Reagents and methods for screening mps i, ii, iiia, iiib, iva, vi, and vii

Patent metadata

Field	Value
Publication number	US-2016298166-A1
Application number	US-201414916526-A
Country	US
Kind code	A1
Filing date	Sep 5, 2014
Priority date	Sep 5, 2013
Publication date	Oct 13, 2016
Grant date	—

Abstract

Reagents, methods, and kits for assaying enzymes associated with lysosomal storage diseases MPS-I, MPS-II, MPS-IIIA, MPS-IIIB, MPS-IV A, MPS-VI, and MPS VII. In one aspect, the invention provides methods for assaying one or more enzymes associated with a lysosomal storage disease. In a first embodiment, the method includes: (a) contacting a sample with a first solution to provide a solution comprising one or more lysosomal enzymes; (b) contacting the one or more lysosomal enzymes in solution with an enzyme substrate for each lysosomal enzyme to be analyzed and incubating the substrates with the enzymes for a time sufficient to provide a solution comprising an enzyme product for each lysosomal enzyme present in the sample.

First claim

1 . A method for assaying for an enzyme associated with a lysosomal storage disease, comprising:
(a) contacting a sample with a first solution to provide a solution comprising one or more lysosomal enzymes; (b) contacting the one or more lysosomal enzymes in solution with an enzyme substrate for each lysosomal enzyme to be analyzed and incubating the substrates with the enzymes for a time sufficient to provide a solution comprising an enzyme product for each lysosomal enzyme present in the sample, wherein the enzyme substrate for each lysosomal enzyme is a compound having a carbohydrate moiety and an aglycone moiety and having the formula:

wherein S is the carbohydrate moiety that when covalently coupled to the aglycone moiety provides a substrate for an enzyme selected from the group consisting of:
(i) alpha-L-iduronidase;
(ii) iduronate 2-sulfatase;
(iii) heparan N-sulfatase;
(iv) N-acetyl-alpha-D-glucosaminidase;
(v) N-acetylgalactosamine 6-sulfate-sulfatase;
(vi) N-acetylgalactosamine 4-sulfate-sulfatase; and
(vii) beta-glucuronidase;
L 2 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, and S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;
L 3 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;
L 4 is optional and when present is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S), and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;
R 1 is a C 1 -C 10 alkyl group or a C 1 -C 10 alkoxy group;
R 2 at each occurrence is independently selected from a C 1 -C 10 alkyl group, a C 1 -C 10 alkoxy group, halogen, nitro, —C(═O)NHR, or —C(═O)OR, where R is C 1 -C 8 alkyl group;
R 3 is a C 1 -C 10 alkyl group or a substituted or unsubstituted C 6 -C 10 aryl group; and
n is 0, 1,2, 3, or 4; and
(c) determining the quantities of one or more of the enzyme products.

2 - 3 . (canceled)

4 . A method for assaying enzymatic activities of one or more lysosomal enzymes, comprising:
(a) contacting a sample with a first solution to provide a solution comprising one or more lysosomal enzymes; (b) contacting the one or more lysosomal enzymes in solution with an enzyme substrate for each lysosomal enzyme to be analyzed and incubating the substrates with the enzymes for a time sufficient to provide a solution comprising a first enzyme product for each lysosomal enzyme present in the sample; (c) subjecting the first enzyme products to a glycohydrolase to provide a second enzyme product for each first enzyme product susceptible to further enzymatic action by the glycohydrolase; and (d) determining the quantities of one or more of the first enzyme products and/or one or more of the second enzyme products.

5 - 7 . (canceled)

8 . The method of claim 1 , wherein the one or more lysosomal enzymes comprises an enzyme selected from the group consisting of:
(a) alpha-L-iduronidase; (b) iduronate 2-sulfatase; (c) heparan N-sulfatase; (d) N-acetyl-alpha-D-glucosaminidase; (e) N-acetylgalactosamine 6-sulfate-sulfatase; (f) N-acetylgalactosamine 4-sulfate-sulfatase; and (g) beta-glucuronidase.

9 - 14 . (canceled)

15 . The method of claim 1 , wherein determining the quantities of the enzyme products comprises mass spectrometric analysis.

16 - 18 . (canceled)

19 . The method of claim 1 , wherein determining the quantities of the enzyme products comprises conducting the products to a mass spectrometer by liquid chromatography or by flow injection.

20 . (canceled)

21 . The method of claim 1 further comprising using the quantities of the enzyme products to determine whether the sample is from a candidate for treatment for a condition associated with one or more lysosomal enzyme deficiencies.

22 . The method of claim 4 , wherein the substrate has a carbohydrate moiety and an aglycone moiety and has the formula:

wherein
S is the carbohydrate moiety that when covalently coupled to the aglycone moiety provides a substrate for an enzyme selected from the group consisting of:
(a) alpha-L-iduronidase;
(b) iduronate 2-sulfatase;
(c) heparan N-sulfatase;
(d) N-acetyl-alpha-D-glucosaminidase;
(e) N-acetylgalactosamine 6-sulfate-sulfatase;
(f) N-acetylgalactosamine 4-sulfate-sulfatase; and
(g) beta-glucuronidase;
L 2 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, and S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;
L 3 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;
L 4 is optional and when present is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S), and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;
R 1 is a C 1 -C 10 alkyl group or a C 1 -C 10 alkoxy group;
R 2 at each occurrence is independently selected from a C 1 -C 10 alkyl group, a C 1 -C 10 alkoxy group, halogen, nitro, —C(═O)NHR, or —C(═O)OR, where R is C 1 -C 8 alkyl group;
R 3 is a C 1 -C 10 alkyl group or a substituted or unsubstituted C 6 -C 10 aryl group; and
n is 0, 1,2, 3, or 4.

23 - 36 . (canceled)

37 . A compound having a carbohydrate moiety and an aglycone moiety and having the formula:

wherein
S is the carbohydrate moiety that when covalently coupled to the aglycone moiety provides a substrate for an enzyme selected from the group consisting of:
(a) alpha-L-iduronidase;
(b) iduronate 2-sulfatase;
(c) heparan N-sulfatase;
(d) N-acetyl-alpha-D-glucosaminidase;
(e) N-acetylgalactosamine 6-sulfate-sulfatase;
(f) N-acetylgalactosamine 4-sulfate-sulfatase; and
(g) beta-glucuronidase;
L 2 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, and S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;
L 3 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;
L 4 is optional and when present is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S), and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;

Assignees

• Univ Washington Through Its Center For Commercialization

Inventors

• Gelb Michael H
• Kumar Arun Babu
• Hocutt Frances
• Spacil Zdenek
• Barcenas Rodriguez Mariana Natali
• Turecek Frantisek
• Scott C Ronald

Classifications

• G01N2333/924

  acting on glycosyl compounds (3.2) · CPC title

• G01N2333/916

  acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4) · CPC title

• C07H15/203 Primary

  Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems · CPC title

• C12Q1/34 Primary

  involving hydrolase · CPC title

• C07H19/01

  sharing oxygen · CPC title