WO2006044633A1 - ENZYME CYCLING BASED ASSAYS FOR ALPHA-METHYLACYL-CoA RACEMASE - Google Patents
ENZYME CYCLING BASED ASSAYS FOR ALPHA-METHYLACYL-CoA RACEMASE Download PDFInfo
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- WO2006044633A1 WO2006044633A1 PCT/US2005/036983 US2005036983W WO2006044633A1 WO 2006044633 A1 WO2006044633 A1 WO 2006044633A1 US 2005036983 W US2005036983 W US 2005036983W WO 2006044633 A1 WO2006044633 A1 WO 2006044633A1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Definitions
- This invention relates generally to the field of alpha-methylacyl-CoA racemase detection.
- the invention provides methods and kits for assaying alpha- methylacyl-CoA racemase in samples.
- Alpha-methylacyl-CoA racemase is a well-characterized enzyme which plays a key role in peroxisomal ⁇ -oxidation of dietary branched-chain fatty acids and C27-bile acid intermediates.
- AMACR catalyzes the conversion of (R)- ⁇ -methyl-branched-chain fatty acyl-CoA esters to their (S)- stereoisomers.
- AMACR was isolated from human liver as a 47-kDa monomer and is mainly localized in peroxisomes with a small amount detected in the mitochondria. Schmitz et al., Eur. J. Biochem. 231:815-822 (1995); Schmitz et al., Eur. J. Biochem. 222:313-23 (1994).
- the isoelectric point for AMACR isolated from human liver is pH 6.1, and the enzyme is optimally active between pH 7 and pH 8.
- AMACR Elevated expression of AMACR has been shown to be a biomarker for several types of cancer such as prostate, colorectal, ovarian, breast, bladder, lung, renal cell carcinoma, lymphoma, and melanoma.
- Rubin et al. JAMA 287: 1662-1670 (2002); Luo et al., Cancer Res. 62:2220-2226 (2002); Sreekumar et al., J Natl. Cancer Inst. 96:834-843 (2004).
- PCT WO 02/27324 and U.S. Appl. Pub. 2002/0123081 describes methods for identifying patients having or at risk of developing prostate cancer and patients having or at risk of developing a cancer arising from metastasis of a prostate cancer to another tissue by measuring the expression or activity of AMACR.
- AMACR. Schmitz et al. (Eur. J. Biochem. 222:313-23 (1994)) describe a radiometric assay for AMACR enzymatic activity, in which 2-methyl[2-(3)H]acyl-CoAs is used as substrate.
- Veldhoven et al. (Biochem. Biophys. Acta 1347:62-68 (1997)) describe an alternative enzymatic assay for AMACR, in which AMACR is combined with 2R-methyl-pentadecanoyl- CoA.
- reaction product a 2S-isomer
- oxidase pristanoyl CoA oxidase
- the invention provides a method for assaying an alpha-methylacyl-CoA racemase in a sample, said method comprises: a) contacting a sample suspected of containing an alpha-methylacyl-CoA racemase with a (2R)-2-methylacyl-CoA to generate a (2S)-2- methylacyl-CoA; b) converting said (2S)-2-methylacyl-CoA from step a) if generated to trans- 2,3-dehydroacyl-CoA in the presence of a (2S)-2-methylacyl-CoA converting enzyme and a first electron acceptor in its oxidized form, whereby reduced form of the first electron acceptor is generated; said trans-2,3-dehydroacyl-CoA is converted back to (2S)-2-methylacyl-CoA in the presence of a trans-2,3-dehydroacyl-CoA converting enzyme and a second electron acceptor in its reduced form to form a cycling reaction system, whereby oxidized form of
- the (2R)-2-methylacyl-CoA is a (2R)-2 -methyl-branched acyl-CoA with chain lengths for the acyl group from C(4) to C(30), from C(8) to C(25), or from C(IO) to C(25).
- the (2R)-2-methylacyl-CoA is (2R)-pristanoyl- CoA.
- the (2R)-2-methylacyl-CoA is (25R)-3 ⁇ , 7 ⁇ , 12 ⁇ -trihydroxy-5 ⁇ - cholestanoyl-CoA.
- the (2S)-2-methylacyl-CoA converting enzyme and the first electron acceptor in its oxidized form are added into the sample first, and the sample is then contacted with (2R)-2-methylacyl-CoA, tran-2,3-dehydroacyl-CoA concerting enzyme, and the second electron acceptor in its reduced form.
- the sample suspected of containing an alpha-methylacyl-CoA racemase is contacted with. (2R)-2- methylacyl-CoA, the (2S)-2-methylacyl-CoA converting enzyme, and the first electron acceptor in its oxidized form.
- the sample suspected of containing an alpha-methylacyl-CoA racemase is contacted with (2R)-2-methylacyl-CoA, the (2S)-2- methylacyl-CoA converting enzyme, the first electron acceptor in its oxidized form, the trans- 2,3-dehydroacyl-CoA converting enzyme, and the second electron acceptor in its oxidized form.
- the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme are different.
- the (2S)-2-methylacyl-CoA converting enzyme is an acyl-CoA dehydrogenase, and the first electron acceptor is selected from the group consisting OfNAD + , NADP + , thio-NAD + , thio-NADP + , acetyl-NAD + , and acetyl-NADP + .
- the (2S)-2-methylacyl-CoA converting enzyme is an acyl-CoA oxidase, and the first electron acceptor is O 2 .
- the trans-2,3-dehydroacyl-CoA converting enzyme is a trans-2-enoyl-CoA reductase
- the second electron acceptor is selected from the group consisting of NADH, NADPH, thio-NADH, thio-NADPH, acetyl-NADH, and acetyl-NADPH.
- the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme are the same.
- both the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme are acyl-CoA dehydrogenase. In some embodiments, both the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme axe trans-2-enoyl- CoA reductase.
- the oxidized form of the first electron acceptor may be selected from the group consisting of NAD + , NADP + , thio-NAD + , thio-NADP + , acetyl-NAD + , and acetyl- NADP +
- the reduced form of the second electron acceptor may be selected from the group consisting of NADH, NADPH, thio-NADH, thio-NADPH, acetyl-NADH, and acetyl-NADPH.
- the methods of the invention may further comprise a step of coupling the oxidized or reduced form of the first electron acceptor or the reduced or oxidized form of the second electron acceptor to a color-producing agent for assessing concentration change of the reduced or oxidized form of the first electron acceptor or the reduced or oxidized form of the second electron acceptor, wherein the concentration change of the oxidized or reduced form of the first electron acceptor or the reduced or oxidized form of the second electron acceptor is assessed by a colorimetric method.
- the sample may be assayed by the methods of the invention includes a biological sample.
- the biological sample is a biological fluid, such as blood, serum, plasma, and urine.
- the biological sample is selected from the group consisting of prostate, colon, ovary, breast, bladder, lung, renal, lymphocytes.
- the methods of the invention may be used for prognosis and/or diagnosis of cancer in an individual.
- the cancer is selected from the group consisting of prostate cancer, colorectal cancer, ovarian cancer, breast cancer, bladder cancer, lung cancer, renal cancer, lymphoma, and melanoma.
- the method of the invention is used for prognosis and/or diagnosis of prostate cancer in an individual by assaying alpha-methylacyl-CoA racemase activity in a blood sample (such as whole blood, plasma, and serum).
- the method further comprises a step of comparing the amount of alpha-methylacyl-CoA racemase in the sample from the individual to a predetermined value, whereby an increase of the amount of alpha-methylacyl-CoA racemase indicates the individual having or at risk of developing cancer.
- the cancer is selected from the group consisting of prostate cancer, colorectal cancer, ovarian cancer, breast cancer, bladder cancer, lung cancer, renal cancer, lymphoma, and melanoma.
- kits for assaying an alpha-methylacyl-CoA racemase in a sample said kit comprises: (2R)-2-methylacyl-CoA, a first electron acceptor in its oxidized form, a (2S)-2-methylacyl-CoA converting enzyme which catalyzes conversion of (2S)-2-methylacyl-CoA to trans-2,3-dehydroacyl-CoA in the presence of the first electron acceptor in its oxidized form, a second electron acceptor in its reduced form, and a trans-2,3- dehydroacyl-CoA converting enzyme which catalyzes conversion of trans-2,3-dehydroacyl- CoA to (2S)-2-methylacyl-CoA in the presence of the second electron acceptor in its reduced form, wherein the first electron acceptor and the second electron acceptor are different.
- the (2R)-2-methylacyl-CoA is a (2R)-2 -methyl-branched acyl-CoA with chain lengths for the acyl group from C(4) to C(30), from C(8) to C(25), or from C(IO) to C(25).
- the (2R)-2-methylacyl-CoA is (2R)-pristanoyl- CoA.
- the (2R)-2-methylacyl-CoA is (25R)-3 ⁇ , 7ot, 12 ⁇ -trihydroxy-5 ⁇ - cholestanoyl-CoA.
- the (2S)-2-methylacyl-CoA converting enzyme and trans-2,3-dehydroacyl-CoA converting enzyme are different.
- the (2S)- 2-methylacyl-CoA converting enzyme is an acyl-CoA dehydrogenase, aad the first electron acceptor is selected from the group consisting OfNAD + , NADP + , thio-NAD + , thio-NADP + , acetyl-NAD + , and acetyl-NADP + .
- the (2S)-2-metliylacyl-CoA converting enzyme is an acyl-CoA oxidase, and the first electron acceptor is O 2 .
- the trans-2,3-dehydroacyl-CoA converting enzyme is a trans-2-enoyl-CoA reductase, and the second electron acceptor is selected from the group consisting of NADH, NADPH, thio-NADH, thio-NADPH, acetyl-NADH, and acetyl-NADPH.
- the (2S)-2-methylacyl-CoA converting enzyme and trans-2,3-dehydroacyl-CoA converting enzyme are the same.
- both the (2S)-2-methylacyl-CoA converting enzyme and trans-2,3-dehydroacyl-CoA converting enzyme are acyl-CoA dehydrogenase or trans-2-enoyl-CoA reductase
- the oxidized form of the first electron acceptor is selected from the group consisting of NAD , NADP + , thio- NAD + , Mo-NADP + , acetyl-NAD + , and acetyl-NADP +
- the reduced form of the second electron acceptor is selected from the group consisting of NADH, NADPH, thio-NADH, thio- NADPH, acetyl-NADH, and acetyl-NADPH.
- the kit may further comprises an instruction indicating use for prognosis and/or diagnosis of cancer including, but not limited to, prostate cancer, colorectal cancer, ovarian cancer, breast cancer, bladder cancer, lung cancer, renal cancer, lymphoma, and melanoma.
- the present invention provides methods for assaying alpha-methylacyl-CoA racemase activity.
- a sample containing alpha-methylacyl-CoA racemase or suspected of containing alpha-methylacyl-CoA racemase is contacted with (2R)-2-methylacyl- CoA. If enzymatically active alpha-methylacyl-CoA racemase is present in the sample, (2R)- 2-methylacyl-CoA is converted into (2S)-2-methylacyl-CoA.
- the method then utilizes a cycling reaction system between (2S)-2-methylacyl-CoA and trans-2,3-dehydroacyl-CoA to generate a detectable signal that corresponds to the enzymatic activity of alpha-methylacyl- CoA racemase.
- the methods described herein can be used for diagnosing cancer.
- the methods can be used for diagnosing prostate cancer using blood sample without biopsy.
- alpha-methylacyl-CoA racemase or "AMACR” (EC 5.1.99.4) refers to an enzyme that catalyzes conversion between (2S)-2-methylacyl-CoA and (2R)-2- methylacyl-CoA. It is intended to encompass derivatives, variants, and analogs of alpha- methylacyl-CoA racemase that do not substantially alter its activity.
- Alpha-methylacyl-CoA racemase can be obtained from any sources, such as human, mouse, bovine, rat, fruit fly, etc.
- (2S)-2-methylacyl-CoA converting enzyme refers to an enzyme which catalyzes formation of trans-2,3-dehydroacyl-CoA from (2S)-2-methylacyl- CoA in the presence of an electron acceptor in its oxidized form. It is intended to encompass derivatives, variants, and analogs of (2S)-2-methylacyl-CoA converting enzyme that do not substantially alter its activity.
- trans-2,3-dehydroacyl-CoA converting enzyme refers to an enzyme which catalyzes formation of (2S)-2-methylacyl-CoA from trans-2,3-dehydroacyl- CoA in the presence of an electron acceptor in its reduced form. It is intended to encompass derivatives, variants, and analogs of trans-2,3-dehydroacyl-CoA converting enzyme that do not substantially alter its activity.
- assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the amount or concentration of the analyte present in the reaction system, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of analyte in the reaction system.
- Assessment may be direct or indirect and the chemical species actually detected need not of course be the analyte itself but may for example be a derivative thereof or some further substance.
- sample refers to anything which may contain an analyte for which an analyte assay is desired.
- the sample may be a biological sample, such as a biological fluid or a biological tissue.
- biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.
- Biological tissues are aggregates of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues.
- biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
- blood sample includes whole blood, serum, and plasma.
- serum refers to the fluid portion of the blood obtained after removal of the fibrin clot and blood cells, distinguished from the plasma in circulating blood.
- plasma refers to the fluid, noncellular portion of the blood, distinguished from the serum obtained after coagulation.
- Fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
- disease or disorder refers to a pathological condition in an organism resulting from, e.g., infection or genetic defect, and characterized by identifiable symptoms.
- contacting means bringing two or more components together.
- Contacting can be achieved by mixing all the components in a fluid or semi-fluid mixture. “Contacting” can also be achieved when one or more components are brought into contact with one or more other components on a solid surface such as a solid tissue section or a substrate. [0040] As used herein, a "cycling reaction system” refers to the process of converting
- the invention provides a method for assaying an alpha-methylacyl-CoA racemase in a sample, said method comprises: a) contacting a sample suspected of containing an alpha-methylacyl-CoA racemase with a (2R)-2-methylacyl-CoA to generate a (2S)-2- methylacyl-CoA; b) converting said (2S)-2-methylacyl-CoA from step a) if generated to trans- 2,3-dehydroacyl-CoA in the presence of a (2S)-2-methylacyl-CoA converting enzyme and a first electron acceptor in its oxidized form, whereby reduced form of the first electron acceptor is generated; said trans-2,3-dehydroacyl-CoA is converted back to (2S)-2-methylacyl-CoA in the presence of a trans-2,3-dehydroacyl-CoA converting enzyme and a second electron acceptor in its reduced form to form a cycling reaction system, whereby oxidized form of
- the detectable signal generated in step c) is a concentration change of the reduced or oxidized form of the first electron acceptor or the reduced or oxidized form of the second electron acceptor in said cycling reaction system.
- the sample is a blood sample (such as whole blood, serum, and plasma).
- the methods for assaying an alpha-methylacyl-CoA racemase in the present invention are based on measuring an enzymatic activity catalyzed by an alpha-methylacyl-CoA racemase.
- Alpha-methylacyl-CoA racemases catalyze reactions which convert (2R)-2- methylacyl-CoA to (2S)-2-methylacyl-CoA.
- the presence or absence and the amount of an alpha-methylacyl-CoA racemase in a sample can Toe determined by determining the (2S)-2-methylacyl-CoA generated in these reactions.
- an exemplary reaction scheme for step a) of the present invention is:
- the acyl group may be derived from any fatty acids.
- the acyl group may be branched acyl (e.g., fatty acyl) witih chain lengths of at least C(4), at least C(6), at least C(8), at least C(IO) or more.
- the chain lengths may be C(4) to C(30), C(4) to C(16), C(8) to C(25), C(8) to C(18), C(IO) to C(25), or C(12) to C(25).
- the acyl group may include aromatic compounds (e.g., ibuprofen) and bile acid intermediates, such as trihydroxycoprostanoyl-CoA.
- the (2R)-2-methylacyl-CoA is (2R)- pristanoyl-CoA.
- the (2R)-2-methylacyl-CoA is (2R, 6R, 10)- trimethylundecanoyl-CoA.
- the (2R)-2-methylacyl-CoA is (2R, 6)- dimethylheptanoyl-CoA.
- the (2R)-2-methylacyl-CoA is (2R)-methyl- pentadecanoyl-CoA.
- the (2R)-2-methylacyl-CoA is (25R)-3 alpha, 7 alpha, 12 alpha-trihydroxy-Sbeta-chlestanoyl-CoA.
- the (2R)-2-methylacyl-CoA in step a) can be of any concentration that is suitable for an alpha-methylacyl-CoA racemase reaction and depends on the activity of the alpha-methylacyl-CoA racemase in the sample.
- concentration of (2R) ⁇ 2- methylacyl-CoA is about 0.01 mM to about 100 mM; about 0.05 mM to about 50 mM, about 0.5 mM to about 10 mM, or about 1 mM to about 5 mM.
- the enzymatic reaction is generally carried out in a condition (such as buffer and temperature) suitable for the completion of the enzymatic reactions.
- the buffer can be a phosphate buffer with a pH of about 6 to about 8; a Tris-HCl buffer with a pH of about 7 to about 9, or a Good's buffer with a pH of about 6 to about 9.
- the reaction may be carried out at a temperature between about 3O 0 C to 37 0 C for 5, 10, 15, 30, or 60 min. The reaction may be terminated before the next step.
- any sample containing or suspected of containing an alpha-methylacyl-CoA racemase can be assayed using the present invention, hi some embodiments, the sample is blood (including whole blood, serum and plasma) or urine, hi some embodiments, the sample is a tissue sample (e.g., prostate, colon, ovary, breast, bladder, lung, renal, and lymphocytes). In some embodiments, the tissue sample is subject to hornogenization to obtain a crude tissue homogenate before the assay is conducted. [0046] In some embodiments, the sample is pre-processed before the assay is carried out. Exemplary processing steps include centrifugation, extraction/washing, cell lysis, freeze/thaw, and sonication. The sample may also be diluted before the assay.
- Step b) of the present invention converts (2S)-2-methylacyl-CoA to trans-2,3- dehydroacyl-CoA in the presence of a first electron acceptor (in its oxidized form) through the action of a (2S)-2-methylacyl-CoA converting enzyme, and then from trans-2,3-dehydroacyl- CoA back to (2S)-2-methylacyl-CoA in the presence of a second electron acceptor (in its reduced form) through the action of a trans-2,3-dehydroacyl-CoA converting enzyme.
- the first electron acceptor and the second electron acceptor are different electron acceptors.
- Step b) thus forms a cycling reaction system generating an. increase of the first electron acceptor in its reduced form and a reduction of the first electron acceptor in its oxidized form, and an increase of the second electron acceptor in its oxidized form and a reduction of the second electron acceptor in its reduced form.
- the increase of an electron acceptor in a specific form or the reduction of an electron acceptor in a specific form can be measured.
- an electron acceptor in a specific form is accumulated and the accumulation is assessed.
- the (2S)-2-mettiylacyl-CoA converting enzyme and the trans ⁇ 2,3-dehydroacyl-CoA converting enzyme are different.
- the (2S)- 2-methylacyl-CoA converting enzyme does not cross-react with the second electron acceptor or binds to the second electron acceptor with a lower affinity than to the first electron acceptor
- the trans-2,3-dehydroacyl-CoA converting enzyme does not cross-react with the first electron acceptor or binds to the first electron acceptor with a lower affinity than to the first electron acceptor.
- Any (2S)-2-methylacyl-CoA converting enzyme that catalyzes formation of trans-2,3-dehydroacyl-CoA from (2S)-2-methylacyl-CoA in the presence of an electron acceptor in its oxidized form can be used.
- the (2S)-2-metlrylacyl-CoA converting enzyme is an acyl-CoA dehydrogenase (EC 1.3.1.8).
- acyl-CoA dehydrogenase EC 1.3.1.8
- Biophys. Acta 164:498-517 (1968)) can be " used.
- the (2S)-2-methylacyl-CoA converting enzyme is an acyl-CoA oxidase (EC 1.3.3.6).
- acyl-CoA oxidase (EC 1.3.3.6) having the amino acid sequence with the following GenBank Accession Nos.
- T52121 (Arabidopsis thaliana), T52120 (Arabidopsis thaliana), 138095 (Homo sapiens), S64224 (Saccaromyces cerevisiae), A54942 (Homo sapiens), 0XRTA2 (Rattus norveqicus), OXRTAl (Rattus norveqicus), OXCKX5 (candida tropicalis) can be used.
- the (2S)-2-methylacyl-CoA converting enzyme is a trans-2-enoyl-CoA reductase (EC 1.3.1.38) .
- any trans-2-enoyl-reductase (EC 1.3.1.38) is a trans-2-enoyl-CoA reductase (EC 1.3.1.38) .
- any trans-2-enoyl-reductase (EC 1.3.1.38) any trans-2-enoyl-reductase
- (2S)-2-methylacyl-CoA from trans-2,3-debrydroacyl-CoA in the presence of an electron acceptor in its reduced form can be used.
- the trans-2,3-dehydroacyl-CoA converting enzyme is a trans-2-enoyl-CoA reductase (EC 1.3.1.38) . Any trans-2-enoyl-CoA reductase known in the art and described herein can be used.
- the trans-2,3-dehydroacyl-CoA converting enzyme is an acyl-CoA dehydrogenase (EC 1.3.1.8). Any acyl-CoA dehydrogenase known in the art and described herein can be used.
- the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme are the same. In one embodiment, both the
- (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme are acyl-CoA dehydrogenase.
- both the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehyclroacyl-CoA converting enzyme are trans-2-enoyl-
- CoA reductase Any acyl-CoA dehydrogenase and trans-2,3-dehydroacyl-CoA converting enzyme known in the art and described herein may be used.
- the first and second electron acceptors can be any electron acceptors that are compatible with the enzymes chosen for trie reactions, provided that the first electron acceptor and the second electron acceptor are different.
- the (2S)-2-methylacyl ⁇ CoA converting enzyme is an acyl-CoA dehydrogenase and the first electron acceptor is selected from the group consisting OfNAD + , NADP + , thio-NAD + , thio-NADP + , acetyl-NAD + , and acetyl-NADP + .
- the (2S)-2-methylacyl-CoA converting enzyme is an acyl-CoA oxidase and the first electron acceptor is O 2 .
- the (2S)-2-methylacyl-CoA converting enzyme is a trans-2-enoyl-CoA reductase, and the first electron acceptor is selected from the group consisting OfNAD + , NADP + , thio-NAD + , thio-NADP + , acetyl-NAD + , and acetyl- NADP + .
- the trans-2,3-dehydroacyl-CoA converting enzyme is a trans-2-enoyl-CoA reductase
- the second electron acceptor is selected from the group consisting of NADH, NADPH, thio-NADH, thio-NADPH, acetyl-NADH, and acetyl-NADPH.
- the trans-2,3-dehydroacyl-CoA converting enzyme is an acyl-CoA dehydrogenase and the second electron acceptor is selected from the group consisting of NADH, NADPH, thio-NADH, thio-NADPH, acetyl-NADH, and acetyl-NADPH.
- the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme may collectively catalyze a cycling reaction between (2S)-2-methylacyl-CoA and trans-2,3-dehydroacyl-CoA that amplifies signals generated in the assay.
- the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3- dehydroacyl-CoA converting enzyme may utilize different electron acceptors and do not cross- react with electron acceptors used by the other enzyme or react with low affinity. Alternatively, the two enzymes may recognize the same electron acceptors. Sufficient amount of oxidized form of the first electron acceptor and reduced form of the second electron acceptor can be added to the cycling reaction to drive both reactions and allow the cycle to go on until sufficient signal is generated.
- the (2S)-2-methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme are different.
- the (2S)-2-methylacyl-CoA converting enzyme is an acyl-CoA dehydrogenase (EC 1.3.1.8)
- the trans-2,3-dehydroacyl-CoA converting enzyme is a trans- 2-enoyl-CoA reductase (EC 1.3.1.38).
- an exemplary reaction scheme for step b) is:
- reaction (2a) represents a reaction catalyzed by an acyl-CoA dehydrogenase
- reaction (2b) represents a reaction catalyzed by a trans-2-enoyl-CoA reductase
- (2S)-2- methylacyl-CoA is (2S)-pristanoyl-CoA
- trans-2,3-dehydroacyl-CoA is trans-2,3- dehydropristanoyl-CoA.
- Sufficient thio-NADP + and NADH can be added to the reaction to generate a cycling reaction, which leads to accumulation of thio-NADPH.
- an acyl-CoA oxidase (EC 1.3.3.6) is used as the (2S)-2 ⁇ methylacyl-CoA converting enzyme, and a trans-2-enoyl-CoA reductase (EC 1.3.1.38) is used as the trans-2,3-dehydroacyl-CoA converting enzyme in the cycling reaction.
- An exempLary reaction scheme is:
- Reaction (3 a) represents a reaction catalyzed by an acyl-CoA oxidase
- reaction (3b) represents a reaction catalyzed by a trans-2-enoyl-CoA reductase
- (2S)-2t- methylacyl-CoA is (2S)-pristanoyl-CoA
- trans-2,3 -dehydroacyl-CoA is trans-2,3- dehydropristanoyl-CoA.
- Sufficient amount of O 2 and NADPH can be added to the reaction to generate a cycling reaction, which leads to accumulation of NADP and H 2 O 2 .
- the following is an exemplary reaction scheme, in which the (2S)-2- methylacyl-CoA converting enzyme and the trans-2,3-dehydroacyl-CoA converting enzyme are the same.
- One such exemplary reaction scheme is:
- Both reaction (4a) and (4b) are catalyzed by the same enzyme, which is an acyl-CoA dehydrogenase (EC 1.3.1.8) or a trans-2-enoyl-CoA reductase (EC 1.3.1.38).
- acyl-CoA dehydrogenase EC 1.3.1.8
- trans-2-enoyl-CoA reductase EC 1.3.1.38
- (2S)-2-methylacyl-CoA is (2S)-pristanoyl-CoA
- trans-2,3 -dehydroacyl-Co A is trans-2,3- dehydropristanoyl-CoA.
- Sufficient thio-NADP + and NADH can be added to the reaction mix to drive both reactions in a cycling reaction that leads to accumulation of thio-NADPH.
- any other combinations of (2S)-2-methylacyl-CoA converting enzymes a ⁇ d trans-2,3-dehydroacyl-CoA converting enzymes may be used to achieve the same cycling effect.
- the enzymatic reactions in steps a) and b) are generally carried out in a condition (such as buffer and temperature) suitable for the completion of the enzymatic reactions.
- the buffer used for step b) and step a) described herein can be the same or can be different.
- Any buffer known in the art suitable for the specific enzymatic reactions in step a) and/or b) can be used.
- the buffer can be a phosphate buffer with a pH of about 6 to about 8; a Tris-HCl buffer with a pH of about 7 to about 9, or a Good's buffer with a pH of " about 6 to about 9.
- the temperature for step b) can be the same or different from step a).
- the temperature in step b) is preferably between about 25 0 C to about 37°C.
- one or more steps described herein are carried out in a separate reaction mixture.
- the end products of one or more steps in the reaction can be partially or completely separated from the reaction mixture before reagents for the nexit step are added.
- steps a) and b) described herein are carried out in a single reaction mixture.
- the enzymes, substrates, or electronic acceptors for the next step are added sequentially to the same reaction mix at the end of a previous step.
- the reaction in a previous step is terminated before reagents for the next step are added.
- some or all reagents for more than one steps are added simultaneously to the reaction mixture.
- reagents for steps a) and b) are mixed with the sample at the same time, hi these embodiments, the sample suspected of containing an alpha-methylacyl-CoA racemase is contacted with (2R)-2- methylacyl-CoA, a (2S)-2-methylacyl-CoA converting enzyme, and the first electron acceptor in its oxidized form, a trans-2,3-dehydroacyl-CoA converting enzyme, and a second electron acceptor in its reduced form.
- reagents for steps a) and some of the reagents for step b) are mixed with the sample at the same time, hi these embodiments, the sample suspected of containing an alpha-methylacyl-CoA racemase is contacted with (2R)-2- methylacyl-CoA, a (2S)-2-methylacyl-CoA converting enzyme, a first electron acceptor in its oxidized form.
- reagents for step b) are mixed with the sample first, and reagents for step a) and other reagents for step b) are added later.
- the (2S)-2-methylacyl-CoA converting enzyme and the first electron acceptor In its oxidized form are added into the sample first, and the sample is then contacted with (2R.)-2- methylacyl-CoA, tran-2,3-dehydroacyl-CoA converting enzyme, and the second electron acceptor in its reduced form.
- the sample with the (2S)-2-methylaoyl- CoA converting enzyme and the first electron acceptor in its oxidized form are incubated to allow conversion of (2S)-2-methylacyl-CoA in the sample into tran-2,3-dehydroacyl-CoA.
- the quantification of alpha-methylacyl-CoA racemase in the sample can t>e achieved by monitoring the subtractive or additive difference in the signal generated in step b).
- the assessment can be carried out continuously or at different time points.
- the concentration change of the reduced or oxidized form of the first electron acceptor is assessed.
- the activity of alpria- methylacyl-CoA racemase is determined by assessing an increase in concentration of the reduced form of the first electron acceptor.
- the activity of alpha- methylacyl-CoA racemase is determined by assessing a decrease in concentration of the oxidized form of the first electron acceptor.
- the concentration change of the reduced or oxidized form of the second electron acceptor is assessed.
- the activity of alpha- methylacyl-CoA racemase is determined by assessing a decrease in concentration of the reduced form of the second electron acceptor.
- the activity of alptia- methylacyl-CoA racemase is determined by assessing an increase in concentration of the oxidized form of the second electron acceptor.
- concentration changes of electron acceptors described herein can be assessed using methods known in the art.
- concentration changes of NA ⁇ DH or NADPH can be determined spectrophotometrically by measuring the absorption at 340 run.
- concentration changes of thio-NADPH can be determined spectrophotometrically by measuring absorption at 405 nm.
- a preferred example is a combination of water-soluble tetrazolltim salt and diaphorase or phenazinemethosulfate.
- These electron transport chromogens are electron acceptors for NADP or NADPH to form a colored formazane pigment, and the formed pigment is colorimetrically measured at the maximum absorption thereof.
- a further assay method for NADH or NADPH is fluorometry wherein NAD or
- NADPH is treated with diaphorase in the presence of a fluorescent reagent such as resazulirx
- a fluorescent reagent such as resazulirx
- Measurement of concentration of reduced and/or oxidized forms of electron acceptors described herein are known in the art. For example, the amount of consumed O 2 or H 2 O 2 formed can be assayed by the use of electronic sensors. Numerous red-ox indicators can be used for this purpose, and a wide range of methods are described in the literature for assaying H 2 O 2 and O 2 in solution. Generated H 2 O 2 can also be measured as a detectable product by reacting with an indicator and H 2 O 2 .
- indicators are reagents which can be measured by spectrophotometric means, color indicators, fluorescent reagents or luminescent reagents.
- H 2 O 2 can be assessed using the non-enzymatic chemiluminescent reactions of peroxioxalate and the acridinium esters.
- Assays may be performed in duplicates with both positive and background controls.
- a standard curve can be obtained by using known amounts of alpha-methylacyl-CoA racemase with known activity. The levels of alpha-methylacyl-CoA racemase in each sample can then be determined by comparing each signal measured to the standard curve.
- the present invention provides an assay with increased sensitivity for detecting alpha-methylacyl-CoA racemase present in a sample, such a blood sample.
- the methods of the invention thus provides a practical means for detecting conditions associated with altered levels of alpha-methylacyl-CoA racemase and monitoring alpha-methylacyl-CoA racemas& levels in an individual.
- the method can be used for prognosis or diagnosis of any disease associated with an inappropriate amount or activity of alpha-methylacyl-CoA racemase, or the effect or activity of such, in a subject.
- the enzymatic assay of the present invention also provides a research tool for the exploration of the role of alpha-methylacyl-CoA racemase in biological processes and various pathological conditions.
- kits for assaying alpha-methylacyl-CoA racemase activities such as a diagnostic kit.
- kits comprise one or more substrates, enzymatic agents and electron acceptors described herein for carrying out the methods of the present invention.
- the kit comprises (2R)-2-methylacyl-CoA, a (2S)-2- methylacyl-CoA converting enzyme, a first electron acceptor in its oxidized form, a trans-2,3- dehydroacyl-CoA converting enzyme, and a second electron acceptor in its reduced form, wherein the first electron acceptor and the second electron acceptor are different.
- kits may also comprise positive and/or negative control standards, as well as necessary reagents for assessing the signals generated by step b), for example, reagents for conducting a colormetic assay may be included.
- the kits may also comprise an apparatus or container for conducting the methods of the invention and/or transferring samples to a diagnostic laboratory for processing, as well as suitable instructions for carrying out the methods of the invention.
- the kits of the invention may be in any suitable packaging.
- the packages discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. Such packages include containers appropriate for use in autoanalyzers.
- Example 1 Alpha-methylacyl-CoA racemase (AMACR) assay using acyl-CoA dehydrogenase and trans-2-enoyl-CoA reductase
- trie (2S)-2-methylacyl-CoA converting enzyme is acyl-CoA dehydrogenase; and the trans-2,3-dehydroacyl-CoA converting enzyme is trans-2-enoyl-CoA reductase.
- the reagents used in this study are set forth in the following Table 1 and Table 2. Table 1. Compositions of Reagent 1
- the (2S)-2-methylacyl-CoA converting enzyme is acyl-CoA oxidase; and the trans-2,3-dehydroacyl-CoA converting enzyme is trans-2-enoyl-CoA reductase.
- the reagents used in this study are set forth in the following Table 3 and Table 4.
- EHSPT N-ethyl-(2-hydroxy-3-sulfopropyl)- methylaniline
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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JP2007536931A JP2008516608A (en) | 2004-10-15 | 2005-10-14 | Enzyme cycling based assay for α-methylacyl-CoA racemase |
CA002583546A CA2583546A1 (en) | 2004-10-15 | 2005-10-14 | Enzyme cycling based assays for alpha-methylacyl-coa racemase |
EP05811858A EP1805322A4 (en) | 2004-10-15 | 2005-10-14 | ENZYME CYCLING BASED ASSAYS FOR ALPHA-METHYLACYL-CoA RACEMASE |
AU2005295677A AU2005295677A1 (en) | 2004-10-15 | 2005-10-14 | Enzyme cycling based assays for alpha-methylacyl-CoA racemase |
Applications Claiming Priority (4)
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US10/966,983 | 2004-10-15 | ||
US10/966,983 US7374897B2 (en) | 2004-10-15 | 2004-10-15 | Enzyme cycling based assays for alpha-methylacyl-CoA racemase |
US11/004,477 | 2004-12-03 | ||
US11/004,477 US7374902B2 (en) | 2004-10-15 | 2004-12-03 | Enzyme cycling based assays for alpha-methylacyl-CoA racemase |
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WO2006044633A1 true WO2006044633A1 (en) | 2006-04-27 |
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EP (1) | EP1805322A4 (en) |
JP (1) | JP2008516608A (en) |
AU (1) | AU2005295677A1 (en) |
CA (1) | CA2583546A1 (en) |
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US4491631A (en) * | 1981-06-25 | 1985-01-01 | Toyo Jozo Kabushiki Kaisha | Assay method for lipid component, assay composition, and process for production of enzyme used therefor |
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US20020123081A1 (en) * | 2000-09-28 | 2002-09-05 | Jennifer Richardson | Methods of use of alpha-methylacyl-CoA racemase in hormone refractory and metastatic prostate cancers |
-
2005
- 2005-10-14 WO PCT/US2005/036983 patent/WO2006044633A1/en active Application Filing
- 2005-10-14 AU AU2005295677A patent/AU2005295677A1/en not_active Abandoned
- 2005-10-14 EP EP05811858A patent/EP1805322A4/en not_active Withdrawn
- 2005-10-14 CA CA002583546A patent/CA2583546A1/en not_active Abandoned
- 2005-10-14 JP JP2007536931A patent/JP2008516608A/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4491631A (en) * | 1981-06-25 | 1985-01-01 | Toyo Jozo Kabushiki Kaisha | Assay method for lipid component, assay composition, and process for production of enzyme used therefor |
Non-Patent Citations (3)
Title |
---|
AMERY ET AL: "Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans.", J LIPID RESEARCH., vol. 41, 2000, pages 1752 - 1759, XP008118632 * |
SCHMITZ ET AL: "Molecular cloning of cDNA species for rat and mouse liver alpha-methylacyl-CoA racemases.", BIOCHEM J., vol. 326, 1997, pages 883 - 889, XP008118718 * |
See also references of EP1805322A4 * |
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EP1805322A1 (en) | 2007-07-11 |
AU2005295677A1 (en) | 2006-04-27 |
JP2008516608A (en) | 2008-05-22 |
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