An update of label-free protein target identification methods for natural active products

Abstract

Natural active products (NAPs) are derived from chemical substances found in nature that have biological activity and medicinal potential. Screening and revealing the protein targets of NAPs is an indispensable link in the pharmacological and toxicological understanding of NAPs. Proteins are the main factors executing cell functions, and cells rely on the function of proteins to complete various activities in the life cycle. The important mechanism of action of drugs is to regulate cell biological activities by interacting with proteins and other macromolecules. At present, the classic way to screen protein targets is based on the molecular label tracing method, which has a long cycle and changes the molecular structure and pharmacological effects of NAPs. Due to the shortcomings of molecular labelling methods, in recent years, scientists have tried to develop a variety of label-free protein target identification methods for NAPs and have made a certain amount of progress. This article reviews the current protein target identification methods for NAPs with the aim of providing a reference for research on NAP protein targets.

Keywords: drug discovery; label-free methods; natural active products; protein stability; protein target identification.

Figure 1

Label-free target identification methods based on shifts in the thermal stability of protein targets. When the proteins are heated, their folded structures denature, and the proteins begin to aggregate. Proteins have intrinsic properties of resistance to thermal denaturation, which can be described by the Tm, the temperature when half of the proteins is denatured. Interactions between proteins and small molecules can alter their free energy and thermal stability. Label-free target identification methods based on shifts in the thermal stability of protein that include DSF, CETSA, TPP (or MS-CETSA), TS-FITGE (or 2DE-CETSA), ITDR-MS-CETSA, STPP and HCIF-CETSA.

Figure 2

Label-free target identification methods based on the difference in chemical denaturant-induced stability of protein targets. Proteins can be denatured by chemicals (i.e., denaturants), such as guanidine salts or urea. Denatured proteins are more susceptible to proteolysis or oxidation than intact proteins. The stability of protein to denaturants can be changed by the combination of small molecules, so as to shift the proteolytic stability and oxidation level of protein. Label-free target identification methods based on the difference in chemical denaturant-induced stability of protein that include PP, SPROX, SILAC-SPROX, SILAC-PP, PePTID, STEPP-PP and CPP.

Figure 3

Label-free target identification methods based on the shift in the limited proteolytic susceptibility of protein targets. The protein conformation is influenced by a variety of factors, including post-translational modifications, disease state, and ligand binding, which may alter the proteolytic susceptibility. DARTS and LiP-MS are label-free target identification methods based on the shift in the limited proteolytic susceptibility of protein.

Figure 4

Label-free target identification methods based on organic solvent-induced shift in the solubility of protein targets. Organic solvents are commonly used to precipitate proteins and remove contaminants. The resistance of the protein targets to organic solvent-induced denaturation and precipitation is stronger after ligand-binding events. DiffPOP and SIP are label-free target identification methods based organic solvent-induced shift in the solubility of protein.

Figure 5

New label-free screening strategies. UPT uses the weak molecular interaction of bait-molecule to non-covalently immobilize it on a polymeric surface to achieve target protein fishing. AS-MS is an affinity-based screening technique for the analysis of interactions between protein targets and small molecules.

Figure 6

Indirect screening strategies for protein targets. (A) Protein degradation methods as dTAG method; (B) Genomic library screening methods as the CRISPR genomic library screening; (C) Differential genomic screening methods and differential proteomic screening methods; (D) Phenotypic-based screening methods; (E) Bioinformatics prediction methods.

Figure 7

Timeline of the different label-free approaches.