Reagents and methods for analysis of proteins and metabolites targeted by covalent probes

Patent metadata

Field	Value
Publication number	US-10969394-B2
Application number	US-201716461486-A
Country	US
Kind code	B2
Filing date	Nov 28, 2017
Priority date	Nov 28, 2016
Publication date	Apr 6, 2021
Grant date	Apr 6, 2021

Abstract

The present application relates to mass spectrometry methods for use in identifying proteins or other biomolecules which are bound irreversibly by test compounds.

First claim

What is claimed is:

1. An analytical method, comprising:
i) contacting a test compound with a polypeptide to form a test-compound-polypeptide conjugate;
ii) analyzing the test compound-polypeptide conjugate using a mass spectrometry assay;
III detecting one or more thiolated ions of the test compound or derivative ions thereof, produced in the mass spectrometry assay; and
identifying that the test compound irreversibly bonds to the polypeptide based on the detection of the one or more thiolated ions, or derivative ions thereof, in the mass spectrometry assay; wherein the thiolated ions or derivative ions thereof are fragment ions.

2. The method of claim 1 , wherein the compound-polypeptide conjugate comprises one or more thioether bonds between the test compound and the polypeptide.

3. The method of claim 1 , wherein step i) comprises contacting the test compound and the polypeptide in the presence of a first solvent component.

4. The method of claim 1 , wherein step i) further comprises contacting the test compound and the polypeptide in the presence of a buffer agent.

5. The method of claim 1 , wherein step i) is performed using a molar excess of the test compound compared to the polypeptide.

6. The method of claim 1 , further comprising contacting the test compound-polypeptide conjugate with an acid in the presence of a second solvent component prior to performing the mass spectrometry assay of step ii).

7. The method of claim 1 , wherein the method further comprises digesting the test compound-polypeptide conjugate prior to the performing the mass spectrometry assay of step ii).

8. The method of claim 7 , wherein the digesting comprises reacting the test compound-polypeptide conjugate with trypsin in the presence of a third solvent component.

9. The method of claim 1 , wherein the polypeptide comprises one or more amino acids residues comprising at least one sulfur atom.

10. The method of claim 9 , wherein the polypeptide comprises one or more cysteine residues.

11. The method of claim 10 , wherein the test compound is identified as irreversibly bonding to one or more cysteine residues of the polypeptide.

12. The method of claim 11 , wherein the test compound comprises one or more groups independently selected from the group consisting of acrylamide groups, dimethylamino acrylamide groups, iodoacetamide groups, chloroacetamide groups, maleimide groups, and reactive C—X groups, wherein X is a halogen.

13. The method of claim 1 , wherein the test compound is selected from the group consisting of JNK-IN-7, HBX-19818, MI-2, TL10-201, THZ1, QL-47, ibrutinib, neratinib, and TL11-113.

14. The method of claim 1 , wherein the polypeptide is a kinase selected from the group consisting of JNK2, JAK3, CDK7, CDK12, ITK, USP-7, TAK1, and EGFR, or a fragment thereof.

15. The method of claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of:

(SEQ ID NO: 1)

L-M-D-A-N-L-C-Q-V-I-Q-M-E;

(SEQ ID NO: 2)

L-V-M-E-Y-L-P-S-G-C-L-R;

(SEQ ID NO: 3)

M-A-P-P-D-L-P-H-W-Q-D-C-H-E-L-W-S-K;

(SEQ ID NO: 4)

H-G-C-L-S-D-Y-L-R-S-Q-R-G-L-F-A-A-E;

(SEQ ID NO: 5)

Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-R-P-N-C-P-V-E-T-L-K;

(SEQ ID NO: 6)

G-C-L-L-D-Y-V-R;

(SEQ ID NO: 7)

F-G-L-C-S-G-P-A-D-T-G-R;

(SEQ ID NO: 8)

Y-M-A-N-G-C-L-sL-N-Y-L-R;

(SEQ ID NO: 9)

I-C-D-F-G-T-A-C-D-I-Q-T-H-M-T-N-N-K;

and

(SEQ ID NO: 10)

Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-(13C6-15N4)R-P-N-C-

P-V-E-T-L-K.

16. An analytical method, comprising:
i) contacting a first mixture comprising one or more test compounds with a second mixture comprising one or more polypeptides to form a third mixture comprising one or more compound-polypeptide conjugates, wherein each of the compound-polypeptide conjugates comprise one or more thioether bonds;
ii) analyzing the third mixture using a mass spectrometry assay;
iii) detecting one or more thiolated ions, or derivative ions thereof, produced in the mass spectrometry assay; and
iv) identifying that one or more of the test compounds binds irreversibly to one or more of the polypeptides based on the detection of the one or more thiolated ions, or derivative ions thereof, in the mass spectrometry assay.

17. An analytical method comprising:
i) reacting one or more acrylamide compounds with a thiol-containing compound to form one or more acrylamide thiolated derivatives;
ii) analyzing the one or more acrylamide thiolated derivatives in a mass spectrometry assay;
iii) generating a database of fragment ion spectra comprising the mass spectra of each of the one or more acrylamide thiolated derivatives;
iv) contacting a first mixture comprising more than one test compound with a second mixture comprising more than one polypeptide to form a third mixture comprising more than one compound-polypeptide conjugate, wherein each of the compound-polypeptide conjugates comprise one or more thioether bonds;
v) analyzing the third mixture using a mass spectrometry assay;
vi) detecting one or more thiolated ions produced in the mass spectrometry assay;
vii) isolating the one or more thiolated ions;
viii) performing a mass spectrometry assay on the one or more isolated thiolated ions;
ix) comparing the mass spectra of the one or more thiolated ions to the database of fragment ion spectra; and
x) identifying that one or more of the test compounds binds irreversibly to one or more of the polypeptides based on the detection of one or more thiolated ions in the mass spectrometry assay of step v) and the identification a mass spectrum in the database of fragment ion spectra that is substantially identical to the mass spectrum of the isolated thiolated ion of step viii).

Assignees

• Dana Farber Cancer Inst Inc

Inventors

• Marto Jarrod A
• Ficarro Scott B
• Adelmant Guillaume

Classifications

• C07D401/12

  linked by a chain containing hetero atoms as chain links · CPC title

• C07C225/14

  the carbon skeleton being unsaturated · CPC title

• C07D403/04

  directly linked by a ring-member-to-ring-member bond · CPC title

• C07C233/33

  with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring · CPC title

• C07C255/60

  at least one of the singly-bound nitrogen atoms being acylated · CPC title