Target discovery-directed pharmacological mechanism elucidation of bioactive natural products

Biotin-labeled strategy for target identification

This approach involves utilizing functional groups (e.g., -OH, -NH₂, -COOH) in natural products to synthesize a biotin-labeled probe for target identification. The biotin-labeled probes are immobilized on the surface of streptavidin-coated solid-phase beads, thereby enabling the selective enrichment of binding proteins from cellular or tissue lysates. The captured proteins are subsequently subjected to separation through gel electrophoresis and analyzed by mass spectrometry (Figure 2). Currently, biotin-labeled probes are extensively utilized for the target identification of natural products and have achieved a relatively advanced stage of development.

For instance, biotin-labeled adenanthin has identified the peroxiredoxin I/II (Prdx I/II) as the target proteins for its anti-leukemia activity [5]. Moreover, pull-down assay with biotin-conjugated artesunate (bio-ATS) demonstrates that artesunate improves the phenotype of polycystic ovary syndrome by targeting the mitochondrial protease LONP1 [6]. Likewise, the biotin-conjugated bruceine A suggests that the inhibition of the galectin-1 protein-mediated inflammatory pathway contributes to the therapeutic effect against diabetic kidney disease [7]. Meanwhile, the construction of a biotin-labeled ainsliadimer A facilitates the identification of the target protein IKKα/β for anti-inflammatory properties [8]. Additionally, biotin-labeled probe also contributes to the identification of extracellular-signal-regulated kinase 2 (ERK2) as the molecular target of natural product strictosamide for anti-inflammatory effect [9].

The development of Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) technology has provided a crucial platform for identifying low-abundance target proteins (Figure 3). This strategy involves the utilization of essential amino acids labeled with natural isotopes (light labeling) and stable isotopes (heavy labeling) to substitute the corresponding amino acids in the cell culture medium. As a result, stable isotope labeled amino acids (e.g., ¹³C and ¹⁵N-labeled arginine or lysine) are integrated into newly synthesized proteins in cells, facilitating accurate quantification through mass spectrometry. For target identification, cell lysates labeled with light isotopes are used to capture the targets using the biotin-labeled probe, while cell lysates labeled with heavy isotopes are employed for competitive analysis by introducing free molecules. Subsequently, the proteins with significant variations are often considered as potential targets by quantitative proteomics analysis [10].

Figure 2:

Target identification via biotin-labeled Strategy.

In recent years, the SILAC-based quantitative proteomics technology has been utilized for the target identification of CDK8 and CDK19 for natural compound CCT251545 [11]. Furthermore, the SILAC-coupled with LC-MS plays a crucial role in uncovering anti-silencing factor 1a (ASF1a) as a cellular target of artone [12]. Similarly, the application of this approach leads to the successfully discovery of peroxiredoxin 6 (PRDX6) as a target for withangulatin A (WA) [13].

Biotin-labeled probes are commonly used in conjunction with protein chip technology for target identification. Protein chips, also known as protein microarrays, are effective tools for high-throughput analysis of protein-ligand interactions, such as protein-protein, protein-nucleic acid, or protein-small molecule. The procedure involves immobilizing recombinant proteins onto the surface of a microarray, followed by co-incubation with biotin-labeled probes. Subsequently, avidin-labeled fluorescence markers are attached to the probes, enabling the tracking of target protein location on the chip for target identification (Figure 4). For instance, hexokinase 2 (HK2) has been identified as a critical target for leukemia therapy using a biotin-labeled arsenic probe and a human protein array chip [14]. Furthermore, the integration of protein chip with surface plasmon resonance strategy has elucidated that geniposide inhibits the progression of hepatocellular carcinoma by specifically targeting TLR4 [15]. Similarly, protein chip technology has also been utilized to identify peroxiredoxin 2 as a potential target of celastrol against gastric cancer [16].

Figure 3:

Target identification via SILAC labeling Strategy.

Alkyne/azide or diazirine-labeled strategy for target identification

In recent years, bioorthogonal reactions have been incorporated in the identification of targets for natural products. Bioorthogonal reactions are highly specific chemical reactions within biological systems that do not interfere with normal biochemical processes [17], 18]. Similar to the biotin-labeled strategy, bioorthogonal reactions involve introducing alkyne or azide labels into the functional groups (-COOH, -NH₂, -OH) of natural products to synthesize probes. Subsequently, the proteins captured by the probe are enriched through the copper(I)-catalyzed Alkyne-Azide cycloaddition (CuAAC) reaction (Figure 5). In recent years, technological advancements have facilitated the expansion of this approach through the integration of photoaffinity labeling (PAL) to investigate transient or weak interactions between ligands and target proteins. Notably, the diazirine is widely employed as the primary photoreactive moiety in PAL. Diazirine undergoes a photochemical reaction upon ultraviolet light, forming a carbene intermediate that enables the covalent crosslinking of target proteins for subsequent affinity purification and mass spectrometry analysis.

Figure 4:

Target identification via human protein Chip.

Currently, bioorthogonal strategies, such as the CuAAC reaction, are extensively employed in the identification of targets for natural products. For instance, a clickable photoaffinity probe of chloroquine (CQ) containing the azide-diazirine bifunctional group has been developed for identifying targets including PfLDH, PfOAT, PfPyrK, PfPGK, and PfTPI. Further investigations have revealed that CQ disrupts the glycolysis and energy metabolism of malaria parasites by directly binding to these target proteins, thereby exerting the antimalarial effect [19]. Similarly, a dual-functional probe derived from benzoxepane derivative 10i has contributed to identifying pyruvate kinase isozyme type M2 (PKM2) as the target protein responsible for anti-inflammatory effect by modulating the glycolysis and NLRP3 signaling pathway [20]. Moreover, alkyne-diazirine photosensitive group also facilitates the identification of retinoblastoma binding protein 4 (RBBP4) as the target protein mediating the anti-tumor properties of protopanaxadiol (PPD) [21]. Additionally, the bioorthogonal approach has been employed to identify disulfide isomerase as the target protein of the diterpenoid compound vinigrol for anti-arthritis effect [22]. Further, the diazirine-labeled metformin bifunctional probe directly binds to PEN2 for the activation of AMP-activated protein kinase (AMPK) to achieve a hypoglycemic effect [23].

Benzophenone-crosslinking strategy for target identification

Benzophenone is a photosensitive group that undergoes carbene formation upon ultraviolet light excitation. The carbene of the activated benzophenone subsequently forms a covalent bond with the carbon-hydrogen bonds of ligands, thereby immobilizing natural products onto the surface of solid-phase beads for target identification (Figure 6). Given that natural products mainly comprise organic molecules, the benzophenone labeling strategy proves effective in solid-phase immobilization of a wide range of natural products for target identification. For example, schisandrol A (SolA), a naturally derived neuroprotective agent, is immobilized onto a solid-phase beads using the benzophenone labeling strategy. Subsequently, ATP6V0d1 has been identified as the crucial target protein responsible for the neuroprotective effects of SolA in neuronal cells [24]. Moreover, the benzophenone-crosslinking strategy has facilitated the identification of the target protein histone H2B for the anti-neuroinflammatory effect of the natural product epoxymicheliolide [25]. Additionally, the targets of natural products such as vancomycin and cyclosporin are also revealed using a similar method based on benzophenone [26], 27]. Meanwhile, benzophenone-crosslinking strategy also aids in identifying target proteins in complex chemical systems, such as herbal extracts, including the extracts of Chrysanthemum indicum L., Caesalpinia sappan L., Albizia julibrissin Durazz., Cistanche deserticola Y.C.Ma, as well as traditional Chinese herbal formula such as Shouhui Laxative Capsule and Jingfang Granules [28], [29], [30], [31].

Figure 5:

Target identification via alkyne/azide or diazirine photoaffinity labeling Strategy.

Figure 6:

Target identification via benzophenone-crosslinking Strategy.

Epoxy-crosslinking strategy for target identification

Epoxy is a reactive group that can undergo covalent bonding with active groups (such as hydroxyl groups, amino groups, thiol groups) in natural products. Therefore, epoxy is an effective chemical tool that can be used to immobilize natural products onto solid microspheres for target identification. Currently, epoxy-activated Sepharose 6 B beads/Sepharose 4 B beads are among the commonly used commercial carriers. Following the immobilization of small molecules onto Sepharose 6 B beads, a blocking agent, such as ethanolamine, is typically employed to neutralize the residual epoxy groups and minimize nonspecific interference. Thus, the epoxy-crosslinking strategy proves useful in uncovering targets for natural products. For example, previously investigation has revealed CRAF and MEK1/2 as the direct targets for the anti-tumor effect of natural product erianin from Drumstick Dendrobium through activated epoxy groups [32]. Similarly, 1β-hydroxyalantolactone, termed as IJ-5 derived from I. japonica Thunb, undergoes chemical crosslinking with epoxy groups-modified beads, leading to the identification of UbcH5 as the crucial target in inhibiting inflammatory gene transcription [33]. Furthermore, sepharose 4 B beads-coupled with gingerenone A indicates the alleviation of ulcerative colitis by targeting IL-17RA [34]. Additionally, Bax is identified as the neuroprotective target of icariin through a similar approach, indicating a promising direction for drug development in diabetic encephalopathy [35].

Targeted protein degradation strategy for target identification

Proteolysis-targeting chimeras (PROTAC) represents a novel strategy utilizing chemical molecules to induce the degradation of specific proteins via the ubiquitin-proteasome system (UPS) [36]. PROTAC molecule comprises an E3 ubiquitin ligase ligand, a target protein ligand, and a linker. The degradation process typically involves ligand binding to target protein, the E3 ligand binding to E3 ubiquitin ligase, and the linker facilitating the proximity of the target protein to the E3 ubiquitin ligase. This process ultimately leads to the degradation of the target protein via a UPS-dependent mechanism.

The conventional PROTAC strategy involves the chemical synthesis of PROTAC molecules to target-specific protein degradation in cells. In recent years, this approach has been employed to explore the target proteins of natural products. Furthermore, targeted degradomics (TGDO), utilizing PROTAC molecular probes from natural compounds for target identification through quantitative proteomics analysis has been proposed (Figure 7).

Figure 7:

Target identification via targeted protein degradation Strategy.

Unlike traditional affinity-based methods, TGDO does not require a strong interaction between the compound and the target protein. Instead, TGDO is dependent on the formation of a ternary complex comprising the target protein, PROTAC, and E3 ligase, thereby ensuring high ligand selectivity. For example, researchers have synthesized the ZCY-PROTAC by conjugating lathyrol with thalidomide to investigate the specific target [37]. Quantitative proteomics analysis demonstrates that MAFF is the protein most efficiently degraded following ZCY-PROTAC treatment. Subsequent investigation indicates that lathyrol facilitates the formation of MAFF-Nrf2 heterodimers while inhibiting MAFF homodimers, thereby leading to the regulation of HO-1 downstream expression for antioxidant and anti-inflammatory effects. Recently, AD4, an artemisinin-based PROTAC, has been synthesized to reveal PCLAF as a crucial target for antitumor activity [38]. Moreover, the anti-tumor target of natural product evodiamine is determined to be REXO4 using TGDO strategy [39]. Notably, an innovative degradation-based protein profiling (DBPP) strategy has been proposed by integrating the PROTAC approach with quantitative proteomics and immunoprecipitation-mass spectrometry. DBPP approach enables the identification of the targets for natural product celastrol, including IKKβ, PI3Kα, and CIP2A, as well as the discovery of potential new targets such as CHK1, OGA, and ERCC6L [40].

Significant progress has been made in the target identification of natural products using chemical labeling strategies. This advancement provides a powerful tool for elucidating the direct targets and pharmacological mechanisms of these compounds. However, many natural products lack available functional groups for chemical modification, greatly limiting the application of these methodologies. To address these constraints, researchers have developed a range of label-free methods for target identification, presenting novel avenues for exploring pharmacological mechanisms.

CETSA

CETSA represents the foremost label-free approach for the identification of drug targets. CETSA operates on the principle that drug binding enhances the stability of protein spatial conformation, thereby distinguishing targets proteins from non-specific targets proteins [41]. In CETSA, cellular or tissue lysates are treated with small-molecule, and then exposed to temperature gradient to induce protein thermal denaturation. Subsequently, the samples are centrifuged to separate soluble components from precipitated proteins. The levels of target proteins in soluble fraction are quantified using western blotting, and the thermal stability is assessed by analyzing their melting curves with mass spectrometry.

Currently, the CETSA strategy is frequently utilized in the identification of target proteins for natural products. For instance, CETSA assay highlights that SHP-2 tyrosine phosphatase plays a crucial role in the anti-tumor activity of geranylnaringenin (CG902), a bioactive ingredient in Artocarpus heterophyllus Lam [42]. Using CETSA strategy, researchers demonstrates that nuciferine directly binds to HBXIP, ameliorating hepatic steatosis and insulin resistance [43]. Similarly, CETSA coupled with mass spectrometry indicates that the podophyllotoxin derivative SU056 targets Y-box binding protein-1 (YB-1) to regulate protein translation, thereby inhibiting triple-negative breast cancer progression [44]. Moreover, CETSA has effectively elucidated the target protein WD Repeat Domain 1 (WDR1) for the glioma inhibitory compound gambogic amide [45]. Importantly, CETSA is used to identify the target protein purine nucleoside phosphorylase for the antimalarial natural product quinine [46]. Additionally, investigations have demonstrated that TLR4 and the tumor-associated NADH oxidase as the direct targets of cucurbitacin B and capsaicin through the CETSA [47], 48].

TPP

TPP strategy represents an advanced version of CETSA, facilitating the effective discovery of low-abundance target proteins (Figure 8) [49]. TPP enables the identification of proteins that show changes in thermal stability in response to ligand binding at different temperatures on a proteomic scale. In this strategy, cells are treated with drugs at varying concentrations and then exposed to heat treatment. Subsequently, the soluble protein components are extracted and labeled with stable isotope labeling reagents tandem mass tags for mass spectrometry analysis, subsequently determining the specific melting temperature of each protein. Thus, TPP strategy aids in identifying proteins that display fluctuations in stability.

At present, TPP has been extensively utilized for the target identification of natural products. For instance, TPP reveals transcription factor specificity protein 1 (Sp1) as a direct target of the natural product murrayafoline A, providing valuable insights for directing therapeutic interventions toward Sp1 in for neuroinflammation [50]. Similarly, TPP facilitates the identification of kurarinone as targeting soluble epoxide hydrolase (sEH), linked to neuroinflammation in Parkinson’s disease [51]. Additionally, TPP has confirmed that oleanolic acid nanomicelles specifically target the 20 S proteasome subunit alpha 6 (PSMA6) to exert antitumor activity and vioprolide A targets nucleolar protein 14 for the treatment of lymphoblastic leukemia [52], 53].

Drug affinity-responsive target stability analysis (DARTS)

In 2009, Lomenick introduced the fundamental concept of DARTS, which is based on the principle that the spatial conformation of the target protein stabilizes upon ligand binding, rendering target protein less susceptible to protease degradation (Figure 9) [54]. Thus, the effect of enzymatic degradation on proteins can be discerned through SDS-PAGE, as well as mass spectrometry analysis. The convenience and efficiency of DARTS assays have facilitated their widespread application in the identification of targets for natural products. For instance, DARTS assay has facilitated the discovery that natural product oridonin exerts anticancer activity by targeting nucleolin [55]. Similarly, investigation suggests that cholesterol 25-hydroxylase binds directly with ARF4, thereby enhancing its activity to improve DKD [56]. Furthermore, DARTS elucidates that diosgenin ameliorates Alzheimer’s disease by targeting the 1,25D₃-membrane-associated, rapid response steroid-binding protein (1,25D₃-MARRS) [57]. Notably, DARTS has revealed that betulinic acid targets glucose-regulated protein 78 (GRP78) to induce endoplasmic reticulum stress-mediated apoptosis in breast cancer cells [58]. DARTS has also played a key role in elucidating that tubocapsenolide A exerts anti-osteosarcoma activity by targeting Src homology two phosphatase 2 [59].

Figure 8:

Target identification via TPP Strategy.

The LiP-MS strategy represents an advanced version of the DARTS assay. LiP-MS provides a proteomics approach that enables the detection of protein structural alterations within complex biological contexts at a proteome-wide scale. LiP-MS analyzes protein hydrolysis patterns to discern the impact of drugs on protein structures. This enables the identification of target proteins and the precise localization of drug-binding sites by comparing the mass spectra of hydrolyzed peptides [60], 61]. For instance, LiP-MS discovers dihydrolipoamide S-acetyltransferase as the target protein of hyperforin, a natural product known to promote energy expenditure for obesity [62].

Likewise, PP is a strategy that capitalizes on the differential sensitivities of folded and unfolded proteins to proteases, allowing for the determination of ligand affinity for protein targets [63]. PP works by selectively digesting unfolded proteins within a mixture that contains both folded and unfolded proteins. This is achieved by exposing protein samples to varying concentrations of chemical denaturants (e.g., urea, guanidine hydrochloride), which establishes an equilibrium between the folded and unfolded states. Subsequently, a brief “pulse” with an excess of nonspecific protease treatment is applied to preferentially digest the unfolded proteins. The resulting data helps to define a functional relationship that links the proportion of folded proteins to the denaturant concentration [64]. Utilizing PP, investigations have successfully monitored the interaction between maltose and maltose-binding protein, obtaining a dissociation constant that aligns with those established through conventional biophysical analyses [63].

Other label-free target identification methodologies

In recent years, advancement in molecular biology, structural biology, and artificial intelligence technologies have driven the development of diverse label-free methodologies. These strategies, based on divergent principles, demonstrate unique capabilities for the discovery of the target proteins and biological mechanisms of natural products. For instance, the stability of proteins from rates of oxidation (SPROX) target identification strategy determines the protein-ligand binding mechanism by detecting the oxidation levels of methionine in target proteins [65]. The SPR provides kinetic information, such as the K_D value of target molecule-ligand binding, by detecting changes in the refractive index on the surface of SPR biosensors and the SPR angle caused by the binding of target molecules to ligands, thus clarifying the mechanism of protein-ligand binding [66].

Moreover, a variety of label-free target identification approaches have been continuously emerged, including chemical denaturation and protein precipitation (CPP) [67], solvent-induced protein precipitation (SIP) [68], mechanical stress-induced protein precipitation (MSIPP) [69], and biolayer interferometry (BLI) [70]. These approaches significantly facilitate the identification of target proteins by assessing changes in mechanical stress and organic solvent tolerance exhibited by proteins upon drug binding.

Figure 9:

Target identification via DARTS Strategy.

Furthermore, connectivity Map (cMap)-based target identification strategy uses gene expression patterns and pattern-matching software to reveal drug action mechanisms [71]. In addition, molecular docking techniques, such as cutting-edge computational methods or artificial intelligence programs like AlphaFold, are employed to model the binding interactions between numerous proteins and drugs to explore potential drug targets [72]. Despite the high-throughput and rapid features offered by these approaches, extensive experiments are still required to validate their applicability and reliability.

Currently, the application of these label-free strategies has garnered widespread attentions, leading to the increasing cases in natural products target identification. For example, the utilization of SPROX indicates Rab1a as the target protein of the N-arylbenzimidazole NAB2, demonstrating its therapeutic potential for neurodegenerative diseases [73]. Nicotine and its metabolite, cotinine, directly target myeloid differentiation protein two to inhibit toll-like receptor four signaling, thereby ameliorating neuroinflammation [74]. Notably, SIP has been successfully applied in the target identification of methotrexate, SNS-032, and staurosporine [75]. Meanwhile, the application of cMap database shows that celastrol and gedunin inhibit the HSP90 signaling pathway for prostate cancer therapy [76]. Specifically, molecular docking analysis demonstrates the interaction between celastrol and STAT3, leading to the mitigation of cardiac dysfunction [77]. In addition, BLI analysis suggests that capsaicin directly binds to KEAP1 to activate NRF2 function, which potentially ameliorates oxidative stress-related diseases [78]. In summary, it is noteworthy that label-free strategies will continue to evolve into diverse avenues and maintain their critical involvement in the discovery of targets for natural products.

Mechanisms of anti-inflammatory natural products

Natural products are known to contain a plethora of compounds with substantial anti-inflammatory and immunosuppressive activities. The identification of targets is crucial for elucidating the underlying mechanisms of action of these natural products. Sappanone A (SA) derived from C. sappan exihibits noteworthy anti-neuroinflammatory properties. A biotin-labeled SA probe is employed to uncover inosine monophosphate dehydrogenase 2 (IMPDH2) as the pivotal target accountable for the anti-neuroinflammatory effects in microglial cells. Subsequent studies revealed that SA covalently binds to the Cys140 in IMPDH2, thereby impeding the neuroinflammatory response through inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways [135]. Moreover, benzoxepane derivative compound 10i exerts anti-neuroinflammatory effects by targeting the M2 isoform of pyruvate kinase (PKM2), thereby inhibiting PKM2-mediated glycolysis and nod-like receptor protein 3 (NLRP3) inflammasome activation [20]. Notably, coptisine chloride alleviates NLRP3 inflammasome activation by directly inhibiting the enzymatic activity of methyltransferase-like 3 [127]. Similarly, berberine exhibits anti-inflammatory properties through the direct inhibition of the eukaryotic translation initiation factor 2 alpha kinase two dimerization, subsequently regulating multiple inflammatory signaling pathways such as c-Jun N-terminal kinase (JNK), NF-κB, protein kinase B (AKT), and NLRP3 [110]. As an effective agent ameliorating ulcerative colitis, vanillic acid binds to carbonic anhydrase IX (CA9) to inhibit CA9/stromal interaction molecule 1-mediated ferroptosis for restoration of intestinal epithelium homeostasis [124]. In addition, the derivative A3-6 of anemoside B4 modulates macrophage polarization by specifically inhibiting the activity of pyruvate carboxylase (PC), thereby exerting a therapeutic effect on colitis [134].

Moreover, ruscogenin exhibits the capability to attenuate LPS-induced pulmonary endothelial barrier dysfunction by directly targeting non-muscle myosin heavy chain IIA for the regulation of toll-like receptor four signaling [94]. Likewise, pristimerin, a quinone methide triterpenoid, alleviates the progression of tendinopathy by modulating absent in melanoma 2-PYD and CARD domain containing (AIM2-PYCARD/ASC) stability via sequestosome 1/p62-mediated autophagic degradation, thus providing a promising autophagy-based therapeutic for tendinopathy [122]. In particular, glycyrrhisoflavone and licoisoflavone A from Huashi Baidu decoction demonstrate potent anti-inflammatory effects by directly inhibiting the cAMP-specific 3′,5′-cyclic phosphodiesterase 4 [113]. Taken together, there may be a heightened emphasis on exploring the potential therapeutic targets and molecular mechanisms of natural products for anti-inflammatory application in the future.

Mechanisms of neuroprotective natural products

Numerous natural products demonstrate neuroprotective properties and serve as a key source for developing potential drugs in brain ischemia and neurodegeneration. Therefore, exploring the targets and molecular mechanisms of natural products contributes to a more understanding of the pathogenesis of these diseases. For example, echinacoside (ECH) is a bioactive compound with potent neuroprotective effects. By using a chemical crosslinking strategy, recent study demonstrates that ECH directly interacts with the casein kinase 2 α′ subunit, subsequently activating the Wnt signaling pathway to promote mitochondrial fusion for a protective effect against brain ischemic damage [83]. Meanwhile, celastrol has the potential to alleviate mitochondrial dysfunction in neurons by interacting with cAMP-activated exchange protein-1 (EPAC-1), thus potentially enhancing the outcome of secondary brain injury resulting from intracerebral hemorrhage [121]. Similarly, scutellarin (SG) mitigates mitochondrial damage by selectively inhibiting the enzymatic activity of pyruvate dehydrogenase kinase 2 (PDK2), subsequently regulating mitochondrial glucose oxidation via the Pdk/pyruvate dehydrogenase complex axis [115]. This offers a promising strategy for neurological disease therapy by ameliorating mitochondrial bioenergetic deficits. Furthermore, cordycepin promotes neuronal survival and plays the key role in combating Alzheimer’s disease by dual-targeting hexokinase II (HKII) and PDK2 to modulate microglial polarization coupled with mitochondrial metabolic reprogramming [136]. Additionally, the natural product P57 induces hypothermia by inhibiting the activity of pyridoxal kinase (PDXK) to promote the accumulation of pyridoxal in hypothalamus, thus exhibiting neuroprotective effect in the ischemic stroke model [103]. Thus, these cases provide a novel perspective for the discovery of target-based neuroprotective agents from natural products.

Mechanisms of anti-tumor natural products

Natural products offer valuable insights for the development of anticancer drugs, thus elucidating the targets and molecular mechanisms that hold great biological significance. Nordihydroguaiaretic acid, a naturally occurring lignan from Larrea tridentata, exhibits potent anti-tumor effects against inflammation-associated colorectal cancer by specifically targeting retinoic acid receptor γ (RARγ). Further investigation has elucidated that nordihydroguaiaretic acid enhances the interaction between RARγ and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to the suppression of TRAF6-mediated NF-kB and interleukin-6-signal transducer and activator of transcription 3 (IL-6-STAT3) activation, thereby inhibiting tumor proliferation [128]. Meanwhile, cucurbitacin B (CuB) induces a conformational change in the insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) via covalent binding to Cys253, disrupting the recognition of downstream m⁶A target genes for anti-tumor effects [92]. Furthermore, studies suggest the potential of bruceine D (BD) as a modulator of the β-catenin/hypoxia-inducible factor-1 alpha axis by targeting inhibitor of β-catenin and T-cell factor for the treatment of hepatocellular carcinoma [87].

Most recently, the anti-tumor compound DHPO induces ferroptosis in gastric cancer by targeting ubiquitin-specific protease 7 (USP7), promoting the ubiquitination and subsequent proteasomal degradation of stearoyl-CoA desaturase [129]. Indole terpenoid derivates (+)-6-Br-JP18 and (+)-6-Cl-JP18, disrupt the spindle microtubule dynamics by specifically binding to the colchicine-binding site of β-tubulin, thereby exerting the anti-tumor effect [123]. Moreover, bufalin functions as a molecular glue that facilitates the assembly of a ternary complex comprising the transcription factor E2 factor 2 (E2F2) and the unconventional ubiquitin ligase ZFP91. This complex promotes the ubiquitination degradation of E2F2, thus offering a potential treatment for hepatocellular carcinoma [89]. Recent study has highlighted the potential implication of quercetin in overcoming T790 M mutation in epidermal growth factor receptor (EGFR^T790M)-driven tyrosine kinase inhibitors resistance by directly targeting glucose-6-phosphate dehydrogenase [104]. In addition, a natural product derived from Chaetomium globosum, XYA-2 specifically binds to the SH2 domain of STAT3, leading to a synergistic inhibition of myelocytomatosis and solute carrier family 39 member 10, ultimately suppressing tumor growth and metastasis in gastric cancer [117]. Therefore, the elucidation of the anti-tumor targets and signaling pathways of these natural products holds practical significance for the subsequent in-depth exploration of novel anti-tumor treatment strategies.

Mechanisms of lipid-lowering natural products

Natural products are an important source of lipid metabolism-regulating drugs. The target identification for natural products greatly contributes to the elucidation of the lipid-lowering mechanisms. For example, artemether (ART), a derivative of artemisinin, specifically targets protein kinase Cδ (PKCδ) to inhibit the palmitoylation modification by blocking the interaction of zinc finger DHHC-type palmitoyltransferase 5. This disruption results in the suppression of subsequent neuroinflammation signaling in the hypothalamus, subsequently activating the neuroendocrine axis, thereby presenting a promising therapeutic target for fatty liver diseases [125]. Furthermore, natural product arbutin (ARB) has been shown to inhibit the demethylase activity of fat mass and obesity-related protein (FTO), resulting in the accumulation of m⁶A methylation in the solute carrier family seven member 11 mRNA and consequently impeding ferroptosis [116]. Concurrently, equisetin, a bioactive compound derived from marine fungus, has been found to directly binds to 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) and presents remarkable inhibition on 11β-HSD1 for anti-obesity effect [95]. In addition, baicalin, a flavonoid from Scutellaria baicalensis, demonstrates unique anti-steatosis activity through directly allosteric activation of carnitine palmitoyltransferase 1, opening new opportunities for pharmacological treatment of diet-induced obesity and associated sequelae [137].

Meanwhile, investigators have discovered that artepillin C (APC) from Brazilian green propolis serves as an inhibitor of cAMP-response element binding protein-CREB regulated transcriptional coactivator 2 (CREB-CRTC2) interaction. APC suppresses the transcription of gluconeogenic and sterol-regulatory element binding protein genes mediated by CREB/CTRC2, resulting in decreased fasting glucose levels, enhanced insulin sensitivity, and reduced lipid levels in both serum and liver [101]. In a recent study, gentiopicroside (GPS), the bioactive secoiridoid glycoside present in Gentiana manshurica Kitagawa, has been determined to decrease lipid synthesis and restore the insulin signaling pathway through direct interaction with the progestin and adipoQ receptor 3 (PAQR3), thereby facilitating the activation of the phosphatidylinositol-3-kinase/AKT axis [102]. Moreover, atractylenolide II (AT II) is a sesquiterpene compound from Atractylodes macrocephala, demonstrating strong lipid-lowering effects by decreasing hepatic sn-1,2-diacylglycerol levels and ameliorating obesity-induced hyperlipidemia, hepatosteatosis, and insulin resistance via targeting diacylglycerol kinase family member DGKQ [105]. Also, aimosaponin AIII contributes to ameliorating diet-induced metabolic dysfunction-associated fatty liver and metabolic dysfunction-associated steatohepatitis in mice by directly targeting to activate hepatic adenosine A1 receptor (A1R) [130]. Puerarin (PU) may provide therapeutic benefits in combating atherosclerosis by modulating the gut microbiota [138]. Future advancements in characterizing drug targets and pharmacological mechanisms in lipid metabolism are expected to propel the field forward and enhance our comprehension of the complex mechanisms governing metabolic syndrome.

Mechanisms of anti-fibrosis natural products

Natural products are increasingly vital for drug discovery targeting fibrotic diseases including liver fibrosis, kidney fibrosis, pulmonary fibrosis, and others. For example, daphnepedunin A (DA) has emerged as a promising candidate for the treatment of renal fibrosis. Mechanistically, DA specially decreases cell division cycle 42 (Cdc42) activity to down-regulate its downstream phosphor-protein kinase Cζ/phosphor-glycogen synthase kinase-3β, thereby promoting β-catenin proteolysis to block classical pro-fibrotic β-catenin signaling [126]. Salvianolic acid B, a component of Salvia miltiorrhiza, also demonstrates a robust affinity to neuraminidase 1 (NEU1) and exhibiting significant protective effects against renal fibrosis [139]. Meanwhile, 2-APQC, analogues derived from camptothecin, is a specific sirtuin 3 (SIRT3) activator that alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis [133]. Similarly, ailanthone ameliorates pulmonary fibrosis by directly targeting mesenchymal homobox 1 (MEOX1) [140]. The matrine-derived compound 6 k regulates liver fibrosis by inhibiting the function of Ewing sarcoma breakpoint region 1 (EWSR1) to affect the expression of liver fibrosis-related genes [106]. In summary, tissue fibrosis is a significant contributor to disability in various diseases. Consequently, the identification of anti-fibrotic targets allows for an in-depth exploration of pharmacological mechanisms and promotes the development of anti-fibrotic agents.

Other pharmacological mechanisms of natural products

The structural variability of natural products contributes to a broad spectrum of biological activities, which are regulated by signal pathways through various functional proteins. Therefore, elucidating the signaling pathways behind the diverse pharmacological activities is crucial for understanding the mechanisms of natural products. For example, forskolin, a natural diterpene from Coleus forskohlii Briq, has demonstrated to promote osteogenesis by targeting transglutaminase 2 (TGM2). Further investigations have revealed that forskolin interacts with amino acid residues, specifically Asn299 and Thr368, in TGM2 via hydrogen bonding, consequently enhancing cell energy metabolism through mitochondrial-associated signaling pathway [107]. Similarly, natural product norlichexanthone (NOR) prevents postmenopausal osteoporosis by targeting estrogen receptor-α (Erα) to inhibit receptor activator of nuclear factor-kappa B ligand signal [86]. Moreover, the flavonoid compound morusin activates the nuclear factor erythroid 2-related factor 2-mediated anti-aging signaling pathway by downregulating cyclin D1 expression and promoting kelch-like ECH-associated protein 1 degradation through targeting tripartite motif-containing 25 (TRIM25) [141]. In addition, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside facilitates epigenetic reprogramming of DNA methylation patterns that regulate hematopoietic stem cells by targeting Ten-eleven-translocation 2 (TET2) [132].

Meanwhile, investigations have suggested that corylin derived from Psoralea corylifolia can alleviate the senescence process in vascular endothelial cells by targeting the Gtr1 protein and suppressing the mTOR signaling pathway [96]. Notably, ellagic acid can inhibit dihydrotestosterone-induced ferroptosis, thereby promoting hair regeneration by targeting β-catenin [131]. Martynoside demonstrates the potential to rescue the impairment of ribosome biogenesis induced by 5-fluorouracil by stabilizing the ribosomal protein L27a (RPL27A) [114]. Additionally, metformin initiates the AMP-activated protein kinase signaling pathway by directly binding to a γ-secretase subunit, PEN2, and subsequently inhibiting the lysosomal proton pump v-ATPase. This mechanism enables metformin to exert its antidiabetic activity [23].

In conclusion, natural products possess a wide range of biological activities and potential medicinal value. Thus, identifying target proteins to elucidate downstream pharmacological signaling pathways is critical for comprehensively understanding how natural products modulate human disease occurrence and beneficial for precise clinical medication.