Activity-Based Protein Profiling for Amino Acid Reactivity Characterization
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Activity-Based Protein Profiling for Amino Acid Reactivity Characterization

Drug development benefits from the discovery of new protein targets, which relies on highly reliable, high-throughput methods for analyzing drug-protein interactions. Currently, over 85% of proteins are still considered undruggable, mainly due to the lack of cavities for drug molecule targeting and corresponding reactive sites. Therefore, characterizing amino acid reactive sites at the proteomic level has become a key aspect of covalent drug design and addressing the challenge of undruggable protein targets.

Activity-based protein profiling (ABPP) is a strategy that utilizes activity-directed chemical probe molecules to detect functional enzymes and protein targets in complex samples. ABPP technology has shown significant potential in the characterization of amino acid reactivity, which will aid in the discovery of new drug targets and the development of lead compounds.


Chemical proteomics can be divided into two major categories: chemical modification proteomics and non-chemical modification proteomics. Non-chemical modification proteomics methods include thermal proteome profiling (TPP) and limited proteolysis (LiP). These methods confirm changes in the stress state of target proteins and their complexes after binding to ligands, but they cannot be applied to target proteins where the binding conformation with drug molecules does not change significantly. Chemical modification proteomics includes methods such as affinity chromatography (AC) and activity-based protein profiling (ABPP). AC is mainly used to study interactions between proteins and biologically active small molecules or protein-protein interactions, but uncertainty about the activity of studied molecules and differences in material ligand binding strength and nonspecific adsorption may interfere with research results.

ABPP methods have been widely used in the study of protein structure and functional states. By directly capturing target proteins with active small molecules, this method can assess the binding state between active small molecules and target proteins. Based on this, drug targets can be discovered, and their binding regions identified using quantitative proteomic techniques. The human body contains 20 naturally occurring amino acids with different reactive activities, and the same protein or type of amino acid is influenced by spatial microenvironments (including hydrogen bonding interactions, local pH, oxidation-reduction conditions, or induction effects), leading to significant differences in their spatial reactivity. Evaluating amino acid reactivity based on ABPP methods and screening for highly reactive amino acids are crucial.

Activity-based Protein Profiling

ABPP is an activity-directed (mainly including reaction activity, binding specificity, etc.) proteomic analysis method. With the development of mass spectrometry technology and quantitative proteomics technology, ABPP methods have been successfully used in studies such as drug target protein screening, becoming a key technology for protein identification and functional characterization. It mainly includes two strategies: direct enrichment based on ABPP and competitive ABPP-based indirect enrichment.

ABPP: First, the probe is covalently labeled with the target protein through the reactive group, and then the probe is connected to the reporting group (including fluorescence, affinity enrichment, etc.) using click chemistry. Subsequently, techniques such as fluorescence spectroscopy and mass spectrometry are used to detect and identify labeled proteins, characterizing the reactive activity of labeled amino acids and discovering more drug target proteins.

Competitive ABPP: First, compound libraries are co-incubated with protein extracts, and then a general-purpose probe molecule is used to label non-library binding sites in proteins. Combined with isotope quantification analysis, the coordination of amino acids with small molecule compounds is characterized, exploring potential lead compounds and their target proteins. This strategy does not require chemical modification of compounds but indirectly selects and identifies different small molecule compound target proteins based on a competitive labeling strategy using universal chemical probes, demonstrating good universality and analytical throughput. Currently, this strategy is mainly used to the study of target proteins of covalently conjugated drug and small molecule drug.

Discovery of drug targets and lead compounds using ABPP.Fig. 1 Discovery of drug targets and lead compounds using ABPP.

ABPP Probes Targeting Amino Acid Reactivity

The core of ABPP technology lies in chemical probe labeling. Activity-based probes (ABPs) mainly consist of three parts: reactive groups, linker arms, and reporter groups. Among them, the reactive group is the foundation of ABP, anchoring drug targets through strong affinity interactions such as covalent bonds. Its reaction features high specificity, high conversion rates, and requires good biocompatibility. Reporter groups are primarily used for subsequent visualization and enrichment detection of labeled proteins. In visualizing labeled proteins, fluorescent groups are common reporter groups, including rhodamine, fluorescein, and other fluorescent reagents. With the development of bioorthogonal reaction technology, biotin and other reporter groups can be introduced post-protein labeling using click chemistry reactions, enabling their visual detection. This strategy facilitates probe synthesis and avoids spatial hindrance that may affect probe reactivity. Linker arms serve as bridges between reactive groups and reporter groups, enhancing probe stability and spatial distribution by regulating chemical reaction activity and spatial hindrance.

In research on amino acid reactivity based on the ABPP strategy, differences in microenvironments where different amino acids of the same protein are located lead to significant differences in their reactivity. Targeting highly reactive amino acids and characterizing proteins will provide new targets for covalent drug screening, making amino acid reactivity screening a new research focus. Currently, efficient labeling probes have been developed for different types of amino acids with nucleophilic/electrophilic activity, and they are used for characterizing amino acid reactivity. For amino acids like cysteine and lysine with high nucleophilic activity, a series of probes with different electrophilic activity types have been developed and applied to characterize amino acid reactivity in vitro protein extracts. In recent years, research on amino acid reactivity based on the ABPP strategy has expanded to other amino acids with low nucleophilic activity.

  • Characterization of Cysteine Reactivity

Cysteine is a target for many clinical covalent drugs, such as afatinib and osimertinib for lung cancer. Based on the strong nucleophilicity of cysteine, developing ABPs with different electrophilicities is the optimal choice. Electrophilic iodoacetamide reacts with the thiol group of cysteine, leading to alkylation of cysteine. Based on this reaction principle, an iodoacetamide-alkyne probe with high reactivity has been developed, and click chemistry reactions with labeled alkynes are used to enrich and identify labeled sites. Furthermore, combining it with stable isotope labeling technology, a tandem bioorthogonal protein labeling characterization technology (isoTOP-ABPP) has been developed. It has been applied to characterize the reactivity of cysteine residues in cell lines such as breast cancer, quantifying 1082 cysteine residues, including 350 highly reactive cysteine residues, which are closely related to protein function.

  • Characterization of Lysine Reactivity

The side-chain amino group of lysine has some nucleophilicity, making it easily covalently linked to probes. More importantly, lysine is often a functional site of many proteins, such as enzyme active sites or located in protein-ligand binding pockets, mediating interactions between proteins and their ligands. Additionally, lysine is the most important site of post-translational modifications in organisms, including acylation, methylation, ubiquitination, and other types of post-translational modifications, directly regulating important biological processes such as epigenetics, cell development, and proliferation. Lysine is considered the most promising covalent drug binding site. Research on lysine-targeted drugs is also gaining attention, such as lysine in the PI3K kinase active site being the target of the natural product wortmannin, leading to the development of PI3K covalent inhibitors, which have entered clinical trials for non-small cell lung cancer, castration-resistant prostate cancer, and glioblastoma.

The active esters generated by carboxylic acid activation are the main reactive groups currently used for lysine labeling. A series of active ester chemical probes have been developed for selective labeling of lysine. Using commercially available hydroxysuccinimide (NHS)-alkyne probes to label lysine residues in mouse liver tissue, this method identified 1639 lysine residues, of which 31% were functional sites, including binding active centers, calcium ion binding sites, and post-translational modification sites. Based on the mild reaction conditions of NHS, which has good biocompatibility, compounds based on NHS-esters are synthesized for lysine screening in target proteins, such as K497 in dihydropyrimidine dehydrogenase (Dpyd) and K211 in aldehyde dehydrogenase 2 (Aldh2), providing candidate targets for targeted drug design.

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