Cinnamaldehyde induces apoptosis and enhances anti-colorectal cancer activity via covalent binding to HSPD1.

Colorectal cancer (CRC) is a common malignant tumor with high morbidity and mortality rates worldwide. Although surgical resection and adjuvant radiotherapy/chemotherapy are the mainstays of CRC treatment, the efficacy is unsatisfactory due to several limitations, including high drug resistance. Accordingly, there is a dire need for new drugs or a novel combination approach to treat this patient population. Herein, we found that cinnamaldehyde (CA) could exert an antitumor effect in HCT-116 cell lines. Target fishing, molecular imaging, and live-cell tracing using an alkynyl-CA probe revealed that the heat shock 60 kDa protein 1 (HSPD1) protein was the target of CA. The covalent binding of CA with HSPD1 altered its stability. Furthermore, our results demonstrated that CA could induce cell apoptosis by inhibiting the PI3K/Akt signaling pathway and enhanced anti-CRC activity both in vitro and in vivo. Meanwhile, CA combined with different chemotherapeutic agents was beneficial to patients resistant to anti-CRC drug therapy.

Ligustilide attenuates airway remodeling in COPD mice by covalently binding to MH2 domain of Smad3 in pulmonary epithelium, disrupting the Smad3-SARA interaction.

Airway remodeling is one of the hallmarks of chronic obstructive pulmonary disease (COPD) and is closely related to the dysregulation of epithelial-mesenchymal transition (EMT). Smad3, an important transcriptional regulator responsible for transducing TGF-ß1 signals, is a promising target for EMT modulation. We found that ligustilide (Lig), a novel Smad3 covalent inhibitor, effectively inhibited airway remodeling in cigarette smoke (CS) combined with lipopolysaccharide (LPS)-induced COPD mice. Oral administration of an alkynyl-modified Lig probe was used to capture and trace target proteins in mouse lung tissue, revealing Smad3 in airway epithelium as a key target of Lig. Protein mass spectrometry and Smad3 mutation analysis via in-gel imaging indicated that the epoxidized metabolite of Lig covalently binds to the MH2 domain of Smad3 at Cys331/337. This irreversible bonding destroys the interaction of Smad3-SARA, prevents Smad3 phosphorylation activation, and subsequently suppresses the nuclear transfer of p-Smad3, the EMT process, and collagen deposition in TGF-ß1-stimulated BEAS-2B cells and COPD mice. These findings provide experimental support that Lig attenuates COPD by repressing airway remodeling which is attributed to its suppression on the activation of EMT process in the airway epithelium via targeting Smad3 and inhibiting the recruitment of the Smad3-SARA heterodimer in the TGF-ß1/Smad3 pathway.

Magnolol alleviates pulmonary fibrosis inchronic obstructive pulmonary disease by targeting transient receptor potential vanilloid 4-ankyrin repeat domain.

Transient receptor potential vanilloid 4 (TRPV4) plays a role in regulating pulmonary fibrosis (PF). While several TRPV4 antagonists including magnolol (MAG), have been discovered, the mechanism of action is not fully understood. This study aimed to investigate the effect of MAG on alleviating fibrosis in chronic obstructive pulmonary disease (COPD) based on TRPV4, and to further analyze its mechanism of action on TRPV4. COPD was induced using cigarette smoke and LPS. The therapeutic effect of MAG on COPD-induced fibrosis was evaluated. TRPV4 was identified as the main target protein of MAG using target protein capture with MAG probe and drug affinity response target stability assay. The binding sites of MAG at TRPV4 were analyzed using molecular docking and small molecule interaction with TRPV4-ankyrin repeat domain (ARD). The effects of MAG on TRPV4 membrane distribution and channel activity were analyzed by co-immunoprecipitation, fluorescence co-localization, and living cell assay of calcium levels. By targeting TRPV4-ARD, MAG disrupted the binding between phosphatidylinositol 3 kinase γ and TRPV4, leading to hampered membrane distribution on fibroblasts. Additionally, MAG competitively impaired ATP binding to TRPV4-ARD, inhibiting TRPV4 channel opening activity. MAG effectively blocked the fibrotic process caused by mechanical or inflammatory signals, thus alleviating PF in COPD. Targeting TRPV4-ARD presents a novel treatment strategy for PF in COPD.

Evaluating the Anti-Melanoma Effects and Toxicity of Cinnamaldehyde Analogues.

Cinnamaldehyde (CA) showed potent activity against melanoma in our previous study, and the structure of unsaturated aldehydes is envisaged to play a role. Nevertheless, its limited drug availability restricts its clinical application. Therefore, a series of CA analogues were synthesized to evaluate their anti-melanoma activities across various melanoma cell lines. These compounds were also tested for their toxicity against the different normal cell lines. The compound with the most potential, CAD-14, exhibited potent activity against the A375, A875 and SK-MEL-1 cells, with IC50 values of 0.58, 0.65, and 0.82 µM, respectively. A preliminary molecular mechanism study of CAD-14 indicated that it could inhibit the p38 pathway to induce apoptosis, and suppress tumor growth by inhibiting the expression of ENO1. Furthermore, an acute toxicity study depicted that CAD-14 has better safety and tolerability than CA in vivo. These findings indicate that CAD-14 might be a lead compound for exploring effective anti-melanoma drugs.

Okicamelliaside targets the N-terminal chaperone pocket of HSP90 disrupts the chaperone protein interaction of HSP90-CDC37 and exerts antitumor activity.

Heat shock protein 90 (HSP90) has been recognized as a crucial target in cancer cells. However, various toxic reactions targeting the ATP binding site of HSP90 may not be the best choice for HSP90 inhibitors. In this paper, an ellagic acid derivative, namely, okicamelliaside (OCS), with antitumor effects was found. To identify potential anti-cancer mechanisms, an OCS photosensitive probe was applied to target fishing and tracing. Chemical proteomics and protein-drug interaction experiments have shown that HSP90 is a key target for OCS, with a strong binding affinity (KD = 6.45 µM). Mutation analysis of the target protein and molecular dynamics simulation revealed that OCS could competitively act on the key Glu-47 site at the N-terminal chaperone pocket of HSP90, where the co-chaperone CDC37 binds to HSP90, affect its stability and reduce the ∆Gbind of HSP90-CDC37. It was demonstrated that OCS destroys the protein-protein interactions of HSP90-CDC37; selectively affects downstream kinase client proteins of HSP90, including CDK4, P-AKT473, and P-ERK1/2; and exerts antitumor effects on A549 cells. Furthermore, tumor xenograft experiments demonstrated high antitumor activity and low toxicity of OCS in the same way. Our findings identified a novel N-terminal chaperone pocket natural inhibitor of HSP90, that is, OCS, which selectively inhibits the formation of the HSP90-CDC37 protein complex, and provided further insight into HSP90 inhibitors for anti-cancer candidate drugs.

Small-Molecule Fluorogenic Probe for the Detection of Mitochondrial Temperature In Vivo.

Mitochondria, as energy factories, participate in many metabolic processes and play vital roles in cell life. Most human diseases are caused by mitochondrial dysfunction, and mitochondrial temperature is an important indicator of mitochondrial function. Despite the biological importance of mitochondria, there are few tools for detecting changes in mitochondrial temperature in living organisms. Here, we report on a thermosensitive rhodamine B (RhB)-derived fluorogenic probe (RhBIV) that enables fluorescent labeling of cell mitochondria at concentrations as low as 1 µM. We demonstrate that this probe exhibits a temperature-dependent response in cell mitochondria. Furthermore, in mice, it has a long half-life (t1/2) and is primarily enriched in the liver. This unique thermosensitive probe offers a simple, nondestructive method for longitudinal monitoring of mitochondrial temperature both in vitro and in vivo to elucidate fundamental physiological and pathological processes related to mitochondrial function.

Micelles self-assembled by 3-O-ß-D-glucopyranosyl latycodigenin enhance cell membrane permeability, promote antibiotic pulmonary targeting and improve anti-infective efficacy.

BACKGROUND: Nanoparticle-based pulmonary drug delivery systems are commonly developed and applied for drug-targeted delivery. They exhibit significant advantages compared to traditional pulmonary drug delivery systems. However, developing the formulation of each drug is a time-consuming and laborious task. RESULTS: In this study, a universal lung-targeting nanoparticle was designed and constructed. The self-assembled micelles were composed of a platycodon secondary saponin, 3-O-ß-D-glucopyranosyl platycodigenin 682 (GP-682), based on its specific amphiphilic structure. The GP-682 micelles exhibited a relatively stable zeta potential with a particle size between 60 and 90 nm, and the critical micelle concentration (CMC) value was approximately 42.3 µg/mL. Preincubation of GP-682 micelles markedly enhanced their cell membrane permeability and improved drug uptake in vitro. The results were visualized using fluorescent dye tracing, transmission electron microscopy (TEM) observations and the lactate dehydrogenase (LDH) release assay. The obtained benefits enhanced the distribution of levofloxacin (Lev) in mouse lung tissue and reduced antibiotics overdosing. The acute lung injury mouse model induced by the Pseudomonas aeruginosa PA 14 strain demonstrated that preinjection of GP-682 micelles before antibiotic administration resulted in a higher survival rate and anti-infective efficacy in vivo. It also caused reductions in pulmonary injury, bacterial invasion and cytokine expression compared with treatment with Lev alone. CONCLUSIONS: GP-682 micelles are another nanoparticle-based pulmonary drug delivery system and provide a new lung-targeting therapy option.

Catalpolaglycone disrupts mitochondrial thermogenesis by specifically binding to a conserved lysine residue of UCP2 on the proton leak tunnel.

BACKGROUND: Catalpol (CAT), a naturally occurring iridoid glycoside sourced from the root of Rehmannia glutinosa, affects mitochondrial metabolic functions. However, the mechanism of action of CAT against pyrexia and its plausible targets remain to be fully elucidated. PURPOSE: This study aimed to identify the specific targets of CAT for blocking mitochondrial thermogenesis and to unveil the unique biological mechanism of action of the orthogonal binding mode between the hemiacetal group and lysine residue on the target protein in vivo. METHODS: Lipopolysaccharide (LPS)/ carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced fever models were established to evaluate the potential antipyretic effects of CAT. An alkenyl-modified CAT probe was designed to identify and capture potential targets. Binding capacity was tested using in-gel imaging and a cellular thermal shift assay. The underlying antipyretic mechanisms were explored using biochemical and molecular biological methods. Catalpolaglycone (CA) was coupled with protein profile identification and molecular docking analysis to evaluate and identify its binding mode to UCP2. RESULTS: After deglycation of CAT in vivo, the hemiacetal group in CA covalently binds to Lys239 of UCP2 in the mitochondria of the liver via an ɛ-amine nucleophilic addition. This irreversible binding affects proton leakage and improves mitochondrial membrane potential and ADP/ATP transformation efficiency, leading to an antipyretic effect. CONCLUSION: Our findings highlight the potential role of CA in modulating UCP2 activity or function within the mitochondria and open new avenues for investigating the therapeutic effects of CA on mitochondrial homeostasis.

Sinapine targeting PLCß3 EF hands disrupts Gαq-PLCß3 interaction and ameliorates cardiovascular diseases.

BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) over-activation is highly involved in cardiovascular diseases (CVDs), with the Gαq-PLCß3 axis acting as a core node of RAAS. PLCß3 is a potential target of CVDs, and the lack of inhibitors has limited its drug development. PURPOSE: Sinapine (SP) is a potential leading compound for treating CVDs. Thus, we aimed to elucidate the regulation of SP towards the Gαq-PLCß3 axis and its molecular mechanism. STUDY DESIGN: Aldosteronism and hypertension animal models were employed to investigate SP's inhibitory effect on the abnormal activation of the RAAS through the Gαq-PLCß3 axis. We used chemical biology methods to identify potential targets and elucidate the underlying molecular mechanisms. METHODS: The effects of SP on aldosteronism and hypertension were evaluated using an established animal model in our laboratory. Target identification and underlying molecular mechanism research were performed using activity-based protein profiling with a bio-orthogonal click chemistry reaction and other biochemical methods. RESULTS: SP alleviated aldosteronism and hypertension in animal models by targeting PLCß3. The underlying mechanism for blocking the Gαq-PLCß3 interaction involves targeting the EF hands through the Asn-260 amino acid residue. SP regulated the Gαq-PLCß3 axis more precisely than the Gαq-GEFT or Gαq-PKCζ axis in the cardiovascular system. CONCLUSION: SP alleviated RAAS over-activation via Gαq-PLCß3 interaction blockade by targeting the PLCß3 EF hands domain, which provided a novel PLC inhibitor for treating CVDs. Unlike selective Gαq inhibitors, SP reduced the risk of side effects compared to Gαq inhibitors in treating CVDs.

Ligustilide covalently binds to Cys703 in the pre-S1 helix of TRPA1, blocking the opening of channel and relieving pain in rats with acute soft tissue injury.

ETHNOPHARMACOLOGICAL RELEVANCE: The natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive. AIM OF THE STUDY: The objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel. METHODS: The therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca2+ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches. RESULTS: The administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca2+ influx through the channel opening and ultimately relieving pain. CONCLUSIONS: Lig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.

Astragaloside IV targets PRDX6, inhibits the activation of RAC subunit in NADPH oxidase 2 for oxidative damage.

BACKGROUND: Radix Astragali Mongolici, as a traditional Chinese medicine, is widely used in the treatment of qi deficiency, viral or bacterial infection, inflammation and cancer. Astragaloside IV (AST), a key active compound in Radix Astragali Mongolici, has been shown to reduce disease progression by inhibiting oxidative stress and inflammation. However, the specific target and mechanism of action of AST in improving oxidative stress are still unclear. PURPOSE: This study aims to explore the target and mechanism of AST to improve oxidative stress, and to explain the biological process of oxidative stress. METHODS: AST functional probes were designed to capture target proteins and combined with protein spectrum to analyze target proteins. Small molecule and protein interaction technologies were used to verify the mode of action, while computer dynamics simulation technology was used to analyze the site of interaction with the target protein. The pharmacological activity of AST in improving oxidative stress was evaluated in a mouse model of acute lung injury induced by LPS. Additionally, pharmacological and serial molecular biological approaches were used to explore the underlying mechanism of action. RESULTS: AST inhibits PLA2 activity in PRDX6 by targeting the PLA2 catalytic triad pocket. This binding alters the conformation and structural stability of PRDX6 and interferes with the interaction between PRDX6 and RAC, hindering the activation of the RAC-GDI heterodimer. Inactivation of RAC prevents NOX2 maturation, attenuates superoxide anion production, and improves oxidative stress damage. CONCLUSION: The findings of this research indicate that AST impedes PLA2 activity by acting on the catalytic triad of PRDX6. This, in turn, disrupts the interaction between PRDX6 and RAC, thereby hindering the maturation of NOX2 and diminishing the oxidative stress damage.

Ligustilide covalently binds to Cys129 of HMGCS1 to ameliorate dyslipidemia.

Dyslipidemia is characterized by elevated levels of total cholesterol and triglycerides in serum, and has become the primary human health killer because of the major risk factors for cardiovascular diseases. Although there exist plenty of drugs for dyslipidemia, the number of patients who could benefit from lipid-lowering drugs still remains a concern. Ligustilide (Lig), a natural phthalide derivative, was reported to regulate lipid metabolic disorders. However, its specific targets and underlying molecular mechanism are still unclear. In this study, we found that Lig alleviated high fat diet-induced dyslipidemia by inhibiting cholesterol biosynthesis. Furthermore, a series of chemical biological analysis methods were used to identify its target protein for regulating lipid metabolism. Collectively, 3-hydroxy-3-methylglutaryl coenzyme A synthetase 1 (HMGCS1) of hepatic cells was identified as a target for Lig to regulate lipid metabolism. The mechanistic study confirmed that Lig irreversibly binds to Cys129 of HMGCS1 via its metabolic intermediate 6,7-epoxyligustilide, thereby reducing cholesterol synthesis and improving lipid metabolism disorders. These findings not only systematically elucidated the lipid-lowering mechanism of Lig, but also provided a new structural compound for the treatment of dyslipidemia.

Ginsenoside CK targeting KEAP1-DGR/Kelch domain disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage.

BACKGROUND: Panax ginseng and Panax notoginseng as traditional Chinese medicines, are widely used in the treatment of qi deficiency, viral or bacterial infection, inflammation and cancer. Ginsenoside CK, an active metabolite of protopanoxadiol among the ginseng saponins, has been shown in previous studies to improve the organism's oxidative balance by regulating the KEAP1-NRF2/ARE pathway, thus slowing the progression of diseases. However, the specific targets and mechanisms of CK in improving oxidative stress remain unclear. PURPOSE: The aim of this study was to determine the potential therapeutic targets and molecular mechanisms of CK in improving oxidative stress injury both in vitro and in vivo. METHODS: LPS was used to induce oxidative damage in RAW 264.7 cells to evaluate the regulatory effects of CK on the KEAP1-NRF2/ARE pathway. Drug affinity responsive target stability technology (DARTS) combined with proteomics was employed to identify CK's potential target proteins. CK functional probe were designed to analyze the target protein using click chemistry. Furthermore, small molecule and protein interaction technologies were used to verify the mechanism, and computer dynamic simulation technology was used to analyze the interaction sites between CK and the target protein. The pharmacological effects and mechanism of CK in improving oxidative damage were verified in vivo by LPS-induced acute injury in mice and physical mechanical injury in rat soft tissues. RESULTS: KEAP1 was identified as the target protein that CK regulates to improve oxidative damage through the KEAP1-NRF2/ARE pathway. CK competitively binds to the DGR/Kelch domain of KEAP1, disrupting the binding between DLG peptide in NRF2 and KEAP1, thereby inhibiting the occurrence of oxidative damage induced by LPS or physical mechanical stress. CONCLUSIONS: CK functions as a natural KEAP1-NRF2 inhibitor, disrupting the binding between KEAP1 and NRF2-DLG motifs by targeting the DGR/Kelch domain of KEAP1, activating the antioxidant transcriptional program of NRF2, and reducing oxidative stress damage.

Magnolol and honokiol target TRPC4 to regulate extracellular calcium influx and relax intestinal smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: Magnolia officinalis Cortex (M. officinalis) is a classical traditional Chinese medicine (TCM) widely used to treat digestive system diseases. It effectively regulates gastrointestinal motility to improve abdominal pain, abdominal distension and other symptoms. Magnolol (MAG) and honokiol (HON) are the main pharmacodynamic components responsible for the gastrointestinal activity of M. officinalis. AIM OF THE STUDY: The transient receptor potential (TRP) family is highly expressed in the gastrointestinal tract and participates in the regulation of gastrointestinal motility, visceral hypersensitivity, visceral secretion and other physiological activities. In this study, the calcium-lowering mechanisms of MAG and HON contributing to the smooth muscle relaxation associated with TRP are discussed. MATERIALS AND METHODS: The relaxation smooth muscle effects of MAG and HON were tested by the isolated intestine tone tests. A synthetic MAG probe (MAG-P) was used to target fishing for their possible target. The distribution of MAG on the smooth muscle was identified by a molecular tracer based on chemical biology. Ca2+ imaging and dual-luciferase reporter assays were used to determine the effects on the target proteins. Finally, the calcium-mediating effects of MAG and HON on smooth muscle cells and TRPC4-knockdown cells were compared to verify the potential mechanism. RESULTS: After confirming the smooth muscle relaxation in the small intestine induced by MAG and HON, the relaxation effect was identified mainly due to the downregulation of intracellular calcium by controlling external calcium influx. Although MAG and HON inhibited both TRPV4 and TRPC4 channels to reduce calcium levels, the inhibitory effect on TRPC4 channels is an important mechanism of their smooth muscle relaxation effect, since TRPC4 is widely expressed in the small intestinal smooth muscle cells. CONCLUSIONS: The inhibition of MAG and HON on TRPC4 channels contributes to the relaxation of intestinal smooth muscle.

Cinnamaldehyde Regulates the Generation of γ-aminobutyric Acid to Exert Sedation via Irreversible Inhibition of ENO1 in the Cerebellar Granular Layer.

SCOPE: Glutamate (Glu) and γ-aminobutyric acid (GABA) are the major excitatory and inhibitory neurotransmitters that control information flow in the brain. GABA dysfunction is a general vulnerability factor for mental illness. Cinnamaldehyde (CA) is found to have sedation in a mental illness model. However, the specific targets and molecular mechanisms related to the sedative effects of CA have not been elucidated. METHODS AND RESULTS: Metabolomics analysis and target fishing showed CA could increase the expression of GABA in vivo, and α-enolase (ENO1) is the primary target protein of CA associated with sedation. CA mainly binds with ENO1 in the cerebellar granular layer of brain, which influences the first transformations of the input signals arriving in the cerebellar cortex. The α,ß-unsaturated aldehyde group of CA blocks the hydroxy group of Ser40, which induces a loss in ENO1 activation. CA also disturbs the glycolysis pathway and influences the tricarboxylic acid cycle and oxidative phosphorylation, which activate gluconeogenesis to provide energy to the brain. This mechanism is verified in zebrafish with ENO1 or glutamic acid decarboxylase (GAD) deficiency. CONCLUSIONS: CA demonstrates sedation and alleviates GABA dysfunction via covalent binding ENO1, which shows the potential to improve the therapy of mental illness.

Dietary Flavone Baicalein Combinate with Genipin Attenuates Inflammation Stimulated by Lipopolysaccharide in RAW264.7 Cells or Pseudomonas aeruginosa in Mice via Regulating the Expression and Phosphorylation of AKT.

Mounting evidence has shown that single-targeted therapy might be inadequate to achieve satisfactory effects. Thus, drug combinations are gaining attention as they can regulate multiple targets to obtain more beneficial effects. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the protein assembly and folding of client proteins and maintains their stability. Interfering with the interaction between HSP90 and its client proteins by inhibiting the latter's activity may offer a new approach toward combination therapy. The HSP90 client protein AKT plays an important role in the inflammatory response syndrome caused by infections. In this study, the dietary flavone baicalein was identified as a novel inhibitor of HSP90 that targeted the N-terminal ATP binding pocket of HSP90 and hindered the chaperone cycle, resulting in AKT degradation. Combining baicalein with genipin, which was extracted from Gardenia jasminoides, could inhibit the pleckstrin homology domain of AKT, significantly increasing the anti-inflammatory effects both in vitro and in vivo. This synergistic effect was attributed to the reduction in AKT expression and phosphorylation. Thus, elucidating the mechanism underlying this effect will provide a new avenue for the clinical application and development of synergistic anti-inflammatory drugs.

Cinnamaldehyde changes the dynamic balance of glucose metabolism by targeting ENO1.

AIMS: Hepatic glucose metabolism involves a variety of catabolic and anabolic pathways, and the dynamic balance of glucose metabolism is regulated in response to environmental and nutritional changes. The molecular mechanism of glucose metabolism in liver is complex and has not been fully elucidated so far. In this study, we hope to elucidate the target and mechanism of cinnamaldehyde (CA) in regulating glucose metabolism. MATERIALS AND METHODS: Molecular image tracing and magnetic capture in combination with an alkynyl-CA probe (Al-CA) was used to show CA covalently binds to α-enolase (ENO1) in both mouse liver and HepG2 cells. Accurate metabolic flow assays subsequently demonstrated that the utilization of glycogenic amino acids and the biosynthesis of tricarboxylic acid (TCA) cycle intermediates were strengthened, which was detected using nontargeted and targeted metabolomics analyses. KEY FINDINGS: Our study shows that CA covalently bonds with ENO1, which affects the stability and activity of ENO1 and changes the dynamic balance of glucose metabolism. The interruption of gluconeogenic reflux by ENO1 enhanced TCA cycle, and eventually led to a decrease in blood glucose and the improvement of mitochondrial efficiency. SIGNIFICANCE: These results provide a detailed description of how CA maintains the dynamic balance of glucose utilization and improves energy metabolism.

Lepidiline A Improves the Balance of Endogenous Sex Hormones and Increases Fecundity by Targeting HSD17B1.

SCOPE: Maca (Lepidium meyenii), a well-known plant from the Andean highlands of Peru, has been used widely as a nutritional supplement to increase sexual function and fecundity. However, the identity of its active ingredients and how they function remain unknown. METHODS AND RESULTS: Chemical substances in maca are identified by UPLC-Q-TOF, and the active ingredients are screened through HotMap coupled with an artificial neural network. Lepidiline A (LA), an imidazole alkaloid, is identified as the key active compound. LA affects the balance of endogenous sex hormones in mice and improves fecundity in Drosophila. Using a molecular LA probe, 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1) is revealed to be the potential target of LA using a fishing-rod strategy. It is demonstrated with experimental data that LA targets HSD17B1 to enhance the enzyme's activity and increases its bioconversion efficiency of actively formed sex hormones including estrogen to 17ß-estradiol and 4-androsten-3,7-dione to testosterone, which ultimately improves reproductive activity. CONCLUSION: LA improves the balance of endogenous sex hormones and increases fecundity by targeting HSD17B1. This underlying mechanism of action provides a useful insight into the application of maca in the regulation of dietary nutrition and healthy fertility.

The flavonoid baicalin improves glucose metabolism by targeting the PH domain of AKT and activating AKT/GSK3ß phosphorylation.

Baicalin is one of the main flavonoids of the dried root of Scutellaria baicalensis Georgi and is reported to exert beneficial effects on the regulation of glucose/lipid metabolism. However, understanding its specific target and unique mechanism for improving glucose utilization is a challenge. In this paper, target fishing with a baicalin probe reveals that baicalin interacts with AKT. An immunofluorescence assay further demonstrates the colocalization of baicalin with AKT in the cytoplasm. A competitive test and virtual docking show that baicalin might bind to the pleckstrin homology domain of AKT. This specific binding hampers AKT membrane translocation, activates the phosphorylation of AKT on Ser473, induces the downstream glycogen synthase kinase 3ß activation, and affects glycogen synthesis.

Genipin, a natural AKT inhibitor, targets the PH domain to affect downstream signaling and alleviates inflammation.

The iridoid compound genipin (GNP) is a geniposide hydrolysate of ß-glucosidase. GNP has many pharmacological effects, including antioxidant, anti-apoptotic, and anti-inflammation effects. However, its exact target and mechanism of action remain poorly understood. In this study, the binding of GNP to AKT protein was demonstrated via a GNP-modified magnetic microspheres (GNP-MMs) capture and immunofluorescence co-localization test. GNP-MMs fishing coupled with competitive testing and AKT plasma transport experiments indicate that GNP may act on the PH domain of AKT, and affect AKT plasma transport. The specific binding directly inhibits phosphorylation of AKT, affecting the downstream activation, and reducing inflammatory responses. The results indicate that GNP targets the PH domain region of AKT, inhibits the phosphorylation of AKT, and attenuates the transduction of AKT based inflammation signal pathway.