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Heliyon 10 (2024) e33917
Available online 29 June 2024
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Review article
Recent advances in identifying protein targets of bioactive
natural products
Xuan Jiang
a
, Kinyu Shon
d
, Xiaofeng Li
e
, Guoliang Cui
d
, Yuanyuan Wu
a
,
Zhonghong Wei
a
, Aiyun Wang
a
,
b
,
c
, Xiaoman Li
a
,
*
, Yin Lu
a
,
b
,
c
,
**
a
Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese
Medicine, Nanjing, 210023, China
b
Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing,
210023, China
c
Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine (TCM) Prevention and Treatment of Tumor, Nanjing University of Chinese
Medicine, Nanjing, 210023, China
d
Department of Gastroenterology, The Second Afliated Hospital of Nanjing University of Chinese Medicine, Nanjing, 210023, China
e
Department of Biochemistry and Molecular Biology, School of Medicine, Nanjing University of Chinese Medicine, Nanjing, 210023, China
ARTICLE INFO
Keywords:
Protein targets
Natural products
Labeling methods
Label-free approaches
Natural products’ innate functions-based
approach
ABSTRACT
Background: Natural products exhibit structural complexity, diversity, and historical therapeutic
signicance, boasting attractive functions and biological activities that have signicantly inu-
enced drug discovery endeavors. The identication 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 modied 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 ap-
proaches to the identication of targets for natural products, as well as the novel target identi-
cation 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, pro-
cedures, 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-modied
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 signicantly increase the opportunities for nding more promising structural
* Corresponding author. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy,
Nanjing University of Chinese Medicine, Nanjing 210023, China.
** Corresponding author. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy,
Nanjing University of Chinese Medicine, Nanjing, 210023, China.
E-mail addresses: lixm@njucm.edu.cn (X. Li), luyingreen@njucm.edu.cn (Y. Lu).
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyon
https://doi.org/10.1016/j.heliyon.2024.e33917
Received 2 January 2024; Received in revised form 28 June 2024; Accepted 28 June 2024
Heliyon 10 (2024) e33917
2
analogues of the natural products, and natural products may interact with several structural
analogues of receptors in humans.
Conclusion: Each approach presents benets 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 identication. This review
provides valuable references for future research in this eld, offering insights into techniques and
methodologies.
1. Introduction
Compared to small-molecule drugs synthesized chemically, natural products offer signicant advantages in terms of structural
novelty, diversity, biocompatibility, and functional versatility. Moreover, they have undergone natural selection and optimization
through long-term evolutionary processes [1]. Approximately 70 % of all approved therapeutic agents originate from natural products
or their derivatives [2]. Thus, natural products are crucial sources of new drugs [3]. Notably, the 2015 Nobel Prize in Physiology or
Medicine awarded to Youyou Tu for discovering the plant natural product artemisinin, which revolutionized malaria treatment,
brought immense pride and optimism to the natural product community worldwide. However, unclear modes of action and challenges
in identifying target proteins hinder the development of natural products in drug discovery. Thus, exploring and identifying targets of
natural products is essential for understanding their mechanisms of action, identifying potential toxicities, and guiding
structure-activity relationship studies. Furthermore, it contributes to the discovery of novel disease-relevant targets, playing a critical
role in innovative drug research based on natural products.
Currently, with the advancement in chemical biology and biophysics, an increasing array of methods has emerged for identifying
targets of natural bioactive molecules. These methods can be broadly classied into labeling and label-free approaches based on
whether natural bioactive constituents are modied into active probes. In probe-based methods, natural bioactive molecules are
transformed into active probes to explore their interactions with targets, whereas label-free methods rely on changes in the biophysical
properties of the target protein upon their binding with the natural bioactive molecule for the identication of the target protein. In
addition, a novel avenue for rapidly exploring the targets of natural products based on their innate functions has also been developed.
This review primarily summarizes these two methodological categories as well as the new approach based on the innate functions of
natural products, providing a systematic elucidation of their principles, workows, and successful cases, as well as the advantages and
disadvantages associated with each approach. This review aims to inspire and provide guidance to researchers in this eld.
Fig. 1. The typical process of afnity 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 identied through quantitative proteomic analysis.
X. Jiang et al.
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Table 1
Recent studies that conducted afnity chromatography-based target identication of natural products.
No. Natural product Structure Category Bioactivity Target protein Reference
1 Artesunate
Quaternary
ammonium salts
Regeneration of pancreatic β cell mass
from
α
cells
Gephyrin [4]
2 Arzanol
Terpenoids Anti-inammatory activity Brain glycogen
phosphorylase
[5]
3 Baicalein Flavonoids Elimination of liver tumor-initiating
stem cell-like cells resistant to mTORC1
inhibition
SAR1B guanosine
triphosphatase
[6]
4 20(S)-
Protopanaxadiol
Saponins Reduction of fatigue CK-MM [7]
5 IJ-5 Terpenoids Anti-inammatory activity Ubiquitin-
conjugating enzyme
UbcH5
[8]
6 Sappanone A Ketones Anti-neuroinammatory activity IMPDH2 [10]
7 Celastrol Terpenoids Intervention of the progression of
hepatocellular carcinoma
ROCK2 [11]
8 Ainsliadimer A Terpenoids Anticancer and anti-inammatory
activity
IKKβ [12]
(continued on next page)
X. Jiang et al.
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4
2. Target identication of natural bioactive components using labeling techniques
Labeling techniques entail chemically modifying natural bioactive components to generate probes with specic markers, such as
uorescent dyes or biotin conjugates. These labeled probes enable the identication of target proteins through corresponding uo-
rescence imaging or enrichment techniques.
2.1. Afnity chromatography
Afnity chromatography stands out as the most traditional method of labeling. As shown in Fig. 1, the typical procedure involves
initially identifying the active functional groups of natural bioactive components and then immobilizing them onto a solid support
matrix, such as agarose beads or resin. Once immobilized, these probes act as baits. Subsequently, the cell or tissue lysate is either
incubated with the solid support matrix loaded with active compounds (when agarose beads are used as the solid support matrix) or
passed through the solid support matrix (when resin materials are used as the solid support matrix). Following this, multiple washes
with inert solvents are performed to remove non-specically bound proteins, leaving the proteins that exhibit afnity binding with the
immobilized natural bioactive components attached to the solid support matrix. Finally, the bound proteins are eluted through various
means, such as heating, using high ionic strength solvents, or adding an excess of free drug to compete with the target protein binding.
The eluted proteins are then subjected to quantitative proteomic analysis for identication. This method has been utilized for the
discovery of target proteins for numerous well-known compounds. For instance, afnity chromatography assays have enabled the
identication of the targets of artesunate [4], arzanol [5], baicalein [6], 20(S)-protopanaxadiol [7], and the herb-derived sesquiter-
pene lactone compound IJ-5 [8] (Table 1). A notable example is the study by Ito et al. [9], where immobilized probes of thalidomide
were employed to identify its target protein, cereblon (CRBN). CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding
protein 1 and Cul4A, thereby inhibiting ubiquitin ligase activity crucial for limb outgrowth in zebrash and chicks, thus contributing to
thalidomide teratogenicity. This research was published in the journal “Science”.
Furthermore, natural bioactive components can be biotinylated. By exploiting the strong afnity interaction between streptavidin
and biotin, the biotinylated natural bioactive components can be captured, enriched, and puried using streptavidin-coated agarose
solid support carriers. This facilitates the capture of target proteins associated with natural bioactive components, which can subse-
quently be identied via mass spectrometry (MS). Tu and colleagues transformed sappanone A, an anti-neuroinammatory active
component from the traditional Chinese medicine Su-Mu, into a chemical probe conjugated with biotin. This probe was employed to
“sh out” the direct target proteins from glial cells, leading to the identication of inosine-5
′
-monophosphate dehydrogenase 2
(IMPDH2) as a key target of sappanone A [10]. This breakthrough was recognized as one of the “Top 10 Medical Advances in China” in
2017. In addition, biotin afnity purication systems have been employed to identify the targets of celastrol, an active component from
the medicinal plant Tripterygium wilfordii Hook F [11], the natural anti-inammatory active molecule ainsliadimer A [12], the marine
natural product pateamine A [13], and adenanthin, a diterpenoid derived from the leaves of Isodon adenanthus [14] (Table 1).
Afnity chromatography facilitates rapid and large-scale enrichment of target proteins. Nevertheless, it faces three primary lim-
itations [15]. Firstly, discerning between high-afnity target proteins and abundant proteins with low afnity poses a challenge.
Secondly, the washing process relies on empirical conditions, wherein overly stringent conditions may signicantly decrease the
number of identied targets, while weak washing conditions could yield false-positive results. Lastly, it fails to capture the interaction
Table 1 (continued )
No. Natural product Structure Category Bioactivity Target protein Reference
9 Pateamine A
Alkaloids Inhibition of eukaryotic translation
initiation
Eukaryotic initiation
factor 4A
[13]
10 Adenanthin Terpenoids Induction of leukemic cell
differentiation
Peroxiredoxins I and
II
[14]
Note: The red-marked portion in the structure refers to the labeling binding site.
X. Jiang et al.
Heliyon 10 (2024) e33917
5
between drugs and proteins within live cells under physiological conditions. Additionally, the biotin afnity tag introduces steric
hindrance, potentially diminishing or even abolishing the activity of natural bioactive components and hindering the entry of probe
molecules into cells.
2.2. Activity-based protein proling (ABPP)
Given the limitations of afnity chromatography, chemists and biologists have focused on exploring target identication methods
suitable for targets exhibiting low abundance and live cells. Currently, the most widely employed and rapidly advancing technique is
ABPP, which was introduced by Dr. Cravatt’s research team at the Scripps Research Institute in the United States in 1999 [16].
Activity-based probes (ABPs) form the cornerstone of ABPP technology. Unlike immobilized probes, ABPs do not necessitate pre-
xation to a matrix material and can be directly applied to live cells, elucidating drug–target interactions under physiological con-
ditions [17]. ABPs comprise three components: a reactive group, a reporter group, and a linker connecting the reactive and reporter
groups (Fig. 2). The reactive group serves as the core of ABPs. Depending on the reactive group, ABPs can be classied as either
covalent or non-covalent probes for natural bioactive components. For example, natural products containing electrophilic moieties,
such as Michael acceptors, β-lactones, β-lactams, epoxides, haloalkyl carbonyl compounds, and isothiocyanates, can form covalent
bonds with the nucleophilic groups of target proteins, facilitating the covalent binding of the probe molecule to the target protein [18].
The design of such probes merely entails incorporating a reporter group via a linker in a region that does not affect the activity of the
natural products.
Fluorescent dyes or biotin are commonly employed as reporter groups in ABPs. However, these labeling molecules suffer from
issues such as large size and poor solubility, signicantly hampering the ability of ABPs to penetrate the cell membrane [15]. To
overcome this limitation, Speers et al. [19] integrated click chemistry with ABPs. In this method, inert reactive groups, such as alkynes
and azides, replace the original reporter groups. These inert reactive groups exhibit minimal interference with the chemical properties
of natural bioactive components, allowing them to bind to target proteins in live cells or cell lysates. Subsequently, through click
chemistry reactions, uorescent dyes or biotin can be appended to the probe–target complex, facilitating the enrichment and
Fig. 2. The structure of ABPs and the workow 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 workow 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.
X. Jiang et al.
Heliyon 10 (2024) e33917
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identication of the target proteins [15].
The typical workow of ABPP entails incubating probes with live cells or cell lysates, facilitating the binding of the small-molecule
probe to the active site of its target enzyme. The probe forms a complex with the enzymes through enzymatic reactions or covalent
binding with the target protein, induced by ultraviolet (UV) irradiation using photoafnity groups. Subsequently, the labeled protein is
enriched and puried using the probe’s reporter group, such as uorescence or biotin. Finally, target identication is achieved using
techniques such as MS.
In ABPP experiments, competitive ABPP groups may be congured by pre-incubating the natural bioactive component with either
cell lysates or live cells prior to introducing the natural bioactive component probe (Fig. 2). Should the natural bioactive component
effectively compete for binding to the enzymatic active site of the target protein, it would competitively impede the labeling process of
the target protein by the probe. Compared to the control group treated solely with the probe, the competitive group exhibits
Table 2
Recent studies that conducted ABPP-based target identication of natural products.
No. Natural
products
Structure Category Bioactivity Target protein Reference
1 Nimbolide
Terpenoids Anticancer
activity
E3 ubiquitin ligase RNF114 [21]
2 Parthenolide Terpenoids Anticancer
activity
Ubiquitin carboxyl-terminal hydrolase
10 (USP10)
[22]
3 Dankastatin B Terpenoids Anticancer
activity
Mitochondrial VDAC3 [23]
4 Salinipostin A Cyclopeptide Antimalarial
activity
Essential
α
/β serine hydrolases [25]
5 Illudin
Terpenoids Anticancer
activity
DNA modication and unselective
protein binding
[26]
6 Withangulatin A Alkaloids Antitumor
activity
phosphoglycerate dehydrogenase
(PHGDH)
[27]
7 Piperlongumine Alkaloids Anticancer
activity
GSTO1 [28]
Note: The red-marked portion in the structure refers to the labeling binding site.
X. Jiang et al.
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Table 3
Recent studies that performed PAL probe-based target identication of natural products.
No. Natural products Structure Category Bioactivity Target protein Reference
1 Matrine
Alkaloids Anticancer activity Annexin A2 [30]
2 Coibamide A Cyclopeptide Anticancer activity Sec61 [31]
3 Betulinic acid Terpenoids Anticancer activity Tropomyosin [32]
4 Harringtonolide Cyclopeptide Suppression of
cancer cell
migration
Receptor for activated C kinase 1 [33]
5 Pseudolaric acid
B
Terpenoids Anticancer activity CD147 [34]
6 Epothilone Macrolides Alleviation of CNS
injuries
The binding site on β-tubulin [35]
7 Quercetin Flavonoids Antitumor activity HSP70, HSP90, RuvB-like 2 ATPases,
and eukaryotic translation initiation
factor 3
[36]
8 Pladienolide Macrolides Antitumor activity Splicing factor SF3b [37]
X. Jiang et al.
Heliyon 10 (2024) e33917
8
diminished uorescence signal intensity or reduced peak intensity in MS, facilitating the identication of potential target proteins
[20].
Spradlin et al. [21] utilized ABPP platforms to reveal that nimbolide, a terpenoid natural product derived from the Neem tree, binds
to a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. This interaction inhibits the
ubiquitination and degradation of tumor suppressors, thereby impeding cancer pathogenicity. Similarly, Zhao et al. [22] identied the
covalently modied target of parthenolide, another terpenoid natural product with potent antitumor activity, using ABPP platforms.
Moreover, ABPP has been employed to explore the mechanisms of action of dankastatin B, which exhibits anticancer activity [23],
spongiolactones, marine natural products with potential anticancer properties [24], the antimalarial natural product salinipostin A
[25], illudin, an anticancer natural product family [26], withangulatin A, a natural small molecule that inhibits the serine synthesis
pathway and proliferation of colon cancer cells [27], and piperlongumine, a cancer-selective killing natural product [28], all of which
possess electrophilic functional groups (Table 2).
This method offers the advantage of robust specicity. ABPs eliminate the need for pre-xation to a substrate material and can be
Note: The red-marked portion in the structure refers to the labeling binding site.
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) quantied 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.
X. Jiang et al.
Heliyon 10 (2024) e33917
9
directly applied to live cells, elucidating drug–target interactions under physiological conditions. However, its effectiveness is con-
strained to capturing target proteins that covalently bind with small molecules, thus limiting its applicability.
2.3. Photoafnity labeling probes (PAL probes)
When designing probes for natural active compounds that bind to targets through non-covalent interactions, it is necessary to
introduce photoreactive groups in addition to linker groups and reporter groups. These photoreactive groups, upon UV irradiation,
generate highly reactive intermediates known as carbenes, which can form stable covalent bonds between the probe and the target
protein. Such probes are categorized as PAL probes. Commonly employed photoafnity groups encompass diazirine, benzophenone,
and aryl azide [29]. Matrine is a plant alkaloid with potent anticancer activities. However, its molecular target(s) and mechanism
remain unknown. Wang et al. [30], employing the PAL approach, for the rst time, identied annexin A2 as a direct-binding target of
matrine in cancer cells. Moreover, photoafnity probes have been utilized to elucidate the targets of various natural compounds, such
as coibamide A, a marine natural product with potent antiproliferative activity against human cancer cells [31], betulinic acid [32],
harringtonolide, a bioactive diterpenoid tropone isolated from Cephalotaxus harringtonia with antiproliferation activity [33], pseu-
dolaric acid B possessing anticancer activity [34], the epothilone binding site on β-tubulin [35], quercetin, a avonoid natural product
found in various foods with a wide range of medicinal effects [36], and pladienolide, a naturally occurring antitumor macrolide [37]
(Table 3).
This method enables the analysis of proteins that cannot be targeted by ABPs. Nevertheless, photoafnity groups may still display
non-specic binding to abundant cellular proteins.
2.4. ABPP coupled with the stable isotope labeling by amino acids in cell culture (SILAC) technique
To enhance quantitative accuracy, Cravatt et al. integrated the SILAC technique and ABPP, yielding ABPP–SILAC. SILAC operates
by substituting specic amino acids in cell culture media with either naturally occurring isotope (light) or stable isotope (heavy)-
labeled amino acids. Following ve to six cell cycles, stable isotope-labeled amino acids are fully incorporated into newly synthesized
proteins, replacing endogenous amino acids. Lysates from light- and heavy-isotope-labeled cells are equitably combined, separated via
gel electrophoresis, and subsequently identied via MS. Each peptide manifests as a pair in MS data, with the light-labeled peptide
containing the light amino acid and the heavy-labeled peptide containing the heavy amino acid. A 1:1 ratio in SILAC peptide pairs
signies unaltered protein abundance in the proteome. If the SILAC peptide pair ratio (light vs. heavy) is large, it indicates a potential
target of the natural product [38].
By integrating competitive ABPP with SILAC, the heavy amino acid-labeled group is subjected to treatment with the natural active
component within the cell lysate (or within live cells), serving as the competitive group, while the light amino acid-labeled group
remains untreated. Subsequently, the two groups are combined in accordance with cell number or protein quantity, followed by the
addition of the natural active component probe to label the target proteins. MS is then employed for target identication purposes
(Fig. 3). A diminished intensity of the heavy-labeled peptide peak implies potential binding of the natural product to the target protein.
Wang and colleagues employed the ABPP–SILAC technique to explore the targets involved in the lipid-lowering effects of baicalin, the
principal active component of Scutellaria baicalensis. They synthesized a baicalin-based photoafnity probe and employed competitive
ABPP–SILAC to capture its targets. LC–MS/MS analysis, complemented by bioinformatics analysis and reverse validation, revealed
carnitine palmitoyltransferase 1A as a target of baicalin in the context of obesity amelioration [39]. In addition, Chen et al. [40]
identied peroxiredoxin 6 as a direct target of withangulatin A in the alleviation of non-small cell lung cancer (NSCLC) via
ABPP–SILAC (Table 4).
Table 4
Recent studies that performed ABPP–SILAC-based target identication of natural products.
No. Natural products Structure Category Bioactivity Target protein Reference
1 Baicalin
Flavonoids Alleviation of obesity Carnitine palmitoyltransferase 1A [39]
2 Withangulatin A Alkaloids Anti-NSCLC effects Peroxiredoxin 6 [40]
Note: The red-marked portion in the structure refers to the labeling binding site.
X. Jiang et al.
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2.5. Proteome microarray assays
Firstly, biotin or uorescent-labeling natural active components were co-incubated with a protein chip. After washing away un-
bound components, the uorescence intensity of each spot on the chip is determined via uorescence scanning or laser confocal
scanning. When biotin-labeling natural active components are employed, they are co-incubated with uorescent-labeling streptavidin
and subjected to uorescence scanning to screen potential targets of the natural active components. Chen and colleagues utilized this
technique to explore the targets of arsenic [41]. Arsenic demonstrates excellent efcacy in acute promyelocytic leukemia treatment
and possesses signicant therapeutic potential for various tumors. However, its broad anticancer mechanisms remain unclear. Re-
searchers co-incubated biotinylated arsenic with a protein chip containing 16,368 proteins, utilizing Cy3-labeling streptavidin to
identify arsenic-binding proteins. Fluorescence scanning revealed enhanced uorescence signals in the probe group compared to the
negative control group. They identied 360 proteins directly interacting with arsenic, including the well-known arsenic target PML.
Previously, fewer than 20 proteins directly interacting with arsenic had been identied worldwide, underscoring the protein chip
platform’s efcacy in rapidly and comprehensively identifying drug targets. The most signicantly enriched proteins are implicated in
the glycolysis pathway. Detailed biochemical and metabolomics analyses have identied hexokinase-2 (HK2) as a key target of arsenic,
functioning as a rate-limiting enzyme in glycolysis. This research was published in “Proceedings of the National Academy of Sciences of
the United States of America”. Liu et al. [42] identied bufalin, a natural product-derived molecular glue, which targets E2F2
degradation, leading to the transcriptional suppression of multiple oncogenes and the growth of hepatocellular carcinoma in vitro and
in vivo. In addition, proteome microarray assays have been employed to identify the targets of coelonin, an active anti-inammatory
component of Bletilla striata [43] (Table 5).
This method offers the advantages of necessitating minimal sample volumes and boasting high throughput, enabling the simul-
taneous analysis of thousands to millions of proteins. Additionally, protein chips are relatively user-friendly. However, they entail high
costs due to the need for a series of expensive, high-end instruments. Furthermore, standardization issues are observed with protein
chips.
3. Target identication and target validation of natural bioactive components using label-free techniques
Despite signicant advancements in afnity-based methods for screening natural product targets, limitations persist [44]. Some
natural products lack suitable modication sites or synthetic methods to introduce such sites. Additionally, exogenous modication
groups may affect the activity of natural active compounds or hinder their binding to true targets, thereby compromising target
identication accuracy. To overcome these challenges, researchers have devised various target screening methods that obviate the
need for structural modications of natural active molecules. Instead, they employ biophysical approaches to assess the impact of the
binding of natural active compounds to target proteins on protein stability, thus facilitating the discovery of targets for natural active
molecules. Moreover, label-free techniques can be categorized into target identication and target validation methods. For example,
drug afnity responsive target stability (DARTS), thermal proteome proling (TPP), and stability of proteins from rates of oxidation
(SPROX) methods are primarily used for target validation, while cellular thermal shift assays (CETSA) and isothermal titration
calorimetry (ITC) are often used for target identication.
Table 5
Recent studies that conducted proteome microarray-based target identication of natural products.
No. Natural
products
Structure Category Bioactivity Target protein Reference
1 Arsenic
Elemental Broad anticancer activity 360 proteins, among which HK2 is a
key target
[41]
2 Bufalin Steroids Suppression of hepatocellular
carcinoma growth
E2F2 [42]
3 Coelonin Alkaloids Anti-inammatory activity PTEN [43]
X. Jiang et al.
Heliyon 10 (2024) e33917
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3.1. Target identication methods
3.1.1. DARTS
In 2009, Lomenick et al. introduced DARTS [45]. The method’s principle posits that upon the binding of a small molecule to a target
protein, the small molecule occupies the recognition site of proteolytic enzymes, impeding their interaction with the cleavage site on
the target protein. Consequently, the target protein exhibits enhanced resistance to proteolytic cleavage and increased stability.
Changes in the resistance to proteolysis pre- and post-small molecule binding can be discerned via electrophoresis. Subsequent
identication of distinct bands on the protein gel can be achieved through MS to ascertain the target proteins of the small molecule. As
a proof-of-principle, researchers selected the FKBP12 protein, a known target of rapamycin, and FK506. Proteolysis was conducted
using subtilisin protease, and the resistance of FKBP12 to proteolysis subsequent to binding with the compound was observed via
SDS-PAGE, thus afrming the scientic validity and feasibility of this method [45].
The typical procedure of this method involves cell lysis, followed by dividing the lysate into two groups (Fig. 4): the active natural
compound group and the solvent group. The lysates of the active natural compound group are incubated with the bioactive compound,
while the lysates of the solvent group are treated with the solvent alone. This allows the bioactive compound to bind to the target
proteins. Subsequently, both the solvent and the bioactive compound groups are further divided into ve to six subgroups, and each
subgroup is treated with a gradient concentration of proteolytic enzyme for protein digestion. Commonly used proteolytic enzymes
include subtilisin protease, thermolysin, and protease from Streptomyces griseus. Finally, various methods, such as SDS-PAGE, 2D-
PAGE, gel staining techniques, and gel or non-gel MS, are employed to detect or identify the bound proteins.
The DARTS technique, which does not necessitate chemical modication of small molecules, offers a convenient means to identify
direct targets of natural products [46]. It has seen widespread application in recent years. Huang et al. [47] employed the DARTS
method and discerned that the antitumor natural active compound batzelladine A targets HSP90. The target of the primary active
compound, andrographolide, in the traditional Chinese medicinal Andrographis paniculata, known for its heat-clearing and detoxifying
properties, was also identied using the DARTS technique. As the concentration of proteolytic enzymes increased, proteins in the
solvent group gradually degraded, while andrographolide inhibited the hydrolysis of the target protein, dynamin-related protein 1
(DRP1), compared to the control group [48]. Moreover, DARTS conrmed the targets of polyphyllin D, a natural product effective
against NSCLC [49], ergolide, a sesquiterpene lactone natural product with anti-inammatory and anticancer activities used to treat
inammatory diseases [50], aconitine, an active compound exhibiting cardiotoxic effects found in Aconitum species [51], crellastatin
A, a cytotoxic sulfated bis-steroid isolated from the Vanuatu Island marine sponge Crella sp. [52], cryptotanshinone, isolated from the
roots of Salvia miltiorrhiza inhibiting the terminal differentiation of human keratinocytes [53], and grape seed extract in anti-colorectal
cancer applications [54] (Table 6).
The limitations of DARTS encompass the following: certain small molecules may bind to the target protein without inducing
signicant conformational changes of the target; certain proteins could exhibit reduced sensitivity to proteolytic enzymes, thereby
resulting in imperceptible alterations in DARTS outcomes; and the capacity to identify targets characterized by low protein abundance
remains limited.
3.1.2. TPP
TPP, a combination of CETSA and multiplexed quantitative MS, enables the monitoring of global protein thermal stability changes
during interactions with small molecules, thereby facilitating high-throughput screening of target proteins [55]. TPP operates on two
principles: the principles of CETSA and multiplexed quantitative MS. Unlike CETSA, TPP characterizes protein thermal stability based
on the melting temperature (Tm), denoting the temperature at which 50 % of the protein unfolds during thermal denaturation.
Typically, proteins bound to small molecules exhibit increased stability and higher Tm values compared to unbound proteins. By
comparing the Tm values and melting curves of proteins with and without small-molecule binding, target proteins can be identied.
Multiplexed quantitative MS primarily refers to tandem mass tag (TMT), a peptide labeling technology developed by Thermo Fisher in
the United States. TMT facilitates the labeling of peptides with up to 10 isotopic tags, enabling the simultaneous comparison of relative
protein levels across 10 different samples. Therefore, TMT serves as a high-throughput analysis method that is particularly suitable for
differential protein analysis of samples subjected to various treatments or treatment durations. Furthermore, through stable isotope
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 ve 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.
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labeling of different samples, all labeled samples can be pooled for subsequent processing, signicantly reducing experimental errors
and considerably improving quantication accuracy.
The typical protocol of this method involves treating cells or cell lysates with or without an active natural compound, followed by
dividing the samples into 10 equal portions and heating them at increasing temperatures to induce protein denaturation and aggre-
gation. The supernatant is then collected and digested using trypsin to generate peptides, which are subsequently labeled using
TMT10. These labeled samples are mixed and analyzed through LC–MS/MS to calculate the Tm values of each protein, thereby
screening for target proteins (Fig. 5). Building upon the potent efcacy of the natural product vioprolide A against human acute
lymphoblastic leukemia cells, Kirsch et al. [56] conducted TPP, conrming nucleolar protein 14, essential in ribosome biogenesis, as a
specic target of vioprolide A in Jurkat cells. Furthermore, in a study published in the journal “Science Translational Medicine”, TPP
was utilized to identify the targets of antimalarial drugs, revealing that the antimalarial drug quinine directly binds to the parasite’s
intracellular purine nucleotide phosphorylases [57]. Chen et al. [58] demonstrated that rhodojaponin VI, a grayanotoxin from
Rhododendron molle, alleviates neuropathic pain by targeting N-ethylmaleimide-sensitive fusion protein through the TPP approach.
Moreover, TPP has been employed to elucidate the targets of tutin-induced epilepsy [59], the natural sesquiterpene lactone alanto-
lactone, which inhibits NSCLC [60], kurarinone, which attenuates MPTP-mediated neuroinammation [61], and arone, which pro-
tects against neuroinammation [62] (Table 7).
This method offers the advantage of monitoring global thermal stability changes in proteins during drug action, enabling high-
throughput detection of target proteins. However, it is unsuitable for heat-insensitive proteins, some of which require extreme tem-
perature conditions to exhibit changes in thermal stability. Additionally, certain proteins may not display signicant alterations in
thermal stability upon ligand binding, rendering their identication unfeasible through TPP. Furthermore, extracting additional
thermodynamic parameters from TPP data, aside from the Tm value, poses challenges. Moreover, the magnitude of the Tm shift
following protein–ligand binding does not consistently correlate with ligand afnity.
Table 6
Recent studies that conducted DARTS-based target identication of natural products.
No. Natural products Category Bioactivity Target protein Reference
1 Batzelladine A Alkaloids Suppression of lung cancer
tumorigenesis
HSP90 [47]
2 Andrographolide Terpenoids Alleviation of parkinsonism DRP1 [48]
3 Polyphyllin D Saponins NSCLC therapy Distruption of HSC70–LAMP2A interaction [49]
4 Ergolide Terpenoids Alleviation of inammatory diseases NLRP3 [50]
5 Aconitine Alkaloids Cardiotoxicity Cytosolic phospholipase A2 [51]
6 Crellastatin A Macrocyclic
depsipeptides
Anticancer activity PARP-1 [52]
7 Cryptotanshinone Diterpenoid quinone Alleviation of keratinopathic ichthyosis FKBP1A [53]
8 Grape seed extract Polyphenolic compounds Anticancer activity Endoplasmic reticulum stress response
proteins
[54]
Fig. 5. Schematic workow 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.
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3.1.3. SPROX
SPROX, introduced by Fitzgerald and colleagues from Duke University’s Department of Chemistry in 2008 [63], represents another
method for detecting natural product-induced target stability. This method capitalizes on the enhanced antioxidant capacity observed
in target proteins upon binding with natural products. Protein oxidation capacity is determined by assessing the oxidation rate of
methionine residues (most proteins typically contain at least one methionine residue, which can serve as a crucial marker for assessing
the global dynamics of protein folding and unfolding equilibrium). The underlying principle is that as the concentration of chemical
denaturants (such as guanidine hydrochloride or urea) increases, the protein unfolds, and increased exposure of its methionine res-
idues to solvent renders them susceptible to oxidation in the unfolded state. Target proteins bound to natural products exhibit
heightened structural stability. They exhibit slower unfolding in the presence of chemical denaturants. The addition of hydrogen
peroxide, aimed at oxidizing methionine residues, results in a decelerated rate of methionine residue oxidation. The oxidation level of
methionine residues is subsequently assessed via MS, and a concentration-dependent curve is generated, depicting the variation in
oxidation and non-oxidized product. The intersection of the two curves denotes the transition midpoint. Binding of the natural product
to the target protein enhances stability, necessitating a relatively higher denaturant concentration to achieve the equivalent oxidation
product levels. Consequently, the transition midpoint shifts towards higher denaturant concentrations. Therefore, the potential target
protein can be inferred by comparing the transition midpoints between the drug and control groups.
The typical procedure of SPROX is as follows (Fig. 6): Initially, a protein lysate is treated with a buffer containing varying denaturant
concentrations in the presence or absence of the active natural product to establish the unfolding/refolding equilibrium of the protein.
Subsequently, hydrogen peroxide is introduced to initiate the oxidation of methionine residues. Upon the completion of oxidation, an
excess of either methionine or hydrogen peroxide scavenger is added to quench the oxidation reaction. Subsequently, the sample is digested
using trypsin, following which the levels of oxidation and non-oxidation in methionine-containing peptide segments are quantied using
MS. Finally, a curve is generated with the quantity of oxidized and non-oxidized methionine-containing peptide segments plotted on the y-
axis and the denaturant concentration plotted on the x-axis. The shift in the transition midpoint is utilized to identify potential target
proteins. The inventors of this technique, the Fitzgerald team, employed SPROX in 2011 to investigate the target proteins of the active
compound resveratrol. They identied six potential target proteins in addition to the known target protein, cytoplasmic aldehyde dehy-
drogenase [64]. In addition, Fitzgerald employed the SPROX technique to analyze the thermodynamics of the geldanamycin–HSP90
interaction [65], as well as elucidate the targets of manassantin A, which exhibits anticancer activity [66] (Table 8).
Table 7
Recent studies that conducted TPP-based target identication of natural products.
No. Natural
products
Category Bioactivity Target protein Reference
1 Vioprolide A Cyclopeptide Anti-human acute lymphoblastic leukemia Nucleolar protein 14 [56]
2 Quinine Alkaloids Antimalarial activity Malarial parasite’s intracellular purine nucleotide
phosphorylase
[57]
3 Rhodojaponin
VI
Saponins Alleviation of neuropathic pain N-ethylmaleimide-sensitive fusion protein [58]
4 Tutin Ketones Induction of epilepsy Calcineurin [59]
5 Alantolactone Terpenoids Inhibition of NSCLC AKR1C1 [60]
6 Kurarinone Flavonoids Attenuation of MPTP-mediated
neuroinammation
Soluble epoxide hydrolase enzyme [61]
7 Arone Terpenoids Protection against neuroinammation Histone-remodeling chaperone ASF1a [62]
Fig. 6. The workow 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.
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This method offers the advantage of facilitating comprehensive assessment of protein folding states and potentially precise mea-
surement of the binding domains and peptide segments engaged in the interaction between natural products and target proteins.
Nonetheless, it is constrained by its ability to solely detect the binding of natural products to proteins containing methionine residues.
In addition, generating the standard curve necessitates a large sample volume, thereby limiting the widespread utilization of this
method.
3.2. Target validation methods
3.2.1. CETSA
In 2013, Martinez Molina et al. introduced CETSA [67], a ligand-induced target stability detection method similar to DARTS. This
technique capitalizes on ligand-induced alterations in target protein, such as alterations in protein conformation, increased hydro-
phobicity within the protein, and chemical cross-linking within or between subunits of the target protein, resulting in enhanced
thermal stability. As the temperature rises, proteins that are not bound to ligands unfold and undergo rapid denaturation, leading to
precipitation. In contrast, ligand-bound proteins maintain their folded state and remain relatively stable, thereby bolstering the
thermal stability of the target protein. Molina and colleagues applied this method to evaluate the thermal melting proles of four
distinct clinical drug targets in cell lysates, each displaying characteristic melting curves. The introduction of drugs capable of binding
to these proteins to the cell lysates resulted in signicant alterations in the melting proles, thereby validating the scientic integrity
and practical utility of CETSA.
The standard procedure of CETSA entails incubating equivalent quantities of cellular lysates or tissue homogenates with small
molecules, followed by heating at various temperatures to induce protein denaturation and aggregation. Proteins bound to drugs
remain relatively stable, whereas unbound proteins undergo rapid denaturation and precipitation with rising temperatures. Relative
quantication of target proteins in the supernatant is performed via western blotting, thereby conrming the target proteins of the
small molecules. Usenamine A, a novel product, was extracted from the lichen Usnea longissima. Yang et al. [68] identied myosin-9 as
the direct target of usenamine A through MS, SPR assays, and CETSA. In addition, CETSA has been employed in combination with other
Table 8
Recent studies that conducted SPROX-based target identication of natural products.
No. Natural products Category Bioactivity Target protein Reference
1 Resveratrol Flavonoids Antioxidant activity Six potential target proteins [64]
2 Geldanamycin Benzoquinone Anticancer activity HSP90 [65]
3 Manassantin A Terpenoids Anticancer activity 28 protein hits [66]
Table 9
Recent studies that conducted CETSA-based target identication of natural products.
No. Natural products Category Bioactivity Target protein Reference
1 Usenamine A Benzofuran
derivatives
Alleviation of parkinsonism Myosin-9 [68]
2 Paederosidic acid Terpenoids Suppression of osteoclast formation
and neuropathic pain
P2Y
14
receptor [69]
3 Echinatin Flavonoids Inhibition of the hemolytic activity
of methicillin-resistant S. aureus
Indirect binding to
α
-hemolysin
[70]
4
α
-Asarone Aromatic
hydrocarbons
Alleviation of ischemic stroke PI3K [71]
5 Nitidine chloride Alkaloids Alleviation of multiple myeloma ABCB6 [72]
6 Pentoxifylline Methylxanthine
derivatives
Alleviation of non-alcoholic fatty
liver
Toll-like receptor 4 (TLR4) [73]
7 Primary active constituents of the Xiang-lian
pill (evodiamine, rutaecarpine, and
stigmasterol)
Alkaloids Anti-pancreatic cancer PTGS2 and PTGS1 [74]
Steroids
8 Toosendanin Terpenoids Anti-colorectal cancer Shh [75]
9 Glytabastan B Flavonoids Prevention and alleviation of
rheumatoid arthritis
ERK2, JNK1 and class I
PI3K catalytic subunit p110
[76]
10 Tubocapsenolide A Terpenoids Therapeutic potential for
osteosarcoma
Src homology 2
phosphatase 2
[77]
11 Primary constituents of Zuojin capsule Alkaloids Anti-colorectal cancer activity CDKN1A, Bcl2, E2F1,
PRKCB, MYC, CDK2, and
MMP9
[78]
Flavonoids
Steroids
Indole
12 Proanthocyanidin A1 Flavonoids Amelioration of chemotherapy-
induced thrombocytopenia
JAK2 [79]
13 Cucurbitacin B Terpenoids Anti-NSCLC activity TLR4 [80]
14 Celastrol and gambogic acid Terpenoids Anti-breast cancer activity ER
α
Y537S mutant [81]
15 Shikonin Terpenoids Anti-triple-negative breast cancer
activity
IMPDH2 [82]
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target identication methods to validate the targets of paederosidic acid, which suppresses osteoclast formation and neuropathic pain
[69], echinatin, a natural compound isolated from licorice, which effectively inhibits the hemolytic activity of methicillin-resistant
Staphylococcus aureus [70],
α
-asarone, an important component of the Huangxiong formula for treating ischemic stroke [71], niti-
dine chloride, an extract from Zanthoxylum nitidum, used in multiple myeloma treatment [72], pentoxifylline, which attenuates
non-alcoholic fatty liver [73], the primary active constituents of the Xiang-lian pill, which suppresses pancreatic tumor [74], too-
sendanin, a natural triterpenoid saponin with anti-colorectal cancer activity [75], glytabastan B, a coumestan isolated from Glycine
tabacina, which is used for the prevention and treatment of rheumatoid arthritis [76], tubocapsenolide A, a withanolide-type steroid
with therapeutic potential for osteosarcoma [77], the primary constituents of the Zuojin capsule, which is used to combat colorectal
cancer [78], proanthocyanidin A1, which ameliorates chemotherapy-induced thrombocytopenia [79], cucurbitacin B, which inhibits
NSCLC [80], celastrol and gambogic acid, which exhibit anti-breast cancer activity [81], shikonin, a natural bioactive component of
Lithospermum erythrorhizon with anti-triple-negative breast cancer activity [82] (Table 9).
One limitation of this method is its dependency on a relatively high concentration of the natural active compound for detection.
Additionally, quantication primarily hinges on western blotting, which exhibits low throughput. Consequently, this method is
typically utilized as a validation tool. Following the capture of small molecule-targeted proteins through alternative techniques, CETSA
is employed for validation purposes.
3.2.2. ITC
ITC enables the direct quantication of heat released or absorbed during biomolecular binding processes. When a compound binds
to a protein, it elicits a heat exchange detectable via ITC. In an ITC experiment, one reactant is placed in a temperature-controlled
sample cell, linked to a reference cell through a heat ow circuit, with both cells in the same external environment. A specic
titrant (such as a natural active compound) is incrementally injected into the sample cell. Upon reaction between the sample and
titrant, heat is either absorbed or released. After each injection of the sample, heat is absorbed or released, causing temperature
changes between the sample and reference cells. A heat-sensitive device detects temperature differences between the two cells upon
binding, providing feedback to a heater to rectify temperature disparities and equilibrate the sample and reference cells. Subsequent
titrant injections gradually increase the molar ratio between the titrant and protein, as the protein reaches saturation. The frequency of
titrant binding events decreases, and the heat change decreases until the number of titrations in the sample cell is in excess relative to
the protein, indicating saturation. Utilizing software integrated with the isothermal titration calorimeter, parameters such as binding
constants, stoichiometry (n), and enthalpy change can be obtained through data tting, thus yielding comprehensive thermodynamic
insights into molecular interactions [83].
Based on the aforementioned principle, the typical procedure entails conguring the reference and sample cells to the desired
experimental temperature. The natural active compound is loaded into a highly precise injection device, such as a syringe, which is
then inserted into the sample cell containing the target protein. The natural active compound is injected into the sample cell in multiple
Fig. 7. The principle and general workow of ITC. Initially, the reference and sample cells are equilibrated to the desired temperature. Subse-
quently, 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 tting the titration curve.
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increments. The slightest temperature variation, on the order of millionths of a degree Celsius, can be detected and quantied if
binding occurs between the natural active compound and the protein. During each injection, the microcalorimeter records all released
heat until equilibrium is reached in the binding reaction, with the measured heat being proportional to the degree of binding. Utilizing
the software provided with the isothermal titration calorimeter, the acquired data can be tted and processed to determine ther-
modynamic parameters (Fig. 7). For instance, polyphenols play an essential role in modulating the gut microbiota and enhancing the
integrity of the mucus barrier. Feng et al. [84] demonstrated that the pyrogallol-containing polyphenols epigallocatechin gallate and
tannic acid strongly bind to the nucleophilic thiol groups of mucins. This binding was shown to serve as cross-linkers within mucin
networks, thereby reinforcing the mucus barrier, via ITC. Xue et al. [85] revealed that thymoquinone, a signicant phytoconstituent of
Nigella sativa used in Alzheimer’s disease therapy, binds spontaneously to human transferrin. The binding constant was determined to
be 0.22 ×10
6
M
−1
, forming a stable complex, using both ITC and spectroscopic methods. In addition, ITC has been employed to
elucidate the targets of glycyrrhizin derivatives, which suppress cancer chemoresistance [86], valonea tannin, which exhibits tyros-
inase inhibition activity [87], bioactive coffee compounds that protect against serotonin degradation for treating depression and
potentially type 2 diabetes [88], neoandrographolide, a novel inhibitor of Rab5 [89], piperine, a bioactive constituent of black pepper,
shown to alleviate experimental allergic encephalomyelitis [90], luteolin, which exhibits antibacterial activity [91], red clover iso-
avones, which inhibit cancer cell metastasis [92], and the molecular interactions of acetylcholinesterase with potential acetylcho-
linesterase inhibitors isolated from the root of Rhodiola crenulata [93] (Table 10).
This technique boasts several advantages, including its rapidity, accuracy, and minimal sample demand. It does not enforce
stringent requirements on the reaction system, such as transparency, turbidity, or viscosity. ITC furnishes detailed and comprehensive
thermodynamic insights into molecular interactions, facilitating precise quantication of molecular interaction levels. However, a
limitation of this method is its low throughput. Consequently, it is commonly employed as a supplementary screening tool in high-
throughput screening protocols.
4. Identication of novel drugs and drug targets based on the innate function of natural products
Notably, there is a novel avenue for rapidly exploring the targets of natural products based on their innate functions [94]. In the
course of evolution, organisms produce specialized secondary metabolites to exhibit important functions, such as defence, survival,
and reproduction, via interacting with specic target proteins in inter-kingdom entities. The structural complementarity between
ligands and their targets provide more opportunities for nding promising ligand analogues. For instance, tabtoxin, a dipeptide
monobactam synthesized by Pseudomonas syringae, inhibits glutamine synthetase (GS) in plants [95]. GS, which has a conserved active
site in both plants and mammals, is a signicant target for cancer therapy [96]. Consequently, structural analogues of tabtoxin hold
promise as potent human GS antagonist for cancer treatment [97,98].
Conversely, ligands may actively interact with several structural analogues of the receptors present in different branches of life.
Inspired by this concept, we can identify therapeutic targets based on the innate functions of natural metabolites. For example, the
steroid hormone ecdysterone and its homologues interact with ecdysone receptors in arthropods. These receptors are composed of EcR
proteins and ultraspiracle protein (USP) heterodimers, functioning as nuclear receptors for ecdysone [99]. In humans, there are several
structural analogues of ecdysone receptors, including liver X receptor
α
(LXR
α
), LXRβ, estrogen receptor
α
(ER
α
), ERβ, retinoid X
receptor
α
(RXR
α
), and bile acid receptors. LXRβ is a promising therapeutic target for inammation [100], cancer [101], dyslipidemia
[102], and diabetes [103], while ER is a target for the treatment of breast cancer [104]. Some ecdysterone analogues have been
experimentally validated in cancer, diabetes, and inammation [105,106]. Therefore, ecdysterone and its analogues may act as se-
lective LXR agonists and ER modulators against human diseases.
Similarly, rhizobitoxin, a plant toxin produced by the fungus Bradyrhizobium elkanii, restrains the activity of β-cystathionase and 1-
aminocyclopropane-1-carboxylate (ACC) synthase to exert its natural effects [107,108]. Structural analysis reveals that the plant
Table 10
Recent studies that performed ITC-based target identication of natural products.
No. Natural products Category Bioactivity Target protein Reference
1 Epigallocatechin gallate and tannic
acid
Polyphenols Reinforcement of the mucus barrier Nucleophilic thiol groups of
mucins
[84]
2 Thymoquinone Isothiocyanates Alleviation of Alzheimer’s disease Transferrin [85]
3 Glycyrrhizin derivatives Terpenoids Suppression of cancer
chemoresistance
Progesterone receptor membrane
component 1
[86]
4 Valonea tannin Polyphenols Tyrosinase inhibition activity Skin whitening properties [87]
5 Coffee compounds Alkaloids Alleviation of depression and
potentially type 2 diabetes
Monoamine oxidase A [88]
Polyphenols
6 Neoandrographolide Terpenoids Anticancer activity Rab5 [89]
7 Piperine Alkaloids Alleviation of experimental allergic
encephalomyelitis
Dihydroorotate dehydrogenase [90]
8 Luteolin Flavonoids Antibacterial activity HK853 [91]
9 Red clover isoavones Flavonoids Inhibition of cancer cell metastasis Actin [92]
10 Constituents isolated from the root
of R. crenulata
Flavonoids Alleviation of Alzheimer’s disease Acetylcholinesterase [93]
Terpenoids
Phenols
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Table 11
Benets and drawbacks of each target identication approach.
Approach Benets Drawbacks
Afnity
chromatography
1. The synthesis of immobilized probes is simple.
2. It facilitates the analysis of all adsorbed proteins without discrimination.
3. It can be easily conducted in individual laboratories.
1. Discrimination between truly high-afnity target proteins and high-abundance proteins with
low afnity is necessary.
2. The elution process relies on empirical knowledge; excessively strong washing conditions can
signicantly reduce the number of identied targets, while weak elution conditions can yield
false-positive results.
3. It does not reveal interactions between drugs and proteins in live cells under physiological
conditions.
4. Biotin afnity tags introduce steric hindrance, potentially reducing or even abolishing the
activity of natural active compounds and impeding probe molecule entry into cells.
ABPP 1. It exhibits strong specicity.
2. ABPs do not require pre-immobilization on matrix material.
3. It can be directly applied to live cells, elucidating drug–target interactions under physiological
conditions.
1. Only proteins capable of forming covalent interactions with bioactive natural products can be
captured.
2. Synthesizing ABPs relies on the structure of the bioactive natural product and necessitates
expertise in medicinal chemistry.
PAL 1. It can facilitate the analysis of proteins that cannot be targeted by ABPs. 1. Photoafnity groups still exhibit non-specic binding to abundant cellular proteins.
2. The synthesis of PAL probes necessitates expertise in medicinal chemistry.
Proteome
microarray
assay
1. It requires minimal sample volumes.
2. High throughput enables parallel analysis of thousands to millions of proteins.
3. Utilizing protein chips is relatively simple.
1. It is relatively costly, necessitating a range of expensive, high-end instruments.
2. Standardization issues with the chips.
DARTS 1. No structural modication of natural bioactive compounds is necessary.
2. It can be easily implemented by individual laboratories.
1. Certain natural products do not signicantly affect the conformation of the target protein upon
binding.
2. Some proteins are less susceptible to proteases, resulting in undetectable changes in DARTS.
3. The ability to identify targets that exhibit low protein abundance is limited.
TPP 1. It monitors changes in protein thermal stability throughout the proteome during the action of
natural bioactive compounds.
2. It enables high-throughput detection of target proteins.
1. Inapplicable to heat-insensitive proteins.
2. The extraction of thermodynamic parameters besides the Tm value from TPP data is
challenging.
3. The magnitude of the Tm shift after protein binding with natural active compounds does not
consistently correlate with binding afnity.
4. Time-consuming and costly.
SPROX 1. It enables the large-scale assessment of protein folding states and potentially precise
determination of binding domains and peptide segments involved in the interactions between
compounds and target proteins.
1. It can only detect binding between proteins containing methionine residues and small
molecules.
2. Generating the standard curve requires a large sample volume.
3. The process is complex, costly, and requires substantial resources.
CETSA 1. No structural modication of natural bioactive compounds is required.
2. It can be easily implemented by individual laboratories.
1. Detection necessitates relatively high concentrations of the natural active compound.
2. The process exhibits low throughput in discovering new targets and is primarily utilized for
target validation.
ITC 1. It provides fast, accurate, and low-sample-volume analysis with minimal requirements for
reaction system transparency, turbidity, and viscosity.
2. It enables precise determination of comprehensive thermodynamic information.
1. It is typically utilized as an auxiliary screening technique in high-throughput screening due to
its low-throughput nature.
2. It requires specialized ITC equipment.
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Heliyon 10 (2024) e33917
18
enzymes β-cystathionase and ACC synthase have structural analogues in human proteins, namely γ-cystathionase and kynurenine
aminotransferase, respectively. Over-activity of γ-cystathionase is linked to various diseases [109], while kynurenine aminotransferase
activity is associated with schizophrenia and cognitive impairment [110]. Hence, these proteins are potential therapeutic targets.
Some known inhibitors share structural similarities with rhizobitoxin [111], suggesting their potential action on these proteins.
Although rhizobitoxin is known to bind ACC synthase, there is no evidence of its binding to kynurenine aminotransferase. However,
structural analogues of rhizobitoxin have been found to suppress kynurenine aminotransferase.
In addition to the functions of metabolites, enzymes that catalyze their synthesis can also be potential therapeutic targets in
humans. For instance, strictosidine synthase, a plant enzyme, has a human structural homolog, regucalcin, which may serve as a
therapeutic target [94]. Although agonists or antagonists for regucalcin have not been studied yet, this concept can be applied to
develop relevant drugs.
5. Conclusions and prospects
Nature has been continuing to be a major source of medicinal products, and especially providing revolutionized structural leads in
the treatment of serious diseases for millennia, such as avonoids, alkaloids, steroidglycosides, sesquiterpene, essential oil and ste-
roids, all of which play an important role in medicinal chemistry. The identication of natural products’ targets is as important as the
knowledge of natural products themselves because clarifying the former provides several new avenues for nding better drugs. Target
identication is the rst step in the early stage of drug discovery, followed by lead optimization.
Based on the foregoing, labeling methods facilitate direct labeling of target proteins while excluding indirectly targeted ones,
thereby signicantly reducing false-positive results in target identication. Nevertheless, these methods entail a high application
threshold and are unsuitable for investigating post-modied compounds with abolished activity, compounds exhibiting chemically
challenging synthesis, or analyzing trace amounts of natural active compounds. Conversely, non-labeling methods can be used to
identify targets of any natural active compound, even in trace amounts or multicomponent mixtures, albeit with lower reliability
compared to labeling methods. Challenges persist in eliminating the inuence of other proteins within the protein complex where the
natural active compound binds. Each method presents distinct advantages and limitations (Table 11). In practical applications, the
selection of appropriate target discovery methods is contingent upon the characteristics of small molecules or target proteins. Typi-
cally, a combination of multiple methods is employed to identify the targets of natural products [112–115]. Furthermore, a recent
review outlined target identication and validation approaches for natural products categorized according to whether the natural
products were labeled [116]. Building upon this, this review claries the principles and processes of each method comprehensively.
Moreover, this review presents chemical structures and modifying moieties relevant to the labeling techniques.
New techniques continuously emerge, leveraging the aforementioned technologies, to aid in the identication of target proteins of
active natural products, thus signicantly impacting novel drug development.
In addition, there is a novel avenue for rapidly exploring the targets of natural products based on their innate functions. If a natural
metabolite (a) has been biosynthesized to fulll a specic role through its interaction with protein (A), the natural target of a, it
presents an ideal strategy to explore therapeutically important human proteins that are structurally analogous to protein A. The
aforementioned target validation approaches can be employed to validate the binding afnity of each target with a. Conversely, the
structural analogues of natural functional metabolite a may interact with protein A with varying degrees of afnity, thereby exerting
similar effects against human diseases.
Funding statement
This work was supported by the Matching Grant of the National Nature Science Foundation of China from Nanjing University of
Chinese Medicine, China (grant number NZY81903857), the National Natural Science Foundation of China (grant number 81903857),
the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (grant number 24KJA360005) and the National
Natural Science Foundation of China (grant number 81961128020).
Ethics declarations
Not applicable.
Data availability
No data was used for the research described in the article.
CRediT authorship contribution statement
Xuan Jiang: Writing – original draft. Kinyu Shon: Visualization. Xiaofeng Li: Visualization. Guoliang Cui: Writing – review &
editing. Yuanyuan Wu: Project administration. Zhonghong Wei: Project administration. Aiyun Wang: Resources, Project admin-
istration. Xiaoman Li: Writing – review & editing, Funding acquisition, Conceptualization. Yin Lu: Resources, Project administration,
Conceptualization.
X. Jiang et al.
Heliyon 10 (2024) e33917
19
Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to
inuence the work reported in this paper.
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