Probe for selectively characterizing enzymes involved in xenobiotic metabolism and method of making and using the same

Patent metadata

Field	Value
Publication number	US-2022282317-A1
Application number	US-202217730004-A
Country	US
Kind code	A1
Filing date	Apr 26, 2022
Priority date	Oct 4, 2017
Publication date	Sep 8, 2022
Grant date	—

Abstract

Activity-based probes that can be used to selectively identify and characterize enzymes that are involved in different phases of xenobiotic metabolism in a host and its microbiota population(s) are described. The activity-based probes described specifically label only their target active enzymes involved in xenobiotic metabolism and therefore provide a measurement of true protein functional activity rather than transcript or protein abundance. The activity-based probes also provide multimodal profiling of these active enzymes. Methods for preparing the activity based probes and exemplary methods for their use also are disclosed.

First claim

We claim:

1 . A method comprising:
exposing a subject or a sample to a probe for a time sufficient to allow the probe to bind to an enzyme involved in xenobiotic metabolism such that a probe-enzyme conjugate is formed; and analyzing the probe-enzyme conjugate using a fluorescent detection technique, a colorimetric detection technique, a mass spectrometry technique, or a combination thereof; wherein the probe comprises a compound of Formula II

wherein
ERG, if present, is

S(O) 2 OH, or an anionic form thereof; P(O)(OH) 2 , or an anionic form thereof; a halogen; or —OPh-CH 2 —ONO 2 ;
EBG has a structure satisfying one or more of Formulas IIA EBG -IIJ EBG

wherein
Y is CH 3 or CF 3 ;
Y′ is O, NO 2 , or —N═NR″, wherein R″ is a dye or other reporting moiety;
m is an integer selected from 0 to 5; and
R′ is selected from aldehyde, ketone, ester, carboxylic acid, acyl, acyl halide, cyano, sulfonate, nitro, nitroso, quaternary amine, CF 3 , alkyl halide, or combinations thereof;

Linker a comprises an aliphatic group, a heteroaliphatic group, an aromatic group, an aliphatic-aromatic group, a heteroaliphatic-aromatic group, a heteroaromatic group, an aliphatic-heteroaromatic group, or a heteroaliphatic-heteroaromatic group;
each of Linker b and Linker c , if present, independently comprises an aliphatic group, a heteroaliphatic group, an aromatic group, an aliphatic-aromatic group, a heteroaliphatic-aromatic group, a heteroaromatic group, an aliphatic-heteroaromatic group, or a heteroaliphatic-heteroaromatic group;
R, if present, is a group comprising a carbamate or a carbonate;
each Tag, if present, independently comprises a functional group or molecule capable of generating a detectable signal;
each p Tag, which is present if a Tag is not present, independently comprises a clickable functional group; and
each of n, m, p, and q independently is 0 or 1.

2 . The method of claim 1 , further comprising exposing the probe-enzyme conjugate to a Tag-containing compound to form a probe-enzyme conjugate comprising a Tag moiety.

3 . The method of claim 1 , further comprising exposing the probe to a light source.

4 . The method of claim 1 , further comprising extracting a subject sample from the subject and analyzing the subject sample using a fluorescent detection technique, a colorimetric detection technique, a mass spectrometry technique, or a combination thereof.

5 . The method of claim 1 , wherein Linker a is a linker group having a structure satisfying one or more of Formulas IIA Linkera -IIH Linkera

wherein
each n′ independently is an integer ranging from 1 to 50;
Z is oxygen or NR′″, wherein R′″ is hydrogen, aliphatic, or aromatic; and
Q is carbon, oxygen or NR′″, wherein R′″ is hydrogen, aliphatic, or aromatic.

6 . The method of claim 1 , wherein at least one of the following applies:
(a) Linker b is present and comprises a —(CH 2 ) n′ — group, wherein n′ is an integer ranging from 1 to 50; an amide group; or a combination thereof; and/or (b) Linker c is present and has a structure satisfying Formula IIA Linkerc or Formula IIB Linkerc

wherein n′ is an integer ranging from 0 to 50; and
Z is oxygen or NR′″, wherein R′″ is hydrogen, aliphatic, or aromatic.

7 . The method of claim 1 , wherein R is present and has a structure of one of Formulas IIA R -IID R

wherein
Z and Z′ independently are oxygen or NR′″, wherein R′″ is hydrogen, aliphatic, or aromatic; and
n′ is an integer ranging from 0 to 50.

8 . The method of claim 1 , wherein one or more Tags are present and wherein each Tag independently is a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye.

9 . The method of claim 1 , wherein the one or more Tags independently are rhodamine, fluorescein, or biotin.

10 . The method of claim 1 , wherein one or more p Tags are present and wherein each p Tag independently is an azide or an alkyne.

11 . The method of claim 1 , wherein the compound has a structure satisfying Formula IIIA

12 . The method of claim 1 , wherein the ERG is present and is iodo; —OPh-CH 2 —ONO 2 ;

13 . The method of claim 1 , wherein Linker a is an ester group, an —O(CH 2 ) n′ NR′″C(O)(CH 2 ) n′ — group, or a —(CH 2 ) n′ — group, wherein each n′ independently is an integer ranging from 1 to 20 and wherein R′″ is hydrogen, aliphatic, or aromatic.

14 . The method of claim 1 , wherein m is 1 and Linker c is an —NR′″C(O)(CH 2 ) n′ — group or an —NR′″C(O)CH 2 [O(CH 2 ) 2 ] n′ OCH 2 — group, wherein each n′ independently is an integer ranging from 1 to 20 and wherein R′″ is hydrogen, aliphatic, or aromatic.

15 . The method of claim 1 , wherein m is 1 and each Tag, if present, independently is a fluorophore, a binding partner of an affinity-based binding pair, a quantum dot, or a dye; or if a p Tag group is present, then each p Tag independently is an alkyne or an azide.

16 . The method of claim 1 , wherein the probe is selected from:

17 . The method of claim 1 , wherein in Formula (II):
ERG is

EBG has a structure selected from

wherein
Y′ is O;
m′ is an integer selected from 0 to 5; and
each occurrence of R′ is independently selected from aldehyde, ketone, ester, carboxylic acid, acyl, acyl halide, cyano, sulfonate, nitro, nitroso, quaternary amine, CF 3 , alkyl halide, or combinations thereof;

Linker a comprises an aliphatic group, a heteroaliphatic group, an aromatic group, an aliphatic-aromatic group, a heteroaliphatic-aromatic group, a heteroaromatic group, an aliphatic-heteroaromatic group, or a heteroaliphatic-heteroaromatic group;
each of Linker b and Linker c , if present, independently comprises an aliphatic group, a heteroaliphatic group, an aromatic group, an aliphatic-aromatic group, a heteroaliphatic-aromatic group, a heteroaromatic group, an aliphatic-heteroaromatic group, or a heteroaliphatic-heteroaromat

Assignees

• Battelle Memorial Institute

Inventors

• Wright Aaron T
• Ramos-Hunter Susan
• Whidbey Christopher

Classifications

• C07C271/28

  to a carbon atom of a non-condensed six-membered aromatic ring · CPC title

• G01N2333/91177

  Glutathione transferases (2.5.1.18) · CPC title

• C12Q2600/106

  Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism · CPC title

• C07D311/16

  substituted in position 7 · CPC title

• C07C305/22

  having oxygen atoms of sulfate groups bound to carbon atoms of six-membered aromatic rings · CPC title