Recent advances in identifying protein targets of bioactive natural products

Abstract

Background: Natural products exhibit structural complexity, diversity, and historical therapeutic significance, boasting attractive functions and biological activities that have significantly influenced drug discovery endeavors. The identification of target proteins of active natural compounds is crucial for advancing novel drug innovation. Currently, methods for identifying targets of natural products can be categorized into labeling and label-free approaches based on whether the natural bioactive constituents are modified into active probes. In addition, there is a new avenue for rapidly exploring the targets of natural products based on their innate functions.

Aim: This review aimed to summarize recent advancements in both labeling and label-free approaches to the identification of targets for natural products, as well as the novel target identification method based on the natural functions of natural products.

Methods: We systematically collected relevant articles published in recent years from PubMed, Web of Science, and ScienceDirect, focusing on methods employed for identifying protein targets of bioactive natural products. Furthermore, we systematically summarized the principles, procedures, and successful cases, as well as the advantages and limitations of each method.

Results: Labeling methods allow for the direct labeling of target proteins and the exclusion of indirectly targeted proteins. However, these methods are not suitable for studying post-modified compounds with abolished activity, chemically challenging synthesis, or trace amounts of natural active compounds. Label-free methods can be employed to identify targets of any natural active compounds, including trace amounts and multicomponent mixtures, but their reliability is not as high as labeling methods. The structural complementarity between natural products and their innate receptors significantly increase the opportunities for finding more promising structural analogues of the natural products, and natural products may interact with several structural analogues of receptors in humans.

Conclusion: Each approach presents benefits and drawbacks. In practice, a combination of methods is employed to identify targets of natural products. And natural products' innate functions-based approach is a rapid and selective strategy for target identification. This review provides valuable references for future research in this field, offering insights into techniques and methodologies.

Keywords: Label-free approaches; Labeling methods; Natural products; Natural products' innate functions-based approach; Protein targets.

Graphical abstract

Fig. 1

The typical process of affinity chromatography. Initially, bioactive natural products are immobilized onto a solid support matrix (such as agarose beads or resin), serving as baits. Subsequently, cell lysates or tissue homogenates are either incubated with the active compound-loaded solid support matrix (when agarose beads serve as the solid support matrix) or passed through the solid support matrix (when resin materials serve as the solid support matrix). Afterward, multiple washes with inert solvents are performed to remove unbound proteins. Finally, the retained proteins are eluted either by heating, utilizing high-ionic-strength solvents, or adding an excess of a free drug to compete with the target protein binding. Subsequently, these proteins are identified through quantitative proteomic analysis.

Fig. 2

The structure of ABPs and the workflow of competitive ABPP. The upper panel depicts the structure of ABPs, which include a reactive group, a reporter group, and a linker facilitating their connection. The middle and lower panels denote the workflow of a competitive ABPP. Initially, cell lysates or live cells are incubated with either the native bioactive natural product or solvent and subsequently labeled with an ABPP probe. Following this, SDS-PAGE or LC–MS/MS are employed to either visualize or identify the targets labeled by the ABPP probe. Targets of the bioactive natural product exhibit diminished signals in the active compound-treated samples compared to those treated with solvent controls.

Fig. 3

The typical process of competitive ABPP–SILAC. The heavy amino acid-labeled group is treated with the natural active compound, serving as the competitive group, while the light amino acid-labeled group remains untreated. Subsequently, the two groups are combined in accordance with either cell number or protein quantity, followed by the addition of the bioactive natural product probe for labeling the target proteins. LC–MS/MS is subsequently employed to identify the proteins captured by the probe. The SILAC ratio (light vs. heavy) quantified for each protein serves as an indicator of the potential target of the active natural compound, with a higher ratio suggesting a stronger likelihood of interaction.

Fig. 4

The typical process of DARTS. Initially, cell lysates are incubated either with a solvent or with a natural active compound. Following this, both groups are subdivided into five to six subgroups. Subsequently, gradient proteolysis is performed for each subgroup. Finally, the samples are subjected to SDS-PAGE for the detection of the target molecule.

Fig. 5

Schematic workflow of TPP. Initially, cell lysates are incubated either with or without the active natural compound. Subsequently, aliquots are incubated at 10 distinct temperatures and then centrifuged. Following this, the proteins are digested with trypsin to generate peptides. After TMT10 labeling, the samples are mixed, fractionated, and subjected to LC–MS/MS analysis to ascertain the Tm values of individual proteins, thereby facilitating the screening of target proteins.

Fig. 6

The workflow of SPROX. Initially, in both the presence and absence of the active natural product, cell lysates are combined with various denaturant-containing buffers. Following equilibration of protein unfolding/refolding, hydrogen peroxide is added to induce the oxidation of methionine residues. Subsequently, the oxidation reaction is quenched using methionine or a hydrogen peroxide scavenger. Finally, proteins in each denaturant-containing buffer are digested into peptides for subsequent quantitative proteomics analysis.

Fig. 7

The principle and general workflow of ITC. Initially, the reference and sample cells are equilibrated to the desired temperature. Subsequently, a highly accurate injection device loaded with the active compound is inserted into the sample cell containing the target protein. The natural active compound is titrated into the sample cell through sequential injections. During each injection, the microcalorimeter measures the released heat until the binding reaction reaches equilibrium. Thermodynamic parameters are derived by fitting the titration curve.