A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2).

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC50 value of 1.6 µM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

The influence of rhein on the absorption of rehmaionoside D: In vivo, in situ, in vitro, and in silico studies.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese Medicine, Rehmannia glutinosa (Gaertn.) DC., as the principle herb of ShengDiHuang Decotion (SDHD), has the effect of cooling blood and hemostasis, and tonifying the yin and kidney. Rheum L., as adjuvant herbs, assist Rehmannia glutinosa (Gaertn.) DC. to promote blood circulation to remove blood stasis. AIM OF STUDY: To study the mechanism of Rhein (RH) involved in the promotion of Rehmannioside D (RD) absorption by pharmacokinetic studies, single-pass intestinal perfusion, Caco-2 cell models, molecular docking technique and western blotting. MATERIALS AND METHODS: Initially, the intestinal absorption of RD in the presence or absence of RH was conducted through pharmacokinetic studies. Thereafter, the intestinal absorption of RD and RH was studied using the single-pass intestinal perfusion and Caco-2 cell models. Finally, using molecular docking technique and western blotting. RESULTS: We found that the promotion of RD absorption by RH was mediated by breast cancer resistance and multidrug resistance-associated protein 2, thereby affecting the permeability of the intestinal epithelium. Additionally, RH and RD can competitively bind to breast cancer resistance and multidrug resistance-associated protein 2, and that RH inhibits the expression of breast cancer resistance and multidrug resistance-associated protein 2 in the ileum to promote the intestinal absorption of RD. CONCLUSION: This study reveals the mechanisms associated with the RH-mediated promotion of RD absorption and provides a basis for further exploring the synergistic effect of Rehmannia glutinosa (Gaertn.) DC and rhubarb.

Systematic insight into the active constituents and mechanism of Guiqi Baizhu for the treatment of gastric cancer.

Traditional Chinese medicine treatment of diseases has been recognized, but the material basis and mechanisms are not clear. In this study, target prediction of the antigastric cancer (GC) effect of Guiqi Baizhu (GQBZP) and the analysis of potential key compounds, key targets, and key pathways for the therapeutic effects against GC were carried out based on the method of network analysis and Kyoto Encyclopedia of Genes and Genomes enrichment. There were 33 proteins shared between GQBZP and GC, and 131 compounds of GQBZP had a high correlation with these proteins, indicating that the PI3K-AKT signaling pathway might play a key role in GC. From these studies, we selected human epidermal growth factor receptor 2 (HER2) and programmed cell death 1-ligand 1 (PD-L1) for docking; the results showed that 385 and 189 compounds had high docking scores with HER2 and PD-L1, respectively. Six compounds were selected for microscale thermophoresis (MST). Daidzein/quercetin and isorhamnetin/formononetin had the highest binding affinity for HER2 and PD-L1, with Kd values of 3.7 µmol/L and 490, 667, and 355 nmol/L, respectively. Molecular dynamics simulation studies based on the docking complex structures as the initial conformation yielded the binding free energy between daidzein/quercetin with HER2 and isorhamnetin/formononetin with PD-L1, calculated by molecular mechanics Poisson-Boltzmann surface area, of -26.55, -14.18, -19.41, and -11.86 kcal/mol, respectively, and were consistent with the MST results. In vitro experiments showed that quercetin, daidzein, and isorhamnetin had potential antiproliferative effects in MKN-45 cells. Enzyme activity assays showed that quercetin could inhibit the activity of HER2 with an IC50 of 570.07 nmol/L. Our study provides a systematic investigation to explain the material basis and molecular mechanism of traditional Chinese medicine in treating diseases.

Antioxidant and Antiproliferative Activity of The Ethanolic Extract of Equisetum Myriochaetum and Molecular Docking of Its Main Metabolites (Apigenin, Kaempferol, and Quercetin) on ß-Tubulin.

Equisetum myriochaetum is a semi-aquatic plant found on riverbanks that is commonly used in traditional medicine as a diuretic agent. Additionally, the genus Equisetum stands out for its content of the flavonoid kaempferol, a well-known antiproliferative agent. Therefore, in this study, E. myriochaetum ethanolic extract was tested in vitro against a cervical cancer cell line (SiHa). Additionally, the antioxidative activity was evaluated through a 2,2-diphenyl-1-picrilhidrazil (DPPH) assay. Finally, a molecular docking analysis of apigenin, kaempferol, and quercetin on the active site of ß-tubulin was performed to investigate their potential mechanism of action. All fractions of E. myriochaetum ethanolic extract showed antioxidative activity. Fraction 14 displayed an antiproliferative capacity with a half maximal inhibitory concentration (IC50) value of 6.78 µg/mL against SiHa cells.

Exploration in the Mechanism of Kaempferol for the Treatment of Gastric Cancer Based on Network Pharmacology.

BACKGROUND: Kaempferol is a natural polyphenol in lots of Chinese herbs, which has shown promising treatment for gastric cancer (GC). However, the molecular mechanisms of its action have not been systematically revealed yet. In this work, a network pharmacology approach was used to elucidate the potential mechanisms of kaempferol in the treatment of GC. METHODS: The kaempferol was input into the PharmMapper and SwissTargetPrediction database to get its targets, and the targets of GC were obtained by retrieving the Online Mendelian Inheritance in Man (OMIM) database, MalaCards database, Therapeutic Target Database (TTD), and Coolgen database. The molecular docking was performed to assess the interactions between kaempferol and these targets. Next, the overlap targets of kaempferol and GC were identified for GO and KEGG enrichment analyses. Afterward, a protein-protein interaction (PPI) network was constructed to get the hub targets, and the expression and overall survival analysis of the hub target were investigated. Finally, the overall survival (OS) analysis of hub targets was performed using the Kaplan-Meier Plotter online tool. RESULTS: A total of 990 genes related to GC and 10 overlapping genes were determined through matching the 24 potential targets of kaempferol with disease-associated genes. The result of molecular docking indicated that kaempferol can bind with these hub targets with good binding scores. These targets were further mapped to 140 GO biological process terms and 11 remarkable pathways. In the PPI network analysis, 3 key targets were identified, including ESR1, EGFR, and SRC. The mRNA and protein expression levels of EGFR and SRC were obviously higher in GC tissues. High expression of these targets was related to poor OS in GC patients. CONCLUSIONS: This study provided a novel approach to reveal the therapeutic mechanisms of kaempferol on GC, which will ease the future clinical application of kaempferol in the treatment of GC.

Structure-based identification of potential novel inhibitors targeting FAM3B (PANDER) causing type 2 diabetes mellitus through virtual screening.

Type 2 diabetes mellitus is a metabolic disorder that requires potent therapeutic approaches. The FAM3B is a cytokine-like protein also referred to as PANcreatic-DERrived factor (PANDER) which mainly exists in pancreatic islets. In the process of identifying potential inhibitors with the aid of structure-based method PANDER protein is identified as a novel therapeutic target against type 2 diabetes mellitus as it involved in the development of type 2 diabetes by negatively regulating the pancreatic ß-cell function and insulin sensitivity in the liver. In the present study, the 3d model of target protein FAM3B was generated by homology modeling technique using the MODELLER9.9 program. The assessment of the structural stability of the 3d model was established by energy minimization technique. The structural quality was evaluated with standard validating protocols. Binding regions of the target protein has been determined by literature and SiteMap tool. In the current study of research, the FAM3B model was subjected to molecular screening with the Asinex-elite database of 14849 output molecules using the Glide virtual screening module in the Schrodinger suite. The final XP descriptor output of 14 molecules was analyzed and prioritized based on molecular interactions at the FAM3B active site. The docking score, binding free energies (Prime MM/GBSA) and bioavailability were undertaken into the consideration to identify lead inhibitors. The identified lead compounds were checked for ADME properties all falling within the permeable ranges. The analysis of results gave the insight to develop the novel therapeutic strategies against type 2 diabetes mellitus.

Detecting In-Situ oligomerization of engineered STIM1 proteins by diffraction-limited optical imaging.

Several signaling proteins require self-association of individual monomer units to be activated for triggering downstream signaling cascades in cells. Methods that allow visualizing their underlying molecular mechanisms will immensely benefit cell biology. Using enhanced Green Fluorescent Protein (eGFP) complementation, here I present a functional imaging approach for visualizing the protein-protein interaction in cells. Activation mechanism of an ER (endoplasmic reticulum) resident Ca2+ sensor, STIM1 (Stromal Interaction Molecule 1) that regulates store-operated Ca2+ entry in cells is considered as a model system. Co-expression of engineered full-length human STIM1 (ehSTIM1) with N-terminal complementary split eGFP pairs in mammalian cells fluoresces to form 'puncta' upon a drop in ER lumen Ca2+ concentration. Quantization of discrete fluorescent intensities of ehSTIM1 molecules at a diffraction-limited resolution revealed a diverse set of intensity levels not exceeding six-fold. Detailed screening of the ehSTIM1 molecular entities characterized by one to six fluorescent emitters across various in-plane sections shows a greater probability of occurrence for entities with six emitters in the vicinity of the plasma membrane (PM) than at the interior sections. However, the number density of entities with six emitters was lesser than that of others localized close to the PM. This finding led to hypothesize that activated ehSTIM1 dimers perhaps oligomerize in bundles ranging from 1-6 with an increased propensity for the occurrence of hexamers of ehSTIM1 dimer units close to PM even when its partner protein, ORAI1 (PM resident Ca2+ channel) is not sufficiently over-expressed in cells. The experimental data presented here provide direct evidence for luminal domain association of ehSTIM1 monomer units to trigger activation and allow enumerating various oligomers of ehSTIM1 in cells.

Agonists of G-Protein-Coupled Odorant Receptors Are Predicted from Chemical Features.

Predicting the activity of chemicals for a given odorant receptor is a longstanding challenge. Here the activity of 258 chemicals on the human G-protein-coupled odorant receptor (OR)51E1, also known as prostate-specific G-protein-coupled receptor 2 (PSGR2), was virtually screened by machine learning using 4884 chemical descriptors as input. A systematic control by functional in vitro assays revealed that a support vector machine algorithm accurately predicted the activity of a screened library. It allowed us to identify two novel agonists in vitro for OR51E1. The transferability of the protocol was assessed on OR1A1, OR2W1, and MOR256-3 odorant receptors, and, in each case, novel agonists were identified with a hit rate of 39-50%. We further show how ligands' efficacy is encoded into residues within OR51E1 cavity using a molecular modeling protocol. Our approach allows widening the chemical spaces associated with odorant receptors. This machine-learning protocol based on chemical features thus represents an efficient tool for screening ligands for G-protein-coupled odorant receptors that modulate non-olfactory functions or, upon combinatorial activation, give rise to our sense of smell.

Phytoestrogens and mycoestrogens interacting with breast cancer proteins.

Breast cancer is a highly heterogeneous disease influenced by the hormonal microenvironment and the most common malignancy in women worldwide. Some phytoestrogens and mycoestrogens have been epidemiologically linked as risk factors or protectors, however their mechanisms of action are complex and not fully understood. The aim of this study was to predict the potential of 36 natural xenoestrogens to interact with 189 breast cancer proteins using AutoDock Vina. In order to validate our protocol, an in silico docking pose and binding site determination was compared with the crystallographic structure and the power of prediction to distinguish between ligand and decoys was evaluated through a receiver operating characteristic curve (ROC) of the resultant docking affinities and in vitro data. The best affinity score was obtained for glyceollin III interacting with the sex hormone binding globulin (-11.9 Kcal/mol), a plasma steroid transport protein that regulates sex steroids bioavailability. Other natural xenoestrogens such as beta-carotene, chrysophanol 8-O-beta-d-glucopyranoside and glyceollin I, also presented good affinity for proteins related to this disease and the validation was successful. This study may help to prioritize compounds for toxicity tests or drug development from natural scaffolds, and to elucidate their mechanisms of action.

A charge-sensing region in the stromal interaction molecule 1 luminal domain confers stabilization-mediated inhibition of SOCE in response to S-nitrosylation.

Store-operated Ca2+ entry (SOCE) is a major Ca2+ signaling pathway facilitating extracellular Ca2+ influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca2+ stores. Stromal interaction molecule 1 (STIM1) is the Ca2+ sensor that activates SOCE following ER/SR Ca2+ depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca2+ concentrations. Low ER Ca2+ levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca2+ channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys49 and Cys56 in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca2+ depletion-dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys49 and Cys56S-nitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that S-nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.

Inhibition of metastasis and angiogenesis in Hep-2 cells by wheatgrass extract - an in vitro and in silico approach.

Metastasis is the major hindrance in the treatment of all cancers, including laryngeal squamous cell carcinoma. Intensive researches are under way to identify the effective natural polyphenols with anti-metastatic ability for cancer treatment. Wheatgrass, an herbal plant has been reported to show anticancer effects. Hence, in this study, we aimed to analyze the anti-metastatic effect of methanol extract of wheatgrass (MEWG). The levels of metastatic marker proteins were determined by western blot. PI3K and AKT levels were determined by real time (RT)-PCR analysis. In silico molecular docking was done to check the interaction of the 14 components (identified by HPLC/GCMS) of MEWG with PI3K and AKT. MEWG effectively decreased the metastatic protein expressions, namely VEGF, MMP-9 and COX-2 and increased TIMP-2. RT-PCR results showed reduced m-RNA levels of both PI3K and AKT when compared to control. Molecular docking studies revealed interaction of most of the identified compounds of the extract with the important residues of PI3K and AKT. These findings indicate that MEWG inhibits metastasis and angiogenesis in Hep-2 cells possibly via PI3K/AKT due to the cumulative effect of polyphenols and other constituent present in extract. The compounds of the extract were also found to be directly involved in inhibition of AKT/PI3K, thus could help to restrain metastasis.

ECOdrug: a database connecting drugs and conservation of their targets across species.

Pharmaceuticals are designed to interact with specific molecular targets in humans and these targets generally have orthologs in other species. This provides opportunities for the drug discovery community to use alternative model species for drug development. It also means, however, there is potential for mode of action related effects in non-target wildlife species as many pharmaceuticals reach the environment through patient use and manufacturing wastes. Acquiring insight in drug target ortholog predictions across species and taxonomic groups has proven difficult because of the lack of an optimal strategy and because necessary information is spread across multiple and diverse sources and platforms. We introduce a new research platform tool, ECOdrug, that reliably connects drugs to their protein targets across divergent species. It harmonizes ortholog predictions from multiple sources via a simple user interface underpinning critical applications for a wide range of studies in pharmacology, ecotoxicology and comparative evolutionary biology. ECOdrug can be used to identify species with drug targets and identify drugs that interact with those targets. As such, it can be applied to support intelligent targeted drug safety testing by ensuring appropriate and relevant species are selected in ecological risk assessments. ECOdrug is freely accessible and available at: http://www.ecodrug.org.

Identification of Potent Chloride Intracellular Channel Protein 1 Inhibitors from Traditional Chinese Medicine through Structure-Based Virtual Screening and Molecular Dynamics Analysis.

Chloride intracellular channel 1 (CLIC1) is involved in the development of most aggressive human tumors, including gastric, colon, lung, liver, and glioblastoma cancers. It has become an attractive new therapeutic target for several types of cancer. In this work, we aim to identify natural products as potent CLIC1 inhibitors from Traditional Chinese Medicine (TCM) database using structure-based virtual screening and molecular dynamics (MD) simulation. First, structure-based docking was employed to screen the refined TCM database and the top 500 TCM compounds were obtained and reranked by X-Score. Then, 30 potent hits were achieved from the top 500 TCM compounds using cluster and ligand-protein interaction analysis. Finally, MD simulation was employed to validate the stability of interactions between each hit and CLIC1 protein from docking simulation, and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) analysis was used to refine the virtual hits. Six TCM compounds with top MM-GBSA scores and ideal-binding models were confirmed as the final hits. Our study provides information about the interaction between TCM compounds and CLIC1 protein, which may be helpful for further experimental investigations. In addition, the top 6 natural products structural scaffolds could serve as building blocks in designing drug-like molecules for CLIC1 inhibition.

Polµ tumor variants decrease the efficiency and accuracy of NHEJ.

The non homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair often requires DNA synthesis to fill the gaps generated upon alignment of the broken ends, a complex task performed in human cells by two specialized DNA polymerases, Polλ and Polµ. It is now well established that Polµ is the one adapted to repair DSBs with non-complementary ends, the most challenging scenario, although the structural basis and physiological implications of this adaptation are not fully understood. Here, we demonstrate that two human Polµ point mutations, G174S and R175H, previously identified in two different tumor samples and affecting two adjacent residues, limit the efficiency of accurate NHEJ by Polµ in vitro and in vivo. Moreover, we show that this limitation is the consequence of a decreased template dependency during NHEJ, which renders the error-rate of the mutants higher due to the ability of Polµ to randomly incorporate nucleotides at DSBs. These results highlight the relevance of the 8 kDa domain of Polµ for accurate and efficient NHEJ, but also its contribution to the error-prone behavior of Polµ at 2-nt gaps. This work provides the first demonstration that mutations affecting Polµ identified in tumors can alter the efficiency and fidelity of NHEJ.

The treatment effects of flaxseed-derived secoisolariciresinol diglycoside and its metabolite enterolactone on benign prostatic hyperplasia involve the G protein-coupled estrogen receptor 1.

Secoisolariciresinol diglucoside (SDG), a lignan extracted from flaxseed, has been shown to suppress benign prostatic hyperplasia (BPH). However, little is known about the mechanistic basis for its anti-BPH activity. The present study showed that enterolactone (ENL), the mammalian metabolite of SDG, shared the similar binding site of G1 on a new type of membranous estrogen receptor, G-protein-coupled estrogen eceptor 1 (GPER), by docking simulations method. ENL and G1 (the specific agonist of GPER) inhibited the proliferation of human prostate stromal cell line WPMY-1 as shown by MTT assay and arrested cell cycle at the G0/G1 phase, which was displayed by propidium iodide staining following flow cytometer examination. Silencing GPER by short interfering RNA attenuated the inhibitory effect of ENL on WPMY-1 cells. The therapeutic potential of SDG in the treatment of BPH was confirmed in a testosterone propionate-induced BPH rat model. SDG significantly reduced the enlargement of the rat prostate and the number of papillary projections of prostatic alveolus and thickness of the pseudostratified epithelial and stromal cells when comparing with the model group. Mechanistic studies showed that SDG and ENL increased the expression of GPER both in vitro and in vivo. Furthermore, ENL-induced cell cycle arrest may be mediated by the activation of GPER/ERK pathway and subsequent upregulation of p53 and p21 and downregulation of cyclin D1. This work, in tandem with previous studies, will enhance our knowledge regarding the mechanism(s) of dietary phytochemicals on BPH prevention and ultimately expand the scope of adopting alternative approaches in BPH treatment.

Systems pharmacology to investigate the interaction of berberine and other drugs in treating polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common multifactorial endocrine disorder among women of childbearing age. PCOS has various and heterogeneous clinical features apart from its indefinite pathogenesis and mechanism. Clinical drugs for PCOS are multifarious because it only treats separate symptoms. Berberine is an isoquinoline plant alkaloid with numerous biological activities, and it was testified to improve some diseases related to PCOS in animal models and in humans. Systems pharmacology was utilized to predict the potential targets of berberine related to PCOS and the potential drug-drug interaction base on the disease network. In conclusion, berberine is a promising polypharmacological drug for treating PCOS, and for enhancing the efficacy of clinical drugs.

Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA--thermodynamic studies.

This work focuses on the pathogenic missense mutation in YY1 protein correlated with insulinomas. Based on in vitro studies, we demonstrate that the mutation does not affect the secondary structure of either zinc fingers or the N-terminal fragment (NTF) of the protein. Apart from a slight increase in the protein's compactness, no changes in the tertiary structure were observed. The introduced mutation significantly alters DNA-binding properties, both the affinity and enthalpy-entropy contribution of the process, which are highly dependent on the recognized sequence. Obtained results indicate concerted rather than a modular mode of sequence recognition by YY1 with the significant impact of a disordered NTF.

Targeting epidermal fatty acid binding protein for treatment of experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease in which dysregulated immune cells attack myelin in the central nervous system (CNS), leading to irreversible neuronal degeneration. Our previous studies have demonstrated that epidermal fatty acid binding protein (E-FABP), widely expressed in immune cells, in particular in dendritic cells (DCs) and T lymphocytes, fuels the overactive immune responses in the mouse model of experimental autoimmune encephalomyelitis (EAE). METHODS: In the present study, we conducted an intensive computational docking analysis to identify novel E-FABP inhibitors for regulation of immune cell functions and for treatment of EAE. RESULTS: We demonstrate that compound [2-(4-acetylphenoxy)-9,10-dimethoxy-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one; designated as EI-03] bound to the lipid binding pocket of E-FABP and enhanced the expression of peroxisome proliferator-activating receptor (PPAR) γ. Further in vitro experiments showed that EI-03 regulated DC functions by inhibition of TNFα production while promoting IL-10 secretion. Moreover, EI-03 treatment counterregulated T cell balance by decreasing effector T cell differentiation (e.g. Th17, Th1) while increasing regulatory T cell development. Most importantly, mice treated with this newly identified compound exhibited reduced clinical symptoms of EAE in mouse models. CONCLUSIONS: Taken together, we have identified a new compound which displays a potential therapeutic benefit for treatment of MS by targeting E-FABP.

Target identification of grape seed extract in colorectal cancer using drug affinity responsive target stability (DARTS) technique: role of endoplasmic reticulum stress response proteins.

Various natural agents, including grape seed extract (GSE), have shown considerable chemopreventive and anti-cancer efficacy against different cancers in pre-clinical studies; however, their specific protein targets are largely unknown and thus, their clinical usefulness is marred by limited scientific evidences about their direct cellular targets. Accordingly, herein, employing, for the first time, the recently developed drug affinity responsive target stability (DARTS) technique, we aimed to profile the potential protein targets of GSE in human colorectal cancer (CRC) cells. Unlike other methods, which can cause chemical alteration of the drug components to allow for detection, this approach relies on the fact that a drug bound protein may become less susceptible to proteolysis and hence the enriched proteins can be detected by Mass Spectroscopy methods. Our results, utilizing the DARTS technique followed by examination of the spectral output by LC/MS and the MASCOT data, revealed that GSE targets endoplasmic reticulum (ER) stress response proteins resulting in overall down regulation of proteins involved in translation and that GSE also causes oxidative protein modifications, specifically on methionine amino acids residues on its protein targets. Corroborating these findings, mechanistic studies revealed that GSE indeed caused ER stress and strongly inhibited PI3k-Akt-mTOR pathway for its biological effects in CRC cells. Furthermore, bioenergetics studies indicated that GSE also interferes with glycolysis and mitochondrial metabolism in CRC cells. Together, the present study identifying GSE molecular targets in CRC cells, combined with its efficacy in vast pre-clinical CRC models, further supports its usefulness for CRC prevention and treatment.