Currently Available Strategies for Target Identification of Bioactive Natural Products

Abstract

In recent years, biologically active natural products have gradually become important agents in the field of drug research and development because of their wide availability and variety. However, the target sites of many natural products are yet to be identified, which is a setback in the pharmaceutical industry and has seriously hindered the translation of research findings of these natural products as viable candidates for new drug exploitation. This review systematically describes the commonly used strategies for target identification via the application of probe and non-probe approaches. The merits and demerits of each method were summarized using recent examples, with the goal of comparing currently available methods and selecting the optimum techniques for identifying the targets of bioactive natural products.

Keywords: drug discovery; natural product; non-probe; probe; target identification.

FIGURE 1

Structural composition of chemical probes. (A) Basic structure of chemical probes. (B) Example of commonly used linkers. (C) Example of commonly used reporter groups.

FIGURE 2

Schematic diagram of the target hooking technique. The NP is first structurally designed to be anchored to an insoluble support. Elution is performed after contact with the cell lysate, and the target proteins interacting with the affinity molecules are retained and identified by high-resolution mass spectrometry (MS).

FIGURE 3

General workflow of the activity-based protein profiling (ABPP) method. The NP is first probed for specific affinity to adsorb the target protein. Affinity purification and elution are performed after contact with the cell lysate, and identified by sodium dodecyl sulfate (SDS)-PAGE and MS.

FIGURE 4

Flow diagram of the click chemistry–activity-based protein profiling (CC-ABPP) strategy. It starts with the synthesis of a NP with a terminal alkyne. After sufficient binding to the target protein, the probe is formed by a click reaction with an azide bearing a fluorescent or radioactive moiety. The target protein is subsequently identified by SDS-PAGE and MS.

FIGURE 5

Flow diagram of the competitive activity-based protein profiling (ABPP) strategy. It allows the precursor compound of the probe to be co-incubated with the proteome before adding the probe to bind with the protein. Then, it is possible to obtain the true target protein by comparing the protein and active site labelled by the probe before and after the addition of the precursor compound.

FIGURE 6

Structures of ABPP and CCCP probes for bioactive natural products.

FIGURE 7

Flow diagram of the combined activity-based protein profiling (ABPP) and stable isotope labeling by amino acids in cell culture (SILAC) strategies. Firstly, it utilizes a set of amino acid isotope markers for two cell populations to be cultured. The probe is added to the heavy group and the light group is used as a control group. The labelled proteins are analyzed and identified by MS against normal proteins after a period of time.

FIGURE 8

Schematic diagram of the drug affinity responsive target stability (DARTS) strategy. The experiment is mainly divided into a small molecule group and a control group. Target proteins bound to small molecules are not readily hydrolysed by proteases.

FIGURE 9

Schematic diagram of the stability of proteins from rates of oxidation (SPROX) strategy. First, two protein samples (with and without ligand) are dispensed into a buffer containing a chemical denaturant , and hydrogen peroxide is added to the protein sample. Then, the oxidation reaction is quenched with an excess of methionine, and the protein sample is precipitated with tricarboxylic acid for subsequent quantitative proteomics to obtain the oxidation ratio of oxidized methionine.

FIGURE 10

Schematic diagram of the thermal proteome profiling (TPP) and cellular thermal shift assay (CETSA) methods. First, two protein samples (with and without ligand) are dispensed into a buffer containing a chemical denaturant. Then,high-resolution MS is performed using neutron-encoded isobaric mass TMT10 as a labelling reagent, and the complete melting curves of heavily expressed soluble proteins are obtained.