Reagents and methods for sirtuin capture

Patent metadata

Field	Value
Publication number	US-9290791-B2
Application number	US-201113698561-A
Country	US
Kind code	B2
Filing date	May 18, 2011
Priority date	May 18, 2010
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

Abstract

The invention provides a method of preparing a sirtuin complex, a method for detecting a sirtuin in a sample, and a method of screening for compounds which inhibit the deacetylase activity of a sirtuin. The method includes (a) providing a sirtuin substrate having the formula:

(b) providing NAD + or an NAD + analog having the formula:

and (c) providing a sirtuin, wherein R 1 -R 4 , A 1 , A 2 , and n are as defined herein.

First claim

The invention claimed is:

1. A method of preparing a sirtuin complex, which method comprises:
(i) providing a first component which is a sirtuin substrate having the formula:

wherein R 1 is hydrogen, optionally substituted alkyl or optionally substituted aryl,
X is O or NR 7 ,
R 2 is selected from azido, nitro, alkylazido, arylazido, alkyarylazido, arylalkylazido, alkyloxyamino, aryloxyamino, alkyaryloxyamino, arylalkyloxyamino, alkynyl, cycloalkynyl, and alkynylarylalkylcarbonyl,
R 3 is alkyl,
n is an integer of 1 to 10,
R 7 is hydrogen or alkyl, and
A 1 and A 2 are the same or different and are amino acid moieties,
(ii) providing a second component which is NAD + or an NAD + analog, wherein the NAD + analog has the formula:

wherein R 4 is alkyloxyamino, aminooxyethyleneoxyamino, aminooxyethyleneoxyazido, aminooxy(polyethyleneoxy)amino, or azido(polyethyleneoxy)amino,
(iii) providing a third component comprising a sirtuin, and
(iv) combining the first, second, and third components such that a sirtuin complex is formed,
wherein the sirtuin complex comprises the sirtuin, the sirtuin substrate, and NAD + or the NAD + analog, and wherein the sirtuin substrate is a substrate for at least one sirtuin selected from SirT1, SirT2, SirT3, SirT4, SirT5, SirT6, and SirT7.

2. The method of claim 1 , wherein the sirtuin substrate is selected from:

3. A sirtuin complex prepared by the method of claim 1 .

4. A method for detecting a sirtuin in a sample, which method comprises:
(i) providing a first component which is a sirtuin substrate having the formula:

wherein R 1 is hydrogen, optionally substituted alkyl or optionally substituted aryl,
X is O or NR 7 ,
R 2 is selected from azido, alkylazido, arylazido, alkyarylazido, arylalkylazido, alkynyl and cycloalkynyl,
n is an integer of 1 to 10,
R 3 is alkyl,
R 7 is hydrogen or alkyl, and
A 1 and A 2 are the same or different and are amino acid moieties,
(ii) providing a second component which is NAD + or an NAD + analog, wherein the NAD + analog has the formula:

wherein R 4 is alkyloxyamino, aminooxyethyleneoxyamino, aminooxyethyleneoxyazido, aminooxy(polyethyleneoxy)amino, or azido(polyethyleneoxy)amino,
(iii) providing a sample suspected of comprising a sirtuin,
(iv) combining the first and second components and sample such that a first mixture is formed, in which the sirtuin, if present in the sample, forms a sirtuin complex,
(v) contacting the first mixture containing the sirtuin complex with a label compound to form a second mixture comprising a labeled sirtuin complex, wherein the label compound is covalently bonded to the sirtuin complex, wherein the label compound is selected from 5-carboxytetramethylrhodamine-azide,

(vi) separating the labeled sirtuin complex from other components of the second mixture to form a third mixture, and
(vii) detecting whether the labeled sirtuin complex is present in the third mixture, wherein the detection of the labeled sirtuin complex in the third mixture indicates the presence of the sirtuin in the sample.

5. The method of claim 4 , further comprising wherein the separating step (vi) comprises the following steps:
(vi-1) contacting the second mixture with a solid phase so as to immobilize the labeled sirtuin complex, and
(vi-2) separating uncomplexed components from the solid phase to form a third mixture comprising the solid phase and the immobilized sirtuin complex,
(vi-3) optionally, subjecting the solid phase to conditions to regenerate the sirtuin from the labeled sirtuin complex to form a fourth mixture, and
(vi-4) optionally, detecting whether the sirtuin is in the fourth mixture, wherein the detection of the sirtuin in the fourth mixture indicates the presence of the labeled sirtuin complex in the third mixture and thus the presence of the sirtuin in the sample.

6. A method of screening for compounds which inhibit the deacetylase activity of at least one sirtuin, which method comprises:
(i) providing a first test mixture comprising (a) a sirtuin substrate, (b) NAD + , and (c) a candidate compound, wherein the sirtuin substrate has the formula:

wherein R 1 is hydrogen, optionally substituted alkyl or optionally substituted aryl,
X is O or NR 7 ,
R 2 is selected from azido, alkylazido, arylazido, alkyarylazido, arylalkylazido, alkynyl, cycloalkynyl, and alkynylarylalkylcarbonyl,
n is an integer of 1 to 10,
R 3 is alkyl,
R 7 is hydrogen or alkyl, and
A 1 and A 2 are the same or different and are amino acid moieties,
(ii) adding a sirtuin to the first test mixture to form a second test mixture,
(iii) incubating the second test mixture for a period of time,
(iv) adding a label compound comprising a second reactive moiety to the second test mixture to form a third test mixture, wherein the first and second reactive moieties react to form at least one covalent bond between the sirtuin substrate and the label compound, wherein the label compound is selected from 5-carboxytetramethylrhodamine-azide,

(v) contacting the third test mixture with a solid phase so as to immobilize components comprising the label compound and to form a test solid phase,
(vi) separating uncomplexed components from the test solid phase,
(vii) subjecting the test solid phase to conditions to regenerate the sirtuin, if present, from the test solid phase and to form a fourth test mixture, and
(viii) determining if the sirtuin is present in a decreased amount in the fourth test mixture relative to the amount of sirtuin added to the first test mixture,
wherein a decreased amount of the sirtuin in the fourth test mixture relative to the amount of sirtuin that would be observed in the absence of the candidate compound indicates that the candidate compound is an inhibitor of the sirtuin.

7. The method of claim 6 , wherein R 1 and R 3 are both alkyl, wherein A 1 and A 2 are both L-alaninyl moieties, and wherein n is 4.

8. The method of claim 6 , further comprising the steps of:
(i′) providing a first control mixture comprising (a) the sirtuin substrate comprising a thioamide moiety and a first reactive moiety and (b) NAD + ,
(ii′) adding the sirtuin to the first control mixture to form a second control mixture,
(iii′) incubating the second control mixture for a period of time,
(iv′) adding the label compound to form a third control mixture, wherein the first and second reactive moieties react to form at least one covalent bond between the sirtuin substrate and the label compound,
wherein the label compound is

Assignees

• Sauve Anthony A
• Cen Yana
• Univ Cornell

Inventors

• Sauve Anthony A
• Cen Yana

Classifications

• C12Q2334/00

  O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases · CPC title

• G01N2333/918

  Carboxylic ester hydrolases (3.1.1) · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title

• C12Q1/34 Primary

  involving hydrolase · CPC title