New Trends in Target Validation: Competitive ABPP Analysis
In the development of new drugs, 'target screening' does not imply 'target confirmation.' Even if a candidate protein is repeatedly identified in differential expression or pathway enrichment analyses, it does not mean it isindeed the direct target of the drug.The real critical questions are:
Is this protein the 'direct target' of the drug action? Is there covalent binding? Is there selective inhibition? Are there potential off-target effects?
Proteomics and molecular biology methods find it difficult to directly answer these questions. However,Competitive Activity-Based Protein Profiling (Competitive ABPP)as an extension and enhancement of ABPP technology, is becoming a new trend and standard in the field of target validation. Biotyech has pioneered the establishment ofa competitive ABPP analysis processwhich has been widely applied in the validation of novel covalent inhibitors, active validation of natural products, and off-target risk assessment of lead compounds.
1. What is Competitive ABPP?
Standard Activity-Based Protein Profiling (ABPP) uses covalent probes (ABPs) to label active proteins, combined with mass spectrometry techniques to identify and quantify these active enzymes, and is commonly used for screening functional proteins and identifying drug targets.
Competitive ABPP is a functional proteomics method that enhances target validation capability by introducing a drug competition mechanism. The basic principle is:Using small molecule drugs to 'compete' with active site probes for binding to the same enzyme active center, to infer whether the drug directly acts on the target enzyme.Competitive ABPP is based on classical ABPP technology but introduces a crucial 'drug first, probe later' competition step in the experimental design. The basic process is as follows:
1. Sample grouping and treatmentincluding drug treatment groups and control groups;
2. Add ABP probes separatelyto perform standard active site labeling;
3、Enrich and digest protein samplesretaining active enzymes bound to the probe;
4、Through mass spectrometry analysisCompare the probe binding levels of each enzyme in the two groups;
5、Based on changes in probe signalsDetermine whether the drug directly competes with the enzyme for binding.
If the signal of a particular enzyme is significantly reduced in the drug group, it indicates that the drug has a strong affinity for the active site of that enzyme, suggesting it as a potential direct target. Conversely, if the signal remains unchanged, it means the enzyme is not affected by the drug or it is a non-targeted binding.
II. Unique Advantages of Competitive ABPP
Compared to proteomics and chemical proteomics methods, competitive ABPP exhibits multidimensional advantages:
1. Function-oriented
Competitive ABPP focuses on theactive staterather than the total amount or expression level, capturing the 'functional targets' truly involved in physiological processes under disease or drug stimulation.
2. Direct visualization of binding events
By comparing the probe binding differences before and after drug intervention, researchers can directly determine whether a drug acts on the catalytic center of a specific enzyme, thus avoiding the cumbersome 'indirect signal interpretation' in traditional validation paths.
3. Simultaneous assessment of selectivity and off-target effects
Competitive ABPP can not only confirm whether a drug acts on the target enzyme but alsoassess its effects on non-target enzymeswithin the same experimental system, providing valuable reference for optimizing drug structure and reducing toxic side effects.
4. Strong technical adaptability
This technique is compatible with cell lysates, tissue homogenates, and even in vivo labeling systems, making it suitable for conducting validation experiments in multiple model systems with good translational capability.
III. Why has competitive ABPP become the 'new standard for target validation'?
1. Clearly Identify 'Direct Binding Targets'
Compared to differential protein screening methods, competitive ABPP can directly determine whether a drug physically binds to a specific enzyme, making it the most reliable method for confirming covalent targets.
2. Analyze Drug Selectivity
Evaluate the affinity and selectivity of a drug for different enzymes through 'competitive inhibition levels,' aiding in structural optimization and side effect control.
3. Detect Off-target Effects
Competitive ABPP can identify non-target enzymes affected by a drug unexpectedly, indicating potential toxicity or interference mechanisms.
4. Adapt to Covalent Drug Development Pathway
In the development of multiple covalent drugs such as Ibrutinib, Neratinib, and Afatinib, competitive ABPP has been widely used for pre-submission target validation data preparation.
Professional Support from Biotyech's Competitive ABPP Platform
As a core platform in the functional proteomics field, Biotyech has established a complete process standard service system in competitive ABPP technology:from probe screening, sample processing, mass spectrometry detection to target data interpretation:
1. Support for various enzyme probes, covering serine proteases, cysteine cathepsins, metalloproteases, etc.;
2、Wide range of sample compatibility, supporting cell lines, animal tissues, human biological samples, etc.;
3、Controllable quantitative accuracy, based on a high-resolution mass spectrometry platform for peptide identification and quantification;
4、Provide binding inhibition analysis report, including inhibition rate calculation, target prioritization, GO/KEGG annotation, and graphical output.
5、Provide multi-round validation design supportto meet experimental needs such as structural adaptation improvements, dosage gradients, and time dynamics analysis.
Traditional target research often relies on multi-round expression validation and functional speculation, making it difficult to quickly establish the direct relationship between drugs and enzyme targets. Competitive ABPP offers a clearer and more logically rigorous validation pathway:using 'blockage' to prove 'binding' and 'differences' to confirm 'direct action'.In the research cognitive chain of 'expression → function → mechanism of action', competitive ABPP is gradually becoming a crucial technical link.
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