression.

Secondary screens of test compounds that appear to modulate methionine synthase reductase activity

After test compounds that appear to have methionine synthase reductase-modulating activity are identified, it may be necessary or desirable to subject these compounds to further testing. At late stages testing will be performed in vivo to confirm that the compounds initially identified to affect methionine synthase reductase activity will have the predicted effect in vivo. Such tests may be performed using cells or animals that have wild-type, mutated, or deleted methionine synthase reductase genes, or wild-type or mutated methionine synthase reductase transgenes.

Therapy

Compounds identified using any of the methods disclosed herein, may be administered to patients or experimental animals with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients or experimental animals. Although intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for antagonists or agonists of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
 


Claim 1 of 21 Claims

1. A method for detecting an increased risk of developing Down's Syndrome in a mammalian embryo or fetus, said method comprising detecting the presence of a polymorphic methionine synthase reductase (MTRR) in said embryo or fetus, or in a future female parent of said embryo or said fetus, wherein detection of a homozygous MTRR polymorphism in said future female parent, said embryo, or said fetus indicates an increased risk of developing Down's Syndrome in said embryo or said fetus, wherein said polymorphism comprises a G instead of an A at position 66 relative to the first nucleotide of the start codon of MTRR.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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