Table 4 Identification methods of drug direct/indirect target
Technology | Principle | Application Characteristics | Limitation | Application | References | |
|---|---|---|---|---|---|---|
Labeling Method | Immobilized probe | Conventional bioactive small molecules are affixed to biocompatible inert resins, which are then used to isolate target proteins from the entire proteome | The synthesis is simple, and all adsorbed protein can be analyzed indiscriminately | The length of the connecting chain and the structural modification of small molecules can affect the binding force between the affinity probe and the target protein | Trapoxin and histone deacetylase (HDAC) specific binding | |
Activity-Based Protein Profiling (ABPP) | Probes are synthesized by retaining the pharmacological activity of their parent molecule and subsequently incubated with live cells, lysates or tissue homogenates, and protein targets are identified using proteomic methods after binding to the target protein | Directly applied to living cells, it reflects the real drug-target interaction under cell physiological conditions | Prone to false positive results | Parthenolide modifies focal adhesion kinase 1 (FAK1) | ||
Photoaffinity probe (PAL) | Chemical probes are prepared by doping photoreactive groups to covalently cross-link to adjacent amino acid residues under UV irradiation. Click chemistry-based enrichment captures labeled proteins for proteomic analysis | It has small volume and high efficiency | Binding of some non-specific proteins, affecting the accuracy of the results | Tropomyosin as a novel target of betulinic acid | ||
Click chemistry | Based on the synthesis of carbon-heteroatom bonds (C-X-C), small molecule probes are obtained by introducing orthogonal reactive groups (alkynes, alkenes, etc.) into the structure of natural products. Subsequently, the probes are bound to proteins in situ using click chemistry in cells. Finally, azide-modified reporter groups (biotin or fluorescent) are attached to the probe-target protein complexes using click reactions. And then the targets are detected, enriched and identified (SDS-PAGE) | Can be carried out in living cells; It has rapid and specific chemical coupling under aqueous conditions; It has little or no effect on the structure and pharmacological activity of small molecules | Some natural product active ingredients do not bind well to their targets; azide is toxic | Artemisinin could successfully pull-down plasmepsinl (PM I), plasmepsin II (PM II), merozoite surface protein 1 (MSP1) and actin | ||
Small-molecule affinity chromatography | Using the affinity between small molecules and proteins, the small molecules are made into solid-phase adsorbent and subsequently adsorbed on the protein with affinity in the protein mixture, and finally, a suitable eluent is selected to elute the bound proteins | High affinity and specificity, suitable for complex organic systems with extremely low target product concentrations | It is difficult to identify proteins that have an affinity for this spatial structure | Isochlorogenic acid A and luteolin-7-O-glucuronide have a stronger affinity with vascular endothelial growth factor receptor 2 (VEGFR2) | ||
Phage Display Technology | Build a phage library and incubate with the target molecule, then remove unbound and non-specific phages, and use buffer to elute and separate the target bound phages. Finally, the eluted phages are infected and amplified in the bacteria | Simple, efficient, and low-cost | Three to five rounds of biological selection are required to separate specific and high affinity peptide binders | Bcl-2 was identified as paclitaxel binding protein | ||
Label-Free | Drug affinity responsive target stability (DARTS) | After the drug is combined with the corresponding protein, it will make the structure of the target protein relatively stable, and the activity of the same enzymatic hydrolysis reaction will change, making it less susceptible to protease decomposition | Small molecules needn’t structural modification, have strong specificity, and can directly use original small molecule compounds or even inconspicuous compound drugs as research objects, making the operation convenient | Identifying targets with low protein abundance has limitations | Aconitine could well bind to cytosolic phospholipase A2 (cPLA2) | |
Cellular thermal shift assay (CETSA) | Incubate small molecules with cell and tissue homogenate, then undergo thermal denaturation, using the principle of target proteins becoming structurally stable after binding to drug molecules, and finally verify using protein immunoblotting | High selectivity | High drug concentrations, commonly used for target identification, are still to be determined for larger and more complex soluble proteins and membrane proteins to be determined | Direct binding of Herbacetin and serum/glucocorticoid regulated kinase 1 (SGK1) | ||
Stability of proteins from rates of oxidation (SPROX) | The denaturation dependence of methionine side chain oxidation in proteins mediated by hydrogen peroxide (H2O2) is used to measure the folding free energy of proteins and the Kd value of protein ligand complexes, similar to the DARTS technique | Enables detection of direct and indirect effects of protein-ligand binding in complex biological mixtures | The rate of oxidation is susceptible to amino acid residues. Protein concentration is affected by chemical denaturants, resulting in a weak oxidation reaction that is not easily detectable | 28 proteins were identified to interact with manassantin A | ||
Thermal proteomic profiling (TPP) | A technique that combines the CETSA method with quantitative proteomics techniques. Using the principle that high temperature can denature proteins, cells are heated before and after administration. Subsequently, the denaturation curve was obtained through quantitative analysis of soluble proteins | Identify direct targets of action indiscriminately from intact cells or tissues. And can calculate the melting temperature (Tm) of the target protein | Only detect the interaction between small molecules and soluble proteins | Nucleolar protein 14 (Nop14) is a specific target of vioprolide A | ||
Limited proteolysis mass spectrometry (LiP-MS) | LiP-MS uses the principle of limited hydrolysis to identify protein targets of small molecule drugs. After binding to the target protein, the small molecule drug will stabilize the rotein structure at the overall or local level, which greatly reduces the possibility of peptides on the protein surface being exposed and cleaved by proteases | No need to design synthetic molecular probes, use drug molecules to directly conduct binding experiments in vitro to obtain direct binding targets and their binding sites | Low abundance proteins are difficult to detect; When small molecule binding causes changes in protein structure, it is difficult to accurately locate the precise region of drug binding | The deubiquitinase UCHL3 is a direct target of farrerol | ||
Other | Proteolysis-targeting chimeras (PROTAC) | Also known as “Targeted Degradomics”. Use the ubiquitin-protease system to develop drugs for target proteins and find drug targets from degraded differential proteins | Inducing the degradation of target proteins does not require strong affinity, which is beneficial for discovering targets with weaker affinity | Off-target effects; fewer PROTAC molecules targeting “undruggable” proteins | MAFF protein is a potential target of lathyrol |