Technical introduction

In cells or tissues, when the target of covalent drugs is discovered, simple chemical modification can be carried out on the active small molecule to connect the reporter groups (biotin, biological orthogonal groups, etc.). On the premise of preserving the original activity of small molecules, the protein targets directly captured in living cells or tissues were obtained through enrichment, enzymolysis, mass spectrometry identification and data analysis to obtain the omics information of the target and the amino acid site information of small molecules.

However, many active molecules are not easy to be reformed, or react with amino acid residues and unstable products, so they are not suitable for mass spectrometry detection. Therefore, competitive site identification is a good solution strategy for this kind of active molecules. The core technology of this method is a general chemical probe. When the active small molecule reacts with amino acid residues to occupy the site, compared with the blank control sample, the general chemical probe will produce signal difference for the labeling of the site. The difference of this part of the labeling signal can be read to obtain the information of the target protein and amino acid site of the active molecule. You can also include on-target and off-target information. This method is applicable to a variety of sample systems such as living cells, tissues and cell lysates. The active small molecules include but are not limited to endogenous metabolites, active components of traditional Chinese medicine, natural products and covalent drugs.

Flow chart

Active molecular probe labeled protein, enrichment, enzymolysis, multiple quantitative labeling, mass spectrometry detection, 

analysis of small molecular action targets and amino acid sites

Service content

Design and modification of covalent drug probes

Omics Analysis of Covalent drug Target Proteins Based on Gel Imaging and biomolecular mass Spectrometry

Analysis of covalent drug action Sites (based on mass spectrometry)

Target validation of covalent drug action (targeted quantitative protein analysis/protein Western Blotting)

Screening of covalent lead compounds

Technical background

Covalent drugs generally refer to drug molecules forming covalent bonds with amino acid residues on target proteins, such as cysteine, lysine, serine, etc. Aspirin is one of the first discovered covalent drug molecules. Natural products are also representative examples, such as rubesin, which has anti-inflammatory biological activity. In recent years, covalent targeted drugs have gradually attracted the attention of drug research and development enterprises. At present, at least 6 covalent drugs targeting kinase targets have been approved by FDA, such as ibrutinib targeting BTK kinase.

Case analysis

Project requirements:

AMG510, developed by Amgen, is the world's first approved drug targeting KRAS-G12C mutation in tumors. The objective of the project was to confirm target specificity and selectivity of AMG510 in tumor cells with the KRAS-G12C mutation.

Solution:

DIA-ABPP, the core technology platform of covalent drugs, can be used to fully screen the target of covalent drugs in cells, accurate to amino acid residues.

Data presentation:

Explanation of the case:

A total of 14597 cysteine sites from 4658 proteins were quantitatively analyzed in four replicates in NCI-H358 cells. Under the condition of 1uM AMG510, the ratio of KRAS_C12 changed most significantly, but the other site KRAS_C80 was not affected. This data demonstrated a high degree of target specificity for AMG510. (* on the right is the site labeled with cysteine)