As a leader in bioactive natural products target identification, our company specializes in identifying bioactive natural products target using sophisticated probe-based approaches. At the heart of our platform is activity-based protein profiling (ABPP)—a powerful chemoproteomic technology that enables the functional characterization of enzymes and protein targets directly in living systems. Our platform enables unbiased, mechanism-driven target discovery and accelerates the translation of natural products into druggable leads.
What is ABPP?
ABPP is a functional proteomic technology that uses active-site directed chemical probes to monitor the availability and functional state of proteins. Unlike traditional proteomics, which measures total protein abundance, ABPP focuses on protein activity. ABPP is particularly effective for studying enzymes and functional proteins such as hydrolases, proteases, oxidoreductases, kinases, and other reactive or regulatory protein classes. Because many natural products exert their biological effects by modulating protein activity rather than expression, ABPP is uniquely suited for uncovering their molecular targets.
Principle of ABPP
The fundamental principle of ABPP relies on the interaction between a chemically designed probe (activity-based probes (ABPs)) and the active site or functional residue of a protein. A typical ABP consists of three core components: a reactive group, a reporter group, and a linker connecting them. Upon application to a biological system, such as cell lysates, living cells, or tissues, the probe selectively labels active proteins. These labeled proteins can then be visualized, enriched, and identified using fluorescence-based methods or mass spectrometry. In competitive formats, bioactive compounds compete with probes for binding, enabling direct identification of compound targets and off-targets. By focusing on protein function rather than abundance, ABPP provides mechanistic insight that is critical for drug discovery.
Fig. 1. Structure of ABPs (upper panel) and the workflow of competitive ABPP (middle and lower panels) [1].
Our ABPP-based Technology Platform
Our ABPP-based technology platform is purpose-built for bioactive natural products and structurally complex small molecules. By integrating chemical probe development, advanced proteomics, and bioinformatics, we offer a comprehensive solution for target identification and mechanism-of-action studies.
Our Probe Design Capabilities
The success of target identification hinges on the quality of the probe. Our probe design strategy is built around the three essential components of ABPs:
- Reactive Group: The reactive group (warhead) determines how the probe engages its target protein. We carefully select reactive groups based on the target protein class, the chemical reactivity of functional residues, and the known or suspected mechanism of action of the parent compound. Our reactive group toolbox includes electrophiles, mechanism-based inhibitors, and latent warheads tailored for specific enzyme families. For natural products with covalent mechanisms, we preserve the native pharmacophore whenever possible. For non-covalent or weakly binding compounds, we incorporate photoactivatable groups to enable covalent capture of transient interactions.
- Reporter Group: This allows for visualization and enrichment. We provide affinity tags (Biotin) for isolation, fluorophores for imaging, and bioorthogonal handles (alkynes/azides) that minimize structural disruption of the natural product.
- Linker: The linker spatially separates the reactive and reporter groups and plays a critical role in probe performance. Our linker designs are optimized for length, flexibility, solubility, and chemical stability.
Our ABPP Strategies
To provide comprehensive insights into complex biological systems, we employ a suite of advanced ABPP strategies:
- Click Chemistry-ABPP (CC-ABPP): CC-ABPP uses probes bearing bioorthogonal handles that are conjugated to reporter tags after biological labeling. This approach minimizes steric hindrance and preserves native target engagement.
- Competitive ABPP: Competitive ABPP identifies direct molecular targets by measuring the ability of a bioactive compound to block probe labeling. Proteins showing reduced labeling in the presence of the compound are inferred to be targets.
- Isotope Tandem Orthogonal Proteolysis-ABPP (isoTOP-ABPP): isoTOP-ABPP combines ABPP with isotope labeling and cleavable linkers to achieve site-specific and quantitative proteomic analysis. This advanced approach enables proteome-wide mapping of reactive residues and covalent binding sites.
Our ABPP Workflow
Step 1
Project Evaluation & ABPP Strategy Design
We begin with a comprehensive assessment of the bioactive compound, including chemical structure, SAR information, and biological phenotype, to define the optimal ABPP strategy (e.g., CC-ABPP, competitive ABPP, or isoTOP-ABPP).
Step 2
Probe Design & Synthesis
Based on the selected strategy, we rationally design and synthesize activity-based probes by minimally modifying the parent compound or scaffold, ensuring preservation of biological activity and target engagement.
Step 3
Proteome Labeling & Competitive Profiling
Probes are applied to complex biological systems (cell lysates, live cells, or tissues). Competitive experiments with the parent compound are performed to distinguish specific targets from nonspecific background labeling.
Step 4
Bioorthogonal Tagging & Target Enrichment
Labeled proteins are conjugated to reporter tags via click chemistry and enriched using affinity-based methods, enabling high-sensitivity detection of probe-engaged proteins.
Step 5
Quantitative Proteomics Analysis
Enriched proteins are digested and analyzed by quantitative LC–MS/MS to identify significantly enriched or competitively inhibited targets.
Step 6
Data Analysis & Target Prioritization
Advanced bioinformatics pipelines are used to integrate quantitative proteomics data and prioritize high-confidence, biologically relevant targets.
Step 7
Orthogonal Target Validation
Top-ranked targets are validated using complementary biochemical and cellular assays, ensuring functional relevance and mechanistic insight.
ABPP is a transformative technology for uncovering the molecular targets and mechanisms of bioactive natural products. Through our ABPP-based technology platform, advanced probe design capabilities, and integrated ABPP strategies, we provide robust solutions for target identification, selectivity profiling, and mechanism elucidation, accelerating natural product-driven drug discovery from phenotype to mechanism.
Online Inquiry
Frequently Asked Questions (FAQ)
Q1: Why is ABPP suitable for natural products?
Q2: How does ABPP compare with affinity pull-down methods?
A: ABPP focuses on functional proteins, reduces false positives, and directly links bioactivity to molecular targets.
Reference
- Jiang X., et al. Recent advances in identifying protein targets of bioactive natural products[J]. Heliyon, 2024, 10(13).