Several flavonoids, amongst which some flavanones, are known not to activate this receptor. As certain flavanones are known to mask bitter taste sensorially, flavanones might act as bitter receptor antagonists. Fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds occurring in green tea. Three flavanones showed inhibitory behavior towards the activation of hTAS2R39 by ECG: 4′-fluoro-6-methoxyflavanone, 6,3′-dimethoxyflavanone, and 6-methoxyflavanone (in order of decreasing potency). The 6-methoxyflavanones also inhibited activation of hTAS2R14 (another bitter receptor activated by ECG), though to a lesser extent. Dose-response curves of ECG at various concentrations of the full antagonist 4′-fluoro-6-methoxyflavanone and wash-out experiments indicated reversible insurmountable antagonism. The same effect was observed for the structurally different agonist denatonium benzoate.Many (dietary) bitter compounds,  For 21 of the 25 the bitter receptors, an agonist, or in some cases dozens of agonists, have been identified Even though bitter taste can be appreciated in some food products, such as beer, coffee, dark chocolate and red wine p-(dipropylsulfamyl)benzoic acid (better known as probenecid). It has been reported to inhibit activation of hTAS2R16, hTAS2R38, and hTAS2R43, and to suppress the bitter taste perception of salicin in sensory tests The small molecule butanoic acid (or GIV 3727) has been reported as inhibitor of six bitter taste receptors Recently, a pharmacophore model for maskers of the bitter taste of caffeine has been developed in-vitro studies on bitter receptor blocking, several molecules are reported to mask bitter taste in-vivoApart from The human bitter taste receptor hTAS2R39 seems to be a bitter receptor for dietary compounds, as many agonists are dietary compounds, such as thiamine (vitamin B1), quinine 4) was 95% pure and compound (6) was 92–95% pure. Each compound was dissolved in DMSO (Sigma-Aldrich) to a 100 mM stock concentration. Trypan blue solution (0.4% w/v) and isoproterenol were purchased from Sigma-Aldrich.Compounds tested were obtained from Extrasynthese , Indofine Chemical Company , Interbioscreen , and Sigma-Aldrich . The majority of compounds were ≥99% or ≥98% pure; compound  with 0.5 mM probenecid (Sigma-Aldrich) was used for dilution of compound-DMSO stock solutions and for calcium imaging assays. The presence of probenecid in the buffer did not lead to inhibition of hTAS2R14 or hTAS2R39. Comparisons of assays with and without the use of probenecid are shown in Tyrode's buffer , geneticin (400 μg/mL) and hygromycin (100 μg/mL) . Cells were grown and maintained at 37 °C and 5% (v/v) CO2.For functional expression of the human bitter taste receptor hTAS2R39, HEK293 T-Rex Flp-In cells  were used, stably expressing the chimeric G-protein α-subunit Gα16-gust44 (cloned into pcDNA4 (Invitrogen)) 3 cells in 100 μL/well and cultured for 24 h. Transcription of the receptors was induced by adding 0.25 μg/mL doxycycline (Sigma-Aldrich). Cells were induced for 24 h and then loaded with the calcium-sensitive fluorescent dye Fluo-4-AM , which was dissolved in Tyrode's buffer containing 5% (v/v) tetracycline-free FBS (Lonza). One hour after loading, cells were washed with Tyrode's buffer and taken up in Tyrode's buffer. Stock solutions of test compounds were prepared in DMSO and diluted to the appropriate concentration in Tyrode's buffer, not exceeding a DMSO concentration of 1% (v/v).Cells were seeded into poly-L-lysine-coated (Sigma-Aldrich) 96-well plates  at a density of 7*102+ release Receptor activation or inhibition was measured via intracellular Ca0), representing receptor activity, was calculated by subtracting the baseline fluorescence (F0) prior to loading from the maximum fluorescence (F) after compound addition, divided by the signal of the baseline in order to normalize background fluorescence 50) and half-maximal inhibitory concentrations (IC50) were calculated. Error bars reflect the standard error of the mean (SEM). Statistical analysis was performed in Graph Pad Prism .Data processing was done as reported previously 80 concentration of 200 μM. Screening for hTAS2R39 inhibition by flavanones was performed with simultaneous application of agonist (200 μM ECG) and putative antagonist (250 μM flavanone). Inhibition was indicated when 80 concentration of the agonist. Another agonist of hTAS2R39, denatonium benzoate, was used in inhibition experiments at 1.7 mM (EC80). hTAS2R14 inhibition was tested with 640 μM ECG or 70 μM genistein as agonists, representing their respective EC80 concentrations. To investigate the specificity of flavanones for inhibition of hTAS2R39 and hTAS2R14, another bitter taste receptor, hTAS2R16 (not known to be activated by flavonoids), was used in inhibition experiments . To distinguish between reversible and irreversible inhibition, washout experiments were performed. Cells were stimulated with 200 μM ECG in the absence and in the presence of each antagonist (∼IC50 concentrations), washed with Tyrode's buffer (80 μL/well), and again stimulated with 200 μM ECG.To determine whether inhibition was specific for the bitter taste receptor, the effect of the antagonists was tested on the β2-adrenergic receptor agonist isoproterenol (50 μM). To this end, the antagonists were applied at ∼IC and 3), 4′-fluoro-6-methoxyflavanone (6), and 6-methoxyflavanone (11). The inhibitory effects of (3) and (6) at screening concentrations were significant. The effect of (11) was not significant, but as there was a trend of reduced ECG responses visible, also (11) was selected for investigation of dose-response behavior.Epicatechin gallate (ECG) (st addition”). st addition”), and subsequently the agonist was added (indicated with the arrow “2nd addition”). In both examples, calcium signals elicited by ECG decreased in the presence of the 6-methoxyflavanones.They were further investigated by two different ways of compound addition: simultaneous and stepwise addition. Simultaneous addition of agonist and antagonist to the receptor reflects the situation of ideal blocker application in food products. Stepwise addition of agonist and antagonist is commonly applied in pharmaceutical research when examining pharmacodynamics of receptor-antagonist interaction 3), 4′-fluoro-6-methoxyflavanone (6), and 6-methoxyflavanone (11) towards hTAS2R39 were investigated using both ways of compound addition. The compound 4′-fluoro-6-methoxyflavanone (6) showed inhibitory activity towards ECG on hTAS2R39 both after simultaneous (6) prior to addition of ECG led to 100% receptor blocking (50) was 102 μM. An overview of inhibition thresholds and IC50 values is given in 6) was added simultaneously with ECG, it had a lower inhibition threshold than when added in the stepwise way. Upon simultaneous addition, a maximal signal reduction of 65% was reached at 63 μM, and further signal reduction could not be observed due to non-specific signals of the compound itself. The same holds for the simultaneous addition of 6,3-dimethoxyflavanone (3) (3), the full efficacy upon simultaneous addition could not be established. As shown in 3) after stepwise addition. In contrast to (6) and (3), the compound 6-methoxyflavanone (11) showed negligible inhibitory activity against ECG on hTAS2R39 when applied simultaneously . 6) towards 1.7 mM denatonium benzoate on hTAS2R39. The same trends as for ECG combined with antagonists were observed. The receptor activation of denatonium benzoate was reduced upon simultaneous addition of (6) up to a concentration of 63 μM , leading to maximal signal reduction of 58%. Stepwise application of (6) led to 100% receptor blocking, as already seen with ECG. The IC50 was calculated to be 55 μM. The results of all antagonists applied with denatonium benzoate are summarized in Next, it was investigated whether the activation of hTAS2R39 by another agonist could also be reduced by the inhibitors identified. Denatonium benzoate (6) and ECG . The increase of signal at increasing concentrations of (6) is of non-specific nature, which can also be seen in the increase of response of non-induced cells, in which the bitter receptor is not expressed. Upon stepwise application, an inhibitory effect was observed. The three methoxyflavanones were also tested with genistein, another agonist of hTAS2R14, at 70 μM (EC80). The results were similar to the results obtained with ECG. An overview of inhibition thresholds and IC50 values on hTAS2R14 is given in It was investigated whether the antagonists identified specifically inhibit hTAS2R39, or also hTAS2R14, as many flavonoids behave similarly towards these two receptors In order to further investigate the specificity of the antagonists identified towards taste receptors, isoproterenol responses were measured. Inhibition of isoproterenol response would indicate non-specific inhibition of β2-adrenergic receptors, endogenous to HEK293 cells. The results Figure 76) on hTAS2R39, this molecule seemed to be the most effective antagonist identified in this study. The mechanism of antagonism was further clarified by measuring dose-response curves of ECG (6). Two effects were observed upon increasing antagonist concentrations: the dose-response curves shifted to the right, and the signal amplitudes decreased. The dose-response curves for the inhibition of ECG and denatonium benzoate showed the same pattern. EC50 values at all antagonist concentrations were calculated and are given in Due to full elimination of agonistic responses by 4′-fluoro-6-methoxyflavanone , 6,3′-dimethoxyflavanone (3), and 6-methoxyflavanone (11) were identified as antagonists (in decreasing order of potency), amongst which (6) fully eliminated the agonistic response. This was observed both for the bitter tea flavonoid ECG and for the synthetic bitter compound denatonium benzoate. The activation of hTAS2R14, another bitter receptor recognizing ECG In this paper we describe, to our knowledge for the first time, the identification of antagonists for hTAS2R39. For hTAS2R39, 4′-fluoro-6-methoxyflavanone (11) and (3) did not lead to full inhibition of the ECG signal on hTAS2R39, the question arises whether these two compounds are antagonists or partial agonists. They were tested for hTAS2R39 agonism as well, but none of them activated the receptor (In view of the fact that the application of (receptor File S4.5) , 6-hydroxyflavanone (8) , 4′-methoxyflavanone (13) , and 7-methoxyflavanone (12)  did not show inhibitory activity. Additionally, the compound 6-methoxyflavone was unable to inhibit hTAS2R39 activation (data not shown), which indicated that absence of a double bond in the C-ring is essential for inhibition.Several flavanones similar to the antagonists identified did not show inhibitory activity towards bitter receptor hTAS2R39. It turned out that only flavanones with a methoxy group on the 6-position of the A-ring, and various B-ring configurations were able to act as antagonists of hTAS2R39, as flavanone (11), which is unsubstituted on the B-ring, showed poor blocking behavior compared to (6) and (3). It might be speculated that size, electronegativity and/or electron withdrawing effect of the B-ring substituent influences the potency of antagonists.Amongst the antagonists identified for hTAS2R39, the difference in substitution of the B-ring determined the blocking potency. Compound (80 concentrations) by 4′-fluoro-6-methoxyflavanone (6) on hTAS2R39, this molecule was investigated further with respect to a possible antagonistic mechanism. Two effects were observed when increasing 4′-fluoro-6-methoxyflavanone concentrations: the dose-response curves shifted to the right, and the signal amplitudes decreased. These phenomena suggest that it can be classified as insurmountable antagonist Due to full elimination of agonistic responses  and (6) seem to be more suitable than (11). (iii) In order to achieve a sensorial effect, blocking of all bitter receptors activated by one compound is desirable. A blocker, that can only inhibit hTAS2R39, but not hTAS2R14, might be too specific to effectively reduce bitterness of ECG. On the other hand, it is not known yet whether these two receptors have an equally important role in the mouth. (iv) The blocker should preferably be of natural origin, and should be available in sufficient quantity. We could not find any natural source of the three inhibitors described in this study, and assume that they are only available synthetically. (v) Only compounds that are known as safe for consumption are of interest for food applications. Due to unknown safety of 4′-fluoro-6-methoxyflavanone, we abstained from sensory tests, which might confirm the function as bitter taste blocker in-vivo. As not all criteria are met by the blockers discovered in the present study, they might not be applicable to food products. Nevertheless, a 6-methoxy substituent on the A-ring of a flavanone has been identified as important for inhibition of hTAS2R39, which might form the basis for other, more suitable, blockers.For application of blockers in food products, several requirements should be met. (i) The blocker should be functional at a low dose. Therefore, an antagonist that has to be applied in equimolar or higher quantity to the agonist, is not efficient. (ii) In order to block the bitter taste of dietary compounds, for practical reasons, it is necessary that a bitter receptor blocker is also functional when applied simultaneously with the bitter compound. Therefore, compounds like (File S1The effect of probenecid.(PDF)Click here for additional data file.File S2Test for toxic effects.(PDF)Click here for additional data file.File S3Investigation of identified inhibitors on hTAS2R16.(PDF)Click here for additional data file.File S4Investigation of agonism on hTAS2R39.(PDF)Click here for additional data file.


Under normal physiological conditions, the hepatocyte growth factor (HGF) and its receptor, the MET transmembrane tyrosine kinase (cMET), are involved in embryogenesis, morphogenesis, and wound healing. The HGF-cMET axis promotes cell survival, proliferation, migration, and invasion via modulation of epithelial-mesenchymal interactions. Hepatocellular cancer (HCC) is the third most common cause of worldwide cancer-related mortality; advanced disease is associated with a paucity of therapeutic options and a five-year survival rate of only 10%. Dysregulation of the HGF-cMET pathway is implicated in HCC carcinogenesis and progression through activation of multiple signaling pathways; therefore, cMET inhibition is a promising therapeutic strategy for HCC treatment. The authors review HGF-cMET structure and function in normal tissue and in HCC, cMET inhibition in HCC, and future strategies for biomarker identification. Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and the third most common cause of global cancer related mortality , 2. HCC Although the transition from normal hepatocyte to HCC is not fully understood, hepatocarcinogenesis is a complex multistep process driven by accumulation of heterogeneous molecular alterations from initial hepatocyte injury to metastatic invasion. Inflammation results in hepatocyte regeneration, which induces fibrosis and cirrhosis through cytokine release. Dysplastic nodules subsequently progress to early HCC through cumulative genetic alterations, while advanced HCC often involves intrahepatic metastasis and portal vein invasion. Molecular alterations implicated in HCC development include mutations in oncogenes and tumor suppressor genes (p53 and p16), epigenetic alterations, chromosomal changes, and aberrant activation of signaling cascades necessary for proliferation, angiogenesis, invasion and metastasis, and survival. Pathogenesis of early and advanced HCC may be modulated through different mechanisms; for example, p53 mutations, p16 gene silencing, and aberrant AKT signaling are more frequently observed in advanced HCC –6. The mThe hepatocyte growth factor (HGF) and its transmembrane tyrosine kinase receptor, cellular MET (cMET) promote cell survival, proliferation, migration, and invasion via modulation of epithelial-mesenchymal interactions. HGF-cMET signaling is critical for normal processes such as embryogenesis, organogenesis, and postnatal tissue repair after acute injury. HGF-cMET axis activation is also implicated in cellular invasion and metastases through induction of increased proliferation (mitogenesis), migration and mobility (motogenesis), three-dimensional epithelial cell organization (morphogenesis), and angiogenesis.in vitro [α-chain containing four kringle domains (K1 to K4) and an amino-terminal loop domain (N), and a β-chain  containing a serine protease homology (SPH) domain [HGF was first discovered in 1984 as a mitogenic protein for rat hepatocytes in vitro . HGF is in vitro . HGF train vitro , 12. Cirin vitro , 14. TheMET protooncogene was first isolated in 1984 from a human osteosarcoma-derived cell line driven by a chromosomal rearrangement TPR-MET, resulting from fusion of translocated promoter region located on chromosome 1q25 and MET sequence located on chromosome 7q31 [TPR-MET rearrangement encodes for a prototype of the cMET receptor tyrosine kinase family. The cMET receptor is expressed predominantly on the surface of endothelial and epithelial cells of many organs, including the liver, kidney, prostate, pancreas, kidney, muscle, and bone marrow [α-chain and transmembrane β-chain. The β-chain contains an extracellular domain, transmembrane domain, and cytoplasmic portion. The extracellular domain includes an amino-terminal semaphorin domain, terminal cysteine-rich PSI domain, and four IPT repeat immunoglobulin domains [HGF is a ligand for the MET receptor, also known as cellular MET or cMET . The METome 7q31 . The TPR domains . The cyt domains .α-chain  and a low affinity site on the β-chain  [α- and β-chain sites have distinct functions. While α-chain N-K1 portion activates cMET and induces MET dimerization [β-chain residues bind cMET once the receptor is already occupied by HGF N-K4 and subsequently induce phosphorylation and downstream signaling [Under normal conditions, HGF interacts with cMET via a paracrine signaling loop and triggers cMET kinase activation within the cytoplasmic portion see . HGF conerminal) , 19. Therization , β-chainγ, STAT3, Shc, Src, Shp2, PI3K, and Ship1 [HGF binding induces autophosphorylation of tyrosine residues Y1234 and Y1235 within the cMET TK domain activation loop. Phosphorylation also occurs at two sites within the carboxyl terminal region (Y1349 and Y1356), forming a multifunctional high affinity binding docking site that recruits a range of intracellular adaptors containing Src homology-2 domains –24. An iThe HGF-cMET pathway serves as a hub for multiple heterogeneous signaling networks and is also modulated by the activation of other receptor TK families such as the epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2, insulin-like growth factor 1 receptor, Raf kinase, and VEGF .The HGF-cMET pathway plays an essential role in mammalian development in terms of morphogenesis, mitogenesis, and motogenesis, and angiogenesis HGF-cMET signaling is likely to be critical in many aspects of adult homeostasis including cardiac and hepatic tissue injury repair , 27, skiHGF or MET genes results in embryonically lethal knockouts with impaired organogenesis of the liver and placenta [MET knockout facilitates liver damage and fibrotic changes with delayed repair [cMET gene amplification, and cMET gene mutations leading to ligand-independent kinase activity [Targeted disruption of the placenta . Preclinplacenta , 32; expd repair . Under nd repair . Despited repair . Mechanid repair , constitactivity –36.cMET-regulated gene expression signature was found to define a subset of human HCC with poor prognosis and aggressive phenotype and correlated with increased vascular invasion, increased microvessel density, and decreased mean survival time [The HGF-cMET axis is implicated in hepatocarcinogenesis through multiple mechanisms, many of which are still being elucidated. Overexpression of HGF  and cMETval time . Higher val time  and prioval time  and posival time ; howeverval time , but mayval time . MET mutations and gene amplifications in HCC pathogenesis is unclear. While gains in 7q have been reported in HCC [The role of d in HCC , gene amd in HCC . Somaticd in HCC . in vivo, tubulogenesis in vitro, and tumoral microvessel density [Crosstalk between cMET and EGFR , 56 and  density . https://ccrod.cancer.gov/confluence/display/CCRHGF/Home. Given the predominant role of dysregulated HGF-cMET signaling in hepatocarcinogenesis, pharmacologic cMET inhibition is a promising therapeutic strategy. Targets for inhibition of the cMET signal transduction pathway include ligand-receptor interaction, cMET kinase activity, and cMET and adaptor protein interaction. HGF-cMET axis inhibitors can be broadly classified into biologic antagonists, c-MET adaptor protein inhibitors, small-molecule downstream pathway inhibitors, and small synthetic MET tyrosine kinase inhibitors (TKI). Of these, TKIs are the most common and the only ones to have completed phase 2 testing in HCC as of March 2013. in vitro and in vivo mice models demonstrate suppression of HGF-induced tumor cell migration and metastasis [Biologic antagonists inhibit cell surface interactions such as ligand-receptor binding or receptor clustering, preventing activation of downstream signaling. These include HGF competitive analogs, MET decoy receptor, and anti-HGF-cMET monoclonal antibodies. HGF competitive analogs compete with ligand for receptor binding without causing cMET dimerization and activation and include NK2 , 61, NK4tastasis , 69. Curin vitro and impaired metastatic spread of human prostate cancer cells in vivo; data in human studies have not been reported to date [Given the importance of adaptor proteins in propagating downstream cMET signaling, cMET adaptor inhibitors offer unique potential for cMET specific inhibition . As desc to date , 75. Small-molecule downstream pathway inhibitors directed towards inhibition of STAT3 phosphorylation showed preliminary tolerability in a phase I trial of advanced solid tumors, but data are not yet available for HCC . Synthetic small MET TKIs inhibit downstream signal transduction by preventing phosphorylation, either via competitive binding of the intracellular adenosine triphosphate (ATP) site in cMET's TK domain, or noncompetitive binding of a cMET region outside of the ATP binding site. While some of the TKIs in development are cMET specific, others target multiple pathways including VEGF, PDGFR, fms-related tyrosine kinase 3 (FLT3), v-kit feline sarcoma viral oncogene homolog protein (KIT), and anaplastic lymphoma kinase (ALK). Preclinical studies and clinical trials show tolerability and efficacy of cMET TKIs across a variety of solid malignancies. As of March 2013, promising results in the phase 2 randomized setting for HCC are available for two cMET inhibitors: tivantinib and cabozantinib. P = 0.63; 6.9 versus 6 weeks, HR = 0.64, P = 0.04, resp.). Patients with high MET expression (defined as 50% or more cells in the tumor specimen with 2+ or 3+ staining intensity) versus low MET expression receiving tivantinib demonstrated a significant improvement in both OS and TTP . No detrimental effect was reported in patients with low MET expression, and common adverse events were neutropenia, asthenia, poor appetite, and anemia. As of November 2012, a phase III trial for tivantinib and HCC patients is in development.Tivantinib (ARQ 197) is an oral low-molecular-weight TKI which is non-ATP competitive. Safety and tolerability without drug-related worsening of hepatic function were previously reported in a phase Ib trial of 20 cirrhotic patients (Child-Pugh A and B) with HCC, with 2 or less prior systemic chemotherapy regimens . RimassaCabozantinib (XL 184) is an unselective oral multikinase TKI targeting cMET, KIT, rearranged during transfection (RET), VEGFR1 and 2 and 3, FLT3, AXL receptor tyrosine kinase (AXL), and Tie family angiopoietin 1 receptor . CabozanIn fact, the inhibition of both VEGF and MET concurrently may be particularly effective. VEGF inhibition leads to increased MET signaling, either from resultant hypoxia or direct interactions between VEGFR2 and MET , 82. ConBased on preclinical trials and phase 2 data for tivantinib and cabozantinib, inhibition of cMET signaling is a promising therapeutic strategy in HCC. Although it is unclear which genetic and molecular abnormalities implicated in HGF-cMET dysregulation are predictive of sensitivity to cMET targeted therapy, ongoing trials must address the challenge of identifying patients most likely to achieve maximal benefit and minimal toxicity. Two current strategies for patient selection based on tumor biomarkers are quantification of tumor cMET content via immunohistochemical (IHC) and immunoassay tissue analysis and assessment of MET sequence status. Pharmacodynamic serum markers are under evaluation in ongoing trials as a strategy to assess clinical response. Novel companion diagnostics are under evaluation in preclinical studies. Tumoral cMET overexpression as measured by commercially available IHC kits appears to correlate with efficacy of cMET inhibitors in HCC and other solid tumors, although more prospective data are necessary for validation. Identification of cMET phosphorylation status is a potentially powerful target for targeted antibodies; two site immunoassays of flash-frozen tissue samples yielding precise measurements of MET content and phosphorylation activation are currently under development . MET mutations, MET amplification, and chromosome 7 polysomy. Preclinical studies of cMET targeted agents demonstrate variable efficacy based on MET mutation location; for example, PF-2341066/4217903 is a selective inhibitor with increased activity against certain cMET ATP binding site mutations in comparison to MET kinase domain activation loop mutations [In vitro studies of SU11274, a small-molecule TK competitive ATP binding site inhibitor, show selective inhibition of two out of four identified MET mutations [cMET is associated with increased clinical response to foretinib (XL 880) in phase 2 gastric cancer data, and cMET copy number correlates with increased clinical response to tivantinib in addition to erlotinib in advanced NSCLC [Another promising stratification strategy is assessment of MET sequence status including utations . In vitrutations . Amplified NSCLC . Ancillary pharmacodynamic and pharmacokinetic marker studies in a variety of solid tumors show variable correlation with clinical response. Preclinical data with crizotinib in gastric cancer cell lines demonstrate variable biomarker modulation of cMET inhibition with cMET dependent gastric cancer cell lines versus cMET-independent lines , 89. CliIn vitro and in vivo animal studies suggest that radiolabelled dyes containing cMET binding peptide successfully targets cMET receptors with higher imaged based tumor uptake [HGF-cMET is an intriguing target for the development of companion diagnostic tools as an adjunct tool for patient selection and stratification for cMET therapy. r uptake . A variar uptake . Inhibition of cMET is a promising therapeutic strategy in HCC. Given the heterogeneous mechanisms underlying cMET dysregulation, there is an urgent and unmet need for the development of predictive biomarkers to identify which subsets of cMET-dependent HCC tumors are most likely to benefit from specific classes of inhibitors. As more agents move into phase 2 and 3 trials for HCC, one important consideration is the emergence of acquired and primary resistance mechanisms from de novo or preexisting mutations. These may be overcome by rational combination therapy directed against multiple pathways, different levels of ligand-receptor-TKI interaction, and the presence of cMET addiction. Innovative clinical trial designs (such as the discontinuation cabozantinib design) with incorporation of enriched patient cohorts, biomarker analyses, pharmacodynamic markers, and companion diagnostics are essential in moving forward.


Dictyostelium is one of the leading lower eukaryotic model systems where stress-induced cellular responses, Wnt-like pathways, and STAT-mediated pathways are well investigated. These Dictyostelium pathways will be reviewed together with their mammalian counterparts to facilitate the comparative understanding of these variant and noncanonical pathways.While studies on metazoan cell proliferation, cell differentiation, and cytokine signaling laid the foundation of the current paradigms of tyrosine kinase signaling, similar studies using lower eukaryotes have provided invaluable insight for the understanding of mammalian pathways, such as Wnt and STAT pathways.  Dictyostelium were initiated in the early 1990s. Their initial efforts were to identify novel tyrosine phosphatase and kinase genes and categorize their functions using null mutants or overexpressors. Studies on the tyrosine phosphorylation of Dictyostelium tyrosine phosphatases were PTP1, PTP2, and PTP3 . Co. CoDicty profile .Dictyostelium cells exhibit adaptive responses to protect themselves. Cell cringing is one such response, which includes remodeling the cortical cytoskeleton through tyrosine phosphorylation of actin and threonine phosphorylation of Myosin II. Although details of the pathway regulating actin tyrosine phosphorylation remain to be determined, more is known about Myosin II regulation: hypertonic stress induced increases in cytosolic cGMP concentration and subsequent activation of Myosin heavy chain kinase triggers the reorganization of Myosin [Upon facing a hypertonic environment, f Myosin . Interesf Myosin . A recenf Myosin .Dictyostelium [The other well-known cellular hypertonic stress response is the alteration of intracellular pH . Given tostelium , NHE1 isostelium , understDictyostelium cells as well. Therefore, in addition to the Tyrosine Kinase Likes (TKLs) being involved in STAT activation and cortical actin reorganization, a tyrosine kinase may also affect NHE1 function in response to hypertonic stresses , and a Tyrosine Kinase Like domain after the TM. −vsk3 cells were able to internalize phagosomes, but failed to enforce their fusion with late endosomes/lysosomes for the maturation [Dictyostelium is one of the leading model hosts. It is established that the members of the TAM receptor protein tyrosine kinases (RPTK)—TYRO3, AXL, and MER—have critical roles in phagocytosis of mammalian cells [Although initially characterized turation , 53. Idean cells , 55. Givan cells . VSK3 liDictyostelium genome contains 66 Tyrosine Kinase Like proteins based on their sequence motives, which resemble both tyrosine kinases and serine/threonine kinases [Dictyostelium TKL group display more divergence, and thus their tyrosine kinase activities need to be experimentally confirmed. The  kinases . Unlike Dictyostelium genome [The presence of a total of thirteen SH2 domain-containing proteins in m genome  also sugAs we understand more of the tyrosine kinase-mediated signaling in lower eukaryotes, we will be better equipped with the knowledge of variant and noncanonical pathways, which in turn will facilitate a better selection of a therapeutic target. Furthermore, we will be in a better position to handle various infectious diseases due to the detailed information regarding the process of phagocytosis from lower eukaryotes.


Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop. Flaviridae family, is a positive single-strand RNA virus. An estimated 3% of world's population is chronically infected with HCV, with 30% of carriers expected to develop serious liver-related diseases, including hepatocellular carcinoma, over a period of 10 to 30 years Hepatitis C virus (HCV), a member of the There are two major classes of NS5B inhibitors: active site nucleotide/nucleoside analogues (Nucs), and nonnucleoside inhibitors (NNIs) that bind to allosteric sites on the enzyme. Nuc inhibitors mimic the natural substrates and function as chain terminators by incorporation into viral RNA. In contrast to Nucs, the NNIs are thought to achieve NS5B inhibition by affecting conformational states of the protein. Crystal structures of NS5B in complex with NNIs, together with resistance analysis, have revealed at least four pockets as allosteric inhibitory sites .570, Despite a wealth of enzymatic and structural information regarding HCV NS5B It has been postulated that the vast majority of crystallographically observed conformations of NS5B, including all inhibitor bound structures, represent a closed, inactive form of the enzyme de novo initiation to productive elongation. While this structure has a more open active site as a result of β-loop truncation and movement of the thumb and fingers domains away from each other to accommodate the double stranded RNA, the primer and template do not seem to be located in a position that would allow proper elongation, when compared to structures of other polymerases . A model that incorporates the unique structural characteristics of the HCV polymerase is required to explain the activity of inhibitors that may prevent the transition to the open form.Several potential conformations of NS5B that would allow binding of double stranded RNA in the active site and polymerization have been proposed, including a large rotation of the thumb domain In this study we characterize the mechanism of action of GS-9669, an allosteric inhibitor of NS5B that binds to the thumb site II and is at present in phase II clinical trials in HCV patients. Our approach relies on enzyme inhibition, calorimetric, and direct binding studies with the use of GS-9669 and two other thumb site II inhibitors, along with other NNIs. We also utilize several truncation mutants of HCV NS5B that disrupt critical interactions at the interface of the thumb domain, β-loop and C-terminus  to invesUnless specified otherwise representative inhibitors for the four allosteric sites and active site nucleotide inhibitor used in this study  were synThe coding sequences of GT1b (con-1 strain) NS5B polymerase with various truncations at the carboxyl terminus were PCR amplified from a plasmid encoding the I389luc-ubi-neo/NS3-3′/ET replicon 600 of 1, the culture temperature was reduced from 37°C to 25°C and induction was immediately initiated by the addition of IPTG to a final concentration of 0.5 mM. After a three hour induction, cells were harvested by centrifugation and stored as frozen pellets at −80°C.BL21(DE3) bacteria were transformed with the NS5B polymerase expression vector pET21a-NS5B(1b) Δ21 C6His, and a 200 mL overnight LB culture was inoculated into a 20 L fermentation vessel , containing 18 L of fresh 2xYT medium supplemented with 100 µg/mL ampicillin. When cell densities reached an ODCell pellets were thawed and resuspended at 10 mL/g cells in Buffer A . The cell suspension was homogenized, filtered through cheesecloth, and passed three times through a microfluidizer  at 18,000 psi. The resulting lysate was centrifuged at 15,500 rpm for 45minutes and the lysate supernatant was loaded onto a Ni-HiTrap column  pre-equilibrated with five column volumes of the Buffer A. Proteins were eluted with a buffer containing 50 mM Tris-HCl, pH 7.6, 300 mM NaCl, 10% glycerol, 3 mM β-mercaptoethanol, and a 25–500 mM imidazole-HCl gradient. Fractions containing NS5B protein were collected, pooled, and dialyzed overnight at 4°C into 50 mM Tris-HCl, pH 7.0, 50 mM NaCl, 10% glycerol, 2 mM dithiothreitol. The sample was then diluted 1 4 with Buffer B  and loaded onto a SP cation exchange column  equilibrated in Buffer B. Recombinant NS5B protein was eluted with 0–1 M NaCl gradient in Buffer B. The enzyme was stored at −80°C in a buffer containing 50 mM Tris-HCl, pH 7.0, 600 mM NaCl, 10% glycerol, 2 mM dithiothreitol.Once the cell pellet was lysed the resulting cell lysate was then loaded onto a Ni HiTrap column  pre-equilibrated with Buffer A and eluted as described previously. To the pooled fractions (NS5B concentration of ∼0.5 mg/mL in 15 mL) 2 mL Sumo Protease was added to obtain a 1 10 (w/w) ratio of protease to NS5B and dialyzed overnight at 4°C against 4 L of 25 mM Tris-HCl, pH 7.8, 250 mM NaCl, 10% glycerol, 3 mM β-mercaptoethanol. The dialysate was centrifuged at 15,000 rpm for 20minutes and the soluble portion was decanted, loaded onto a Ni HiTrap column pre-equilibrated with 2 column volumes of Buffer A, the flow-through fraction was collected and concentrated to 1 mL prior to loading onto a size exclusion column  pre-equilibrated with 100 mL buffer 50 mM Tris-HCl, pH 7.0, 600 mM NaCl, 10% glycerol, 2 mM DTT. Eluted fractions containing NS5B Δ55 were pooled and concentrated to 4 mg/mL, flash-frozen and stored at −80°C.2+ to Mg2+. Active site side chains (particularly D225 and S282) were manipulated into conformations consistent with the Norwalk structure (3BSO) Poliovirus polymerase (3OL7) 33P-CTP into an RNA product using a heterologous RNA template. The assay was performed at multiple inhibitor concentrations to determine inhibitor concentration resulting in 50% inhibition of enzymatic activity (IC50). The compound was serially diluted 3-fold in 100% DMSO from a starting DMSO stock concentration of 4 mM, and then diluted 1 40 in the assay buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol. Finally, 4 µL of this dilution was added to a 96 well reaction plate containing 25 nM NS5B Con1 constructs and 4 ng/µL RNA template  and incubated for 60minutes at 30°C. The total reaction volume was 40 µL with 0.25% DMSO final concentration. The reaction samples were transferred to a 96 well white Whatman Unifilter 350 plate , washed three times with 125 mM sodium phosphate dibasic, once with water, once with 100% ethanol and then air-dried. 100 µL of Microscint-20 (PerkinElmer) scintillation fluid was added to each well and the plate was read on the Top Count scintillation reader (PerkinElmer). Data were further processed using the following protocol: raw data points were normalized by subtraction of the average background control and divided by the average DMSO control, yielding % Activity compared to DMSO. Duplicate measurements were averaged and the mean values and standard deviations were used in the fitting procedure. The IC50 was determined by applying the Sigmoidal Dose Response Model equation, fit  =  A+((B-A)/(1+(X/C)?D)), where A is the lowest % Activity, B is the highest % Activity, C is the IC50, D is a Hill slope and X is an inhibitor concentration. The 10 point dose response curves were generated in XLFit , using model 205.The HCV NS5B polymerase assay measures the incorporation of 2, 10 mM KCl, 1 mM EDTA, 1 mM TCEP, and 0.01% P-20. This was followed by immobilization of the NS5B constructs Δ21, Δ55, and Δ21-Δ8 in designated channels of the GLH chip. Prior to immobilization, constructs were diluted in 10 mM HEPES, pH 7.5, 150 mM NaCl. This was considered to be a more gentle buffer than the low pH acetate buffers containing no salt traditionally used to chemically immobilize proteins. Gentler immobilization conditions were chosen in an attempt to maximize the activity of the Δ55 and Δ21-Δ8 surfaces. Final immobilization levels ranged between 9000–11000 RU. Surfaces were deactivated by a 5-minute injection of ethanolamine. The L1 channel served as a negative control surface and was activated and deactivated with no protein immobilized. Compounds were prepared by first serially diluting in 100% DMSO. Samples were then transferred into running buffer containing no DMSO to yield the desired concentration of compound and a final DMSO concentration of 5% . A DMSO concentration series (ranging from 4.5% to 5.5% DMSO) was also injected so that a DMSO calibration curve to correct for excluded volume effects could be generated. Compounds were run at a flow rate of 100 µL/min and for a contact time of 90seconds. Dissociation times varied for different compounds and ranged between 1 and 90minutes. Data were analyzed using ProteOn software (BioRad). Sensorgrams were double-referenced and corrected for excluded volume effects and then fitted to a simple kinetic 1 1 binding model yielding the on- and off-rate constants ak and dk, the equilibrium dissociation constant KD, as well as the theoretical maximal response (Rmax).All SPR experiments were performed on a Biorad ProteOn XPR36 using GLH chips . With distilled water as running buffer, each chip was pre-conditioned using a 1-minute pulse each of 0.5% SDS, 50 mM NaOH, 100 mM HCl, and 10% DMSO in both the horizontal and vertical directions. Channels were activated with a 5-minute injection of EDC/sulfo-NHS in running buffer composed of 50 mM HEPES, pH 7.5, 5 mM MgClm – during thermal denaturation based on the change in fluorescence intensity of environmentally sensitive dye Sypro Orange  2, 1 mM EDTA, 1.5% DMSO. The assay was performed in the absence and the presence of compounds. Various constructs of NS5B at ∼5 µM concentration were preincubated with 1.5% DMSO or inhibitor at 25 µM for 5minutes at room temperature. To prevent bleaching 1 1000 diluted Sypro Orange fluorophore was added to the reaction mixture just prior to thermal denaturation. All concentrations were final after mixing. Thermal denaturation was carried out within 5–10minutes of dye addition by increasing the temperature from 25°C to 95°C with 0.017°C/second ramp. Fluorescence intensity data (excitation at 490 nm and emission at 575 nm) were collected at 0.07°C intervals and analyzed with Protein Thermal Shift Software (Life Technologies) using the first derivative approach to calculate Tm. For each compound delta Tm (ΔTm) was calculated by subtracting the Tm value obtained for protein alone (apo) from the Tm value obtained in the presence of compound (bound).Differential Scanning Fluorimetry (DSF) measures the midpoint of unfolding transition of protein – T2, 1 mM EDTA, 1 mM TCEP, 5% glycerol. The protein was then diluted in dialysate to a final concentration of 5 µM and compound  was added to a final concentration of 25 µM yielding 1.5% final concentration of DMSO. Matched dialysate buffer was used as a reference. The samples were scanned from 10°C–85°C at a rate of 60°C/hour using a filtering period of 8seconds and with 15minutes of pre-equilibration prior to the start of each run. Data were first normalized for protein concentration, then baseline corrected and buffer subtracted using Origin 7.0 AutoSampler DSC software . Melting transitions were irreversible and were analyzed with cursor initiated DSC peak fit function using the non-two- state unfolding model within the Origin 7.0 software to enable deconvolution of multiple transitions, determination of the relative change in enthalpy (ΔH) for the whole melting transition of each sample and for individual transitions, and the apparent thermal unfolding midpoint (Tm apparent) for each transition. ΔH was estimated from the area under the curve by integrating the change in the heat capacity with respect to the temperature . %ΔH for the deconvoluted transitions was calculated as the % of Area for each peak with respect to the total ΔH, or total Area, for the entire melting transition for each sample All DSC measurements were performed on a Microcal VP-Capillary DSC platform . Protein samples were dialyzed overnight at 4°C in a buffer composed of 50 mM Hepes, pH 7.0, 200 mM KCl, 1 mM MgClet al. To obtain additional insights into the conformational changes involved in the transition of NS5B from closed to open form, we built a model of elongating genotype 1b NS5B based on the primer-template bound structure published by Mosley This model is in good agreement with mutational data for thumb site II inhibitors, which mapped viral resistance mutations towards this class of compounds to the vicinity of the β-loop as well as to the binding 570), Δ39 (NS5B552), Δ47 (NS5B544) and Δ55 (NS5B536) C-terminal truncations; Δ21-AAA (NS5B570-LWF), in which hydrophobic residues of the C-terminal LWF motif that interact with the β-loop in the closed conformation were removed; and the β-loop deletion Δ21-Δ8 (NS5B562)  .The three thumb site II inhibitors effectively inhibited RNA elongation by Δ21 , and retIn contrast, the potency of control inhibitors 3′dCTP (a nucleotide NS5B chain terminator) and two thumb site I NNIs remained unchanged against all the constructs . The palD) for binding of the thumb site I inhibitor to Δ55 and Δ21-Δ8 construct relative to Δ21 was observed in agreement with potent inhibition of enzymatic activity of NS5B mutant proteins by this class of compounds. For the palm site I inhibitor, a large increase in KD for binding to Δ55 relative to Δ21 was observed, in agreement with the profoundly weaker inhibitory potency against this construct. A KD for palm site 1 inhibitor binding to Δ21-Δ8 could not be determined due to super-stoichiometric responses observed at the higher concentrations of compound that were required to detect signal on this surface. This indicates that the high affinity binding observed for this compound on the Δ21 surface has at best been reduced to very weak association. This data is also consistent with reduced inhibitory potency of the palm site I inhibitor against Δ21 and diminished activity for Δ21-Δ8.To determine if loss of inhibition was simply due to decreased affinity of the inhibitors to the mutant NS5B constructs, Surface Plasmon Resonance (SPR) measurements were conducted for Δ21, Δ55, and Δ21-Δ8 . No signBinding of the palm site II inhibitor to Δ55 was marked by significant deviations from simple 1 1 kinetics. The origin of this complexity is puzzling given the unequivocal fitting of sensorgrams collected for the thumb site II inhibitor filibuvir to a simple 1 1 model on all three surfaces. Rather than fit the binding data of the palm site II inhibitor to Δ55 and Δ21-Δ8 using a complex kinetic model, we note a qualitative differences in the sensorgrams obtained for those two protein constructs: namely, an obvious disappearance of a single slow dissociation component that is clearly present in sensorgrams collected for the Δ21 surface, suggesting a decrease in affinity of palm site II inhibitor towards Δ55 and Δ21-Δ8. Such a reduction in affinity is consistent with the 44- to 81-fold weaker inhibitory potency of the palm site II inhibitor against Δ55 and Δ21-Δ8, respectively, in comparison to Δ21. In contrast, thumb site II compounds show only a modest reduction in affinity for the Δ55 and Δ21-Δ8 constructs despite dramatic reductions in inhibitory potency. Thus, among the NNIs the thumb site II inhibitors display a unique profile: disruption of the interaction between the LWF motif and β-loop in the thumb domain by deletion or mutation has little to no impact on inhibitor binding, yet leads to significant loss of inhibitory potency. This indicates a critical role of both structural elements in communication between the inhibitor - occupied thumb site II and the NS5B catalytic site.m for each construct along with the change in Tm relative to apo Δ21 (ΔTm). With successive removal of key structural elements involved in the regulation of RdRp activity, a progressive decrease in the midpoint of thermal unfolding temperature relative to Δ21 is observed  relative to apo protein. Stabilization of a folded form of a protein by small molecule binding, and the attendant Tm upshift (ΔTm), derives from the contribution of the free energy of binding to the free energy of (un)folding according to a two-state unfolding model The impact of binding of thumb site II inhibitors on the mutant constructs was explored by determining the changes in Tm for all NS5B mutant constructs relative to the Tm for apo protein, showing that this class of inhibitors retains the ability to bind and stabilize all protein constructs. This is consistent with the SPR binding data for Δ21, Δ55, and Δ21-Δ8. Interestingly, the Tm upshift is greatest for the destabilized Δ55 and Δ21-Δ8 constructs, despite the lack of inhibition of their enzymatic activity by this inhibitor class. This indicates that binding and stabilization of the polymerase in the absence of the C-terminal tail or the β-hairpin is not sufficient to confer inhibition of RdRp activity by the thumb site II inhibitors.In the presence of the thumb site II inhibitors GS-9669, lomibuvir, and filibuvir there is an increase in Tm and relative Tm shifts that are measurable by DSF, DSC also allows for the deconvolution of multiple transitions of the protein as it goes from the folded to the unfolded state. Although NS5B undergoes an irreversible non-two-state melting transition, all DSC experiments were performed under identical conditions allowing for comparison of thermal unfolding profiles, relative stabilities and apparent Tm.Differential scanning calorimetry (DSC) was employed for a subset of constructs and construct – inhibitor pairs to understand the relationship between the inhibitor binding and protein thermal and conformational stability. Excellent agreement between melting temperatures determined by DSC and DSF for apo Δ21, Δ39 and Δ47 proteins was observed . In addim1) and a leading shoulder transition 2 with Tm2 around 2°C lower .The melting profile of apo Δ21 NS5B showed an asymmetric peak that can be described by two distinct transitions: a major transition 1  3′dCTP nucleotide inhibitor, (B) thumb site II inhibitor GS-9669, (C) thumb site I-A inhibitor and (D) palm site II inhibitor HCV-796. On average a 89-fold increase in IC50 is observed for GS-9669 and Δ55 in comparison to potency towards Δ21, while the active site inhibitor and thumb site I-A inhibitor are unaffected by removal of C-terminal residues  and the potency of palm site II inhibitor against Δ55 is decreased on average by 44-fold in comparison to Δ21.(TIF)Click here for additional data file.Figure S2SPR sensorgrams for binding of NNIs to Δ21, Δ55 & Δ21-Δ8 NS5B constructs. Kinetic traces obtained for respective NS5B constructs (in columns) binding to various allosteric inhibitors  are shown. (A) Thumb site I-B ; (B) Thumb site II - GS-9669 ; (C) Thumb site II - Lomibuvir ; (D) Thumb site II - Filibuvir ; (E) Palm site I inhibitor A-837093 ; (F) Palm site II - HCV-796 . All data sets with exception of data collected for palm site I inhibitor A-837093 on Δ21-Δ8 (E) and palm site II - HCV-796 on Δ55 and Δ21-Δ8 (F) were analyzed using a simple 1 1 kinetic binding model with ProteOn software (solid lines represent the best fit). Association (ka) and dissociation (kd) rate constants and equilibrium dissociation constant (KD) obtained for each fit are provided in Table S1 in (TIF)Click here for additional data file.Figure S3Unfolding profiles and thermal unfolding midpoints obtained from DSF and DSC techniques are comparable. (A) DSC thermal unfolding transitions obtained for apo Δ21, Δ39, and Δ47 NS5B truncation mutants . DSC unfolding profiles are similar to melting curves obtained by DSF (B) Correlation between Tm determined for apo Δ21, Δ39, and Δ47 by DSC and DSF.d by DSF . (B) Cor(TIF)Click here for additional data file.Figure S4ms for melting transitions of NS5B are independent on protein concentration.T DSC experiments were performed for 2.5, 5, and 7.5 µM of NS5B Δ21 . (A) Overlay of raw Cp data. (B) The best fit of data in (A) was obtained using a non-two-state unfolding model with two transitions: a major transition 1  and the leading shoulder transition 2 . For clarity only the overlay of individual first and second transitions fitted for three NS5B Δ21 concentrations is shown. (C) Average Tm of the first  and leading shoulder  transitions at each protein concentration (D) ΔH for the first (dark blue bar) and second leading shoulder (cyan bar) transitions and total ΔH of unfolding (yellow bar) are shown.(TIF)Click here for additional data file.Figure S5Unique unfolding transitions observed for NS5B in the presence of GS-9669 depend on the formation of protein-inhibitor complex. DSC experiments were performed for constant concentration of NS5B (5 µM) and increasing concentrations of GS-9669 . Traces in graphs are colored as follows: raw DSC Cp (solid black line); overall fit (solid red line); apo major transition with Tm1° (dashed blue line), apo second transition with Tm2° (solid cyan line); major transition for NS5B bound to GS-9669 with Tm1′ ; transition 3 which is visible only in the presence of inhibitor with Tm3′ (solid green line). (A) Unfolding profile for apo Δ21 shown with fit to non-two step unfolding model with two transitions; (B)–(D) Unfolding transitions for NS5B in the presence of increasing concentrations of GS-9669 . Disappearance of the major transition with Tm1° characteristic of apo Δ21 (dashed blue line) is concomitant with fractional increase of major transition for protein in complex with thumb site II inhibitor  and appearance of third transition with Tm3 (solid green line) visible only in the presence of thumb site II inhibitor; (E) Overlay of the major transitions (with Tm1) in the unfolding profiles of Δ21 in the absence  and presence of increasing concentrations  of thumb site II inhibitor. Both peaks are anti-correlated with dashed and solid lines indicating transitions for unbound and bound NS5B at each concentration, respectively. (F) ΔH of each transition in the melting profile plotted as % area under a peak, showing anti-correlative signature of the major transition for unbound NS5B  and bound in complex with GS-9669  and the appearance of third transition in the presence of thumb site II inhibitor (solid green bar). %ΔH for the second transition is also shown .(TIF)Click here for additional data file.File S1(DOC)Click here for additional data file.


One of the possible mechanisms by which resveratrol affects these disease states is by affecting the cellular signaling network involving protein kinase C (PKC). PKC is the family of serine/threonine kinases, whose activity is inhibited by resveratrol. To develop PKC isotype selective molecules on the resveratrol scaffold, several analogs (2–5) of resveratrol with a long aliphatic chain varying with number of unsaturated doubled bonds have been synthesized, their cytotoxic effects on CHO-K1 cells are measured and their effects on the membrane translocation properties of PKCα and PKCε have been determined. The analogs showed less cytotoxic effects on CHO-K1 cells. Analog 4 with three unsaturated double bonds in its aliphatic chain activated PKCα, but not PKCε. Analog 4 also activated ERK1/2, the downstream proteins in the PKC signaling pathway. Resveratrol analogs 2–5, however, did not show any inhibition of the phorbol ester-induced membrane translocation for either PKCα or PKCε. Molecular docking of 4 into the activator binding site of PKCα revealed that the resveratrol moiety formed hydrogen bonds with the activator binding residues and the aliphatic chain capped the activator binding loops making its surface hydrophobic to facilitate its interaction with the plasma membrane. The present study shows that subtle changes in the resveratrol structure can have profound impact on the translocation properties of PKCs. Therefore, resveratrol scaffold can be used to develop PKC selective modulators for regulating associated disease states.Resveratrol ( The activity of resveratrol has been linked to cell-surface receptors, membrane signaling pathways, intracellular signal-transduction machinery, nuclear receptors, gene transcription, and metabolic pathways PKC 2+ and diacylglycerol (DAG) for activation; novel isoforms  that require only DAG and atypical isoforms  that require neither Ca2+ nor DAG The PKC family has been divided into three main groups: conventional isoforms  that require Cain vitro purified proteins. Resveratrol regulates cellular PKCα and PKCδ to inhibit growth and induce apoptosis in gastric cancer cells The biological effects of resveratrol on PKCs have been studied both in the cellular system and in The mechanism of the effects of resveratrol on the activities of purified recombinant PKC isozymes induced by association with model lipid vesicle membranes was investigated using an in vitro assay system in which the cofactor and activator-concentration dependencies for activation were systematically varied In the present study, we describe the synthesis of several resveratrol derivatives having unsaturated aliphatic chain and their effects on the translocation properties of PKCα and PKCε in the presence and absence of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Our results show that chemical modification of one of the hydroxyl groups of resveratrol with aliphatic carbon chain reduced its cytotoxicity on CHO-K1 cells. Modification with a linolenyl chain completely abolished resveratrol's inhibitory effects on PKC. Instead, the molecule activated PKCα and the downstream protein ERK1/2 in the PKC signaling pathway.1) and its derivatives (2–5) in various organic solvents are listed in 2 in different solvent. Resveratrol (1) showed broad absorption maxima in the range of 304–318 nm with two humps at around 306 nm and 319 nm. This band did not show any significant wavelength shift when the solvent was switched from polar ethanol to nonpolar hexane. For the resveratrol derivatives 2–4, absorbance of the 319 nm band was slightly higher than the 306 nm band in ethanol and acetonitrile and the absorbance of the 306 nm band was higher than the 319 nm band in hexane and water. For compound 5, the 306 nm band was higher than the 319 nm bands in ethanol, acetonitrile and hexane, whereas in water it showed a 275 nm band.The absorption and emission maxima of resveratrol  for 24 h. Results indicated that PKCα expression was significantly reduced (∼60%) when the cells were treated with 1 as compared to the untreated cells  at 25–100 µM for 1 h with either PKCα or PKCε did not show any PKC membrane translocation. Under the similar condition however, 100 nM TPA caused almost complete membrane translocation of both PKCα and PKCε  and TPA (100 nM) for 1 h, there was ∼45% and ∼20% reductions in the TPA-induced membrane translocation of PKCα and PKCε, respectively , activated PKCα. This property of 4 is quite different from resveratrol which did not activate but inhibited the stimulated activity of PKCα. All the molecules were inert towards PKCε both in the activation and inhibition processes. For resveratrol, we detected 20% inhibition for PKCε and 45% inhibition for PKCα. While our data is consistent with the inhibition data reported earlier for PKCα in PC-3 cells, our PKCε inhibition data is not in agreement with earlier study in PC-3 cell in which the authors reported no inhibition of PKCε. Contrasting results were however reported with purified protein. While Stewart et al We have thoroughly investigated the activity of PKCα and PKCε by measuring their translocation to the plasma membrane in response to the synthetic derivatives. We observed increase in PKCα in the membrane fraction while the amount in the cytosolic fraction remained similar to the control  inhibited TPA-induced PKC activation and competed with phorbol ester but not with the calcium, led Slater et al 4 in the activation of PKCα suggests that 4 could bind to its C1 domain. The energy minimized structure of 4 shown in 4 for PKCα and PKCε. These differences in the residues in the C1B domains are also responsible for the difference in the binding affinity for phorbol ester, phorbol 12, 13-dibutyrate (PDBu) and DAG. For example, αC1B showed lower binding affinity for both phorbol ester and DAG than εC1B The observation that resveratrol  That 4 shows reduced cytotoxicity, selectivity towards PKCα and also activated the downstream ERK1/2 either by the Ras →Raf →MEK1/2 →ERK1/2 or the Raf →MEK1/2 →ERK1/2 pathways, this compound can be used as a potential drug for disease states involving PKCα, such as cardiac contractility, atherogenesis, cancer and arterial thrombosis That compound In summary, our results demonstrated that resveratrol moiety can be modified suitably as PKC selective modulators. Development of newer synthetic molecules around the resveratrol scaffold and studying their mechanism for isotype selectivity is warranted before any analog can be used as a drug candidate for a particular disease state.1H NMR and 13C NMR spectra were recorded on a QE-300 spectrometer. Unless otherwise specified, all NMR spectra were obtained in deuterated chloroform (CDCl3) and referenced to the residual solvent peak. Chemical shifts are reported in parts per million, and coupling constants in hertz (Hz). Multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), m (multiplet) and br (broadened). Mass spectra were obtained on either a VG 70-S Nier Johnson or JEOL mass spectrometer.Resveratrol, TPA and all other reagents were purchased from Sigma and used without further purification. Progress of chemical reaction was monitored through thin layer chromatography (TLC) on pre-coated glass plates  purchased from EMD chemicals. 2–4 is outlined in 1′ was prepared following our previously published methods 2–4, compound 1′ was dissolved in anhydrous pyridine at 0°C and treated with the corresponding acid chloride , which was prepared by the treatment of the corresponding acid  in anhydrous dichloromethane with thionyl chloride  and catalytic amount of DMF. Acid chloride was distilled under vacuum and used immediately for the next step. The reaction mixture was allowed to stir for 1 h at room temperature and then heated at 60 C for another 2 h. After cooling the mixture to room temperature, excess pyridine was removed in high vacuum. The compound was immediately used for the demethylation step. Demethylation was done by following the methods described earlier The synthetic procedure of 2): Yield: 77%, 1H NMR  δ 7.47 , 7.06 , 7.00 , 6.88 , 6.55 , 6.26 , 5.33 , 4.91 , 2.55 , 2.01 , 1.75 , 1.42–1.23 , 0.86 ; 13C NMR (CDCl3) δ 172.6, 157.0 (2C), 150.01, 139.9, 130.0, 129.8, 128.6 (2C), 128.2, 127.5, 121.9, 121.0 (2C), 106.2 (2C), 102.2, 34.2, 32.0, 29.7, 29.6 (2C), 29.4, 29.1 (2C), 28.9 (2C), 28.8 (2C), 26.8, 26.7, 13.1. ES-MS: 516 [M+Na].4-((E)-3,5-dihydroxystyryl)phenyl oleate (3): Yield: 70%, 1H NMR (CDCl3) δ 7.46 , 7.06 , 7.00 , 6.88 , 6.51 , 6.25 , 5.36 , 5.07 , 2.76 , 2.55 , 2.04 , 1.75 , 1.45–1.23 , 0.88 ; 13C NMR (CDCl3) δ 172.69, 157.06 (2C), 150.3, 139.9, 134.4, 130.3, 130.1, 128.5, 128.2 (2C), 128.1, 127.6 (2C), 121.9 (2C), 106.2 (2C), 102.4, 34.2, 31.8, 29.6, 29.4, 29.2 (2C), 29.1, 27.2 (2C), 25.7, 24.8, 22.2, 14.1; ES-MS: 514 [M+Na].-4-((E)-3,5-dihydroxystyryl)phenyl octadeca-9,12-dienoate (4): Yield: 68%, 1H NMR (CDCl3) δ 7.47 , 7.06 , 6.98 , 6.90 , 6.54 , 6.26 , 5.36 , 4.95 , 2.80 , 2.55 , 2.01 , 1.75 , 1.42–1.34 , 0.96 ; 13C NMR (CDCl3) δ 172.4, 157.04 (2C), 150.37, 140.0, 134.5, 132.0, 130.3, 128.6, 128.3, 128.3, 128.2, 127.8 (2C), 127.5, 121.9 (2C), 106.2 (2C), 102.3, 34.5, 29.6, 29.2 (2C), 29.1, 27.2, 25.7 (2C), 25.6, 21.0, 14.3; ES-MS: 512 [M+Na].-4-((E)-3,5-dihydroxystyryl)phenyl octadeca-9,12,15-trienoate (5) was synthesized and characterized as described earlier The saturated analog (E)-5-(4-(hexadecyloxy)styryl)benzene-1,3-diol (1) and its derivatives (2–5) (1–10 µM) were recorded in different solvents at room temperature. Spectral maxima were determined from the fit of Gaussian function .The UV-Vis absorption  and fluorescence emission spectra  of resveratrol  and F12 medium supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS) and 100 unit/ml antibiotics. Before treatment with compounds, cells were starved in without FBS media for 12 h at 60–70% confluency.4 cells per well. Cells were either treated with DMSO (1%), resveratrol or derivatives (1–100 µM) for 48 h. Cell viability was determined using Vybrant® MTT cell proliferation assay kit  as per the manufacturer's recommendations.The cells were plated overnight in a 96-well plate  at a density of 10Compound-treated cells were washed and harvested in PBS. Cell lysis was carried out in lysis buffer  with brief sonication . Cell debris was removed by centrifuging the sample at 3500 rpm for 10 minute at 4°C. Protein concentration was measured using the BCA protein assay kit . Cell lysate (25 µg protein/lane) was subjected to SDS-PAGE and immunoblot to detect protein expression. Cell lysate (200 µg protein/100 µl) was centrifuged at 40,000 rpm for 2 h at 4°C to separate out soluble (cytosolic) and pellet (membrane) fraction. Pellet fraction was incubated in lysis buffer (100 µl) containing 1% Triton X-100 for 1 h in ice, centrifuged at 40,000 rpm for 1 h and the supernatant was collected as the membrane fraction. The cytosolic and membrane fractions (30 µl) were subjected to SDS-PAGE (7%) and transferred to nitrocellulose membrane. Membranes were blocked with TBST  buffer containing 10 mg/ml BSA and then washed three times with the TBST buffer. Membranes were probed with primary antibody for overnight at 4°C and HRP-conjugated secondary antibody at room temperature for 1 h. Antibody dilutions were used as follows: anti-rabbit PKCα, 1∶500; anti-rabbit PKCε, 1∶500; anti-rabbit ERK, 1∶1000; anti-rabbit phopho-ERK, 1∶500; anti-rabbit β-actin, 1∶2000 and anti-rabbit HRP-conjugated, 1∶5000 . The blots were stripped and probed with β-actin and secondary antibody to check for equal loading. Protein bands were visualized using ECL (enhanced chemiluminescence) reagent  and analyzed by AlphaImager® Gel Documentation system .Activation of ERK1/2 was determined by treating the CHO-K1 cells either with 10 µM of the resveratrol derivatives for 24 h or 100 nM TPA for 1 h. ERK1/2 phosphorylation was measured by whole cell lysate immuno-blot analysis using phosphor-ERK1/2 specific antibody.4 was energy minimized using Chem3D pro 12.0.2 (Cambridgesoft) with 1000 iterations. Homology model for PKCεC1B and docking studies were performed using the methods described earlier The chemical structure of


Their function is inhibited by LIMK-mediated phosphorylation at Ser-3. Previous in vivo cross-linking with different probes, a cofilin oligomer (65 kDa) could be detected in platelets and endothelial cells. The cofilin oligomer did not contain actin. Notably, ADF that only depolymerizes F-actin was present mainly in monomeric form. Furthermore, we found that formation of the cofilin oligomer is regulated by Ser-3 cofilin phosphorylation. Cofilin but not phosphorylated cofilin was present in the endogenous cofilin oligomer. In vitro, formation of cofilin oligomers was drastically reduced after phosphorylation by LIMK2. In endothelial cells, LIMK-mediated cofilin phosphorylation after thrombin-stimulation of EGFP- or DsRed2-tagged cofilin transfected cells reduced cofilin aggregate formation, whereas inhibition of cofilin phosphorylation after Rho-kinase inhibitor (Y27632) treatment of endothelial cells promoted formation of cofilin aggregates. In platelets, cofilin dephosphorylation after thrombin-stimulation and Y27632 treatment led to an increased formation of the cofilin oligomer.We found that after in vivo.Based on our results, we propose that an equilibrium exists between the monomeric and oligomeric forms of cofilin in intact cells that is regulated by cofilin phosphorylation. Cofilin phosphorylation at Ser-3 may induce conformational changes on the protein-protein interacting surface of the cofilin oligomer, thereby preventing and/or disrupting cofilin oligomer formation. Cofilin oligomerization might explain the dual action of cofilin on actin dynamics  The actin depolymerization factor ADF/cofilin family of proteins (15–19 kDa) is expressed in all eukaryotic cells and is essential for enhancing actin filament turnover. These proteins weakly sever actin filaments without capping their ends, thereby increasing the number of free filament ends where polymerization and depolymerization occur. They also enhance the rate of monomer dissociation from the pointed ends Actin dynamics regulate cell functions. For example, actin cytoskeleton rearrangement is essential for platelet shape change, secretion, and aggregation, and it underlies endothelial cell contraction, migration, and proliferation Besides actin filament depolymerization, cofilin can also accelerate spontaneous assembly of actin monomers The function of cofilin is regulated by LIM-kinase mediated phosphorylation at Ser-3 in vitro mediated by disulphide bonds, and that in contrast to the monomer, cofilin oligomers are unable to depolymerize actin filaments but rather induce actin polymerization and filament bundling in vitro analysis has shown that the function of cofilin depends on the concentration of cofilin: low cofilin concentrations favor actin filament severing, whereas high cofilin concentrations favor actin polymerization A previous study showed that cofilin possesses an intrinsic tendency to form oligomers in vivo and, if so, whether it is regulated by cell stimulation. We also pondered the possible role of LIMK-mediated cofilin phosphorylation and phosphatase-mediated cofilin dephosphorylation in the regulation of cofilin oligomerization. Our results indicate that cofilin exists as both a monomer and an oligomer in endothelial cells and platelets. Rho-kinase/LIMK-mediated phosphorylation of cofilin at Ser-3 inhibited cofilin oligomerization in vitro and in vivo, whereas cofilin dephosphorylation enhanced cofilin oligomerization in vivo.In the present study, we addressed the question of whether cofilin oligomer formation occurs in vivo, we used membrane-permeable homobifunctional, maleimide cross-linkers of different lengths (BMOE and BMH) for conjugation between sulfhydryl groups (-SH). BMOE and BMH probes have spacer arms of 8Åand 13Å in length, respectively. Human cofilin has four cysteine residues at positions 39, 80, 139, and 147. By using these probes, we optimized the in vivo cross-linking experiments by varying the concentration of cross-linkers and the incubation times. We found that cofilin exists as a monomer and an oligomer in endothelial cells and platelets  of cofilin. The (His)6-tagged cofilin oligomers showed different molecular masses  were subjected to mass spectrometry. Beside of actin we could identify 14-3-3ζ (27.7 kDa) in the cofilin immunoprecipitates. It is however, unlikely that these proteins are part of the 65 kDa cofilin oligomer: 14-3-3ζ is known to interact only with phospho-cofilin in vivo cross-linked cell lysates of platelets and endothelial cells with a specific anti-phospho-cofilin antibody. We could not detect phospho-cofilin in the cofilin oligomer; cofilin phosphorylation was detected only in the cofilin monomer  was drastically reduced after phosphorylation  or BMOE cross-linking, and performed immunoblotting experiments with anti-EGFP and anti-cofilin antibodies. A band of ∼100 kDa could be identified by both anti-EGFP and anti-cofilin antibodies .We conclude that LIMK-mediated cofilin phosphorylation after thrombin stimulation of endothelial cells inhibits and dissolves cofilin aggregate formations. In contrast, inhibition of LIMK-mediated cofilin phosphorylation promotes cofilin aggregate formation in endothelial cells. The regulation of cofilin aggregate formation by phosphorylation is a reversible process.p<0.05)  . Pretreap<0.05) , and a fp<0.05) . These oin vivo and also in vitro, whereas cofilin dephosphorylation enhanced the cofilin oligomer formation.In the present study, we have found that cofilin exists as a monomer and 65 kDa oligomer in endothelial cells and platelets. LIMK-mediated phosphorylation of cofilin at Ser-3 inhibited the formation of the cofilin oligomer In vitro chemical cross-linking has been widely used to study protein-protein interactions. In contrast, far fewer studies have been performed to study protein-protein interactions with membrane permeable cross-linkers in vivoin vivo cross-linking studies, we found that in vitro cross-linked recombinant cofilin exists as dimer, tetramer, and higher molecular weight oligomers. These findings are in agreement with the results of a previous study in vivo, oligomerization of endogenous cofilin is highly regulated to perform specific cell functions. Cofilin dimer formation in vivo might be a transient stage with a very short half-life that forms stable cofilin tetramers. The cofilin tetramer might be the functional unit of the cofilin oligomer.An interesting question is, why a cofilin dimer was not detectable in intact cells. We could, however, obtain some evidence for the formation of a cofilin-EGFP dimer in transfected endothelial cells . A possiin vivo, a minimum of two cysteine residues must be present as a free sulfhydryl, and the distance between the free cysteine residues should not be greater than 8 Å. Our data suggests that at least two cysteine residues are present on the protein-protein interacting surface of the cofilin oligomer in vivo. Previously, it has been shown that cofilin oligomerizes in vitro through intermolecular disulphide bonds that involved Cys39 and Cys147 To cross-link the cofilin oligomer by the BMOE probe in vivo without stabilizing the oligomers with chemical cross-linkers. However, it has been clearly shown by us and others that in vitro cofilin can form dimers, and high MW oligomers in vitro is stabilized by S-S bonds in vitro results, S-S bonds are not involved in cofilin oligomer formation in vivo: No cofilin oligomers are detected after cell lysis and protein separation under non-reducing conditions.It was not possible for us to observe cofilin oligomers in vitro and in vivo, respectively. This is in contrast to a previous study showing that oxidation of cofilin induced the formation of an intramolecular disulfide bond and an inability of cofilin to be phosphorylated at Ser-3 We found that monomeric cofilin (endogenous and recombinant His-tagged) had a higher electrophoretic mobility using SDS-PAGE under non-reducing conditions, as compared to reducing conditions, similar to other disulphide bond containing proteins reported previously As already mentioned, human ADF and cofilin both show 72% sequence homology. Interestingly, ADF is a more potent actin-depolymerizing protein than cofilin in vitro; and c) cofilin phosphorylation regulated the formation of cofilin aggregates in endothelial cells and cofilin oligomers in platelets in vivo. Cofilin modeling data (not shown) indicate that cofilin phosphorylation at Ser-3 induces conformational changes in the protein-protein interacting surface of the cofilin oligomer, thereby preventing and/or and disrupting the cofilin oligomer formation.The reversible phosphorylation of Ser-3 residue by LIMKs is a well-established regulatory mechanism of cofilin function. Based on our results, we propose that an equilibrium between cofilin monomers and cofilin oligomers exists in cells, which is regulated by phosphorylation at Ser-3. We found that a) only cofilin but not phosphorylated cofilin was present in the endogenous cofilin oligomer; b) phosphorylation of recombinant His-tagged cofilin by GST-LIMK2 inhibited the formation of BMOE-cross-linked cofilin oligomers ∼100 kDa could be identified by both anti-EGFP and anti-cofilin antibodies . Cofilin binds F-actin cooperatively and could, at the concentrations where cross-linking occurs, bind and saturate small regions of F-actin leading to the 65 kDa cofilin tetramer. However, if this is the case, actin should also be cross-linked with the cofilin oligomer, which is not the case (see above). Moreover, the distance between cofilin molecules on actin fibers is much higher than the 8Å and 2.3–2.7 Å distances required for protein cross-linking by BMOE and formaldehyde, respectively, suggesting that the 65 kDa tetramer formation on F-actin is not possible Apparently the situation Besides actin, other proteins such as Memo (33 kDa), Aip1 (65 kDa), Cap1 (51.67 kDa) and Nck1 (42.86 kDa) were reported to directly interact with cofilin or present in the complex of actin and cofilin To further support our findings in endothelial cells, we studied platelets, where we quantified the formation of the 65 kDa cofilin oligomer. We have shown in an earlier study that cofilin is maximally dephosphorylated 30 sec after thrombin stimulation of platelets in vitro increase actin polymerization Based on our results and previous studies, we propose the following model of regulation of cofilin function . Cells cEGFP or DsRed2) at the 3′ end of the cofilin gene, the full-length coding sequence of cofilin was amplified by PCR from a cDNA pool of human umbilical vein endothelial cells (HUVEC) and then cloned into the XhoI and BamHI sites of the pEGFP-N1, pDsRed2-N1 vector (Clontech Inc). A phospho-mimetic mutant (cofilin-S3D-EGFP) was prepared by using Quick-Change II site–directed mutagenesis Kit  as per manufacturer’s instructions. To prepare FLAG-tagged cofilin plasmid, EGFP gene was deleted and FLAG tag (DYKDDDDK) coding oligonucleotide was inserted in cofilin-EGFP plasmid by using Quick-Change II site-directed mutagenesis kit. To prepare His-tagged cofilin (cofilin-(His)6) construct, cDNA of cofilin was cloned into the BamH1 and NcoI sites of the pQE60 vector (Qiagen GmbH).To attach the reporter gene .HUVECs were obtained and cultured as described previously 6 cells in 20 µl of complete medium. Cross-linker  or solvent (DMSO) was added to the cells and then incubated at 37 °C for one hour in a CO2 cell incubator. The cross-linking reaction was stopped by adding 2× Laemmli buffer and subjected to immunoblotting.Confluent endothelial cells were treated with trypsin and EDTA to prepare a cell suspension. The cells were pelleted by centrifugation, resuspended in 0.5 ml of complete endothelial growth medium, and incubated on ice for 30 minutes. Endothelial cells were distributed in sterile polypropylene tubes at a density of approx. 1.0×106) were centrifuged at 100 x g for 5 minutes. Cell pellet was resuspended in 4.5 ml complete endothelial growth medium. Formaldehyde was used as cross-linking agent based on a previous study HUVECs in suspension  were stimulated with thrombin (Sigma) in the presence or absence of the Rho-kinase inhibitor Y27632 . RGDS  was added prior to thrombin-stimulation in order to avoid platelet aggregation. Aliquots (100 µl) of unstimulated and stimulated platelet suspensions were lysed in an equal volume of ice cold 2x lysis buffer  with or without the chemical cross-linker BMOE or BMH (0.2 mM) for 20 minutes on ice. The lysates were then transferred to an equal volume of 2x SDS-PAGE sample buffer for immunoblotting.Blood was taken from human volunteers. Approval was obtained from the Ethic Commission of the Medical Faculty of the University of Munich. Informed consent was provided according to the Declaration of Helsinki. Platelets from acetylsalicylic acid–treated human blood were isolated as described previously Western blotting was done as described previously Densitometric analysis of the proteins was performed using the public domain NIH ImageJ (version 1.32j) software. The films were scanned into TIF format using a ScanJet 5300C (Hewlett-Packard). The optical density of proteins in unstimulated control samples was set to 100%. Data are presented as mean ±S.E. of three independent experiments.In later stages of the study we used Odyssey Infrared Imager for scanning the membranes. Briefly, after primary antibody incubation, the secondary goat-anti-rabbit 800 antibody and goat-anti-mouse 680 antibody  were used with a dilution of 1:10,000. Protein signal was detected using the Odyssey Infrared Imager with application Software 3.0.30 (LI-COR Biosciences).E.coli cells (Qiagen) and grown at 37 °C. The expression of recombinant cofilin was induced by adding 1 mM isopropyl ß-d-thiogalactoside at an A600 of ∼0.6 for three hours. Bacteria were harvested by centrifugation and resuspended in 10 ml of ice-cold lysis buffer (PBS buffer containing complete mini protease inhibitors (Roche Diagnostics), 10 mM imidazole, lysozyme (1 µg/ml); pH 8.0) and incubated on ice for 30 minutes. The bacterial cells were lysed by sonication, and then the lysate was incubated with DNAse I (5 µg/ml) on ice for 15 minutes. Cell debris was removed by centrifugation at 60,000xg, and the supernatant was loaded on Ni-NTA Superflow column (GE lifesciences) pre-equilibrated with lysis buffer. The column was then washed thoroughly with lysis buffer and then bound His-tagged cofilin was eluted with elution buffer . Eluted fractions containing high amounts of purified His-tagged proteins (>97%) were selected, pooled, desalted, and concentrated using Centricon® Plus-20 (Millipore).The pQE-60-cofilin plasmid was transformed in M15 in vitro cross-linking with BMOE.GST beads (GE Life sciences) were washed twice with kinase buffer  and then used as 50% slurry to couple LIMK2. GST-LIMK2  was incubated with 30 µl of a 50% GST-bead slurry in a volume of 100 µl kinase buffer at 30°C for one hour. LIMK2 bound GST beads were pelleted at 13,000 rpm for 15 sec, washed once with kinase buffer, and resuspended in 100 µl kinase buffer containing ATP (10 mM). His-tagged cofilin  were added in kinase buffer with GST-LIMK2, and the reaction mixture was incubated for 1 hour at 37 °C. The GST-LIMK2 beads were pelleted by centrifugation at 13,000 rpm for 15 sec, and the supernatant containing phosphorylated His-tagged cofilin was transferred to a new tube and subjected to Different concentrations of the purified recombinant human His-tagged cofilin and phosphorylated His-tagged cofilin or equal concentrations of actin (Cytoskeleton Inc.) and human cofilin (without His-tag) were taken in reaction buffer  with a final volume of 50 µl. Cross-linker  or solvent (DMSO) were added in reaction buffer and incubated one hour at room temperature. The reaction was stopped by adding 2x Laemmli buffer and subjected to SDS-PAGE.2, 0.1 mM DTT, and 0.2 mM phenylmethylsulfonyl fluoride) to a final GuHCI concentration of 1 M. Cell debris were removed by centrifugation at 14,000xg for 15 min at 4 °C, and then 150 µl of 50% protein A–Sepharose slurry was added to the supernatants and incubated for 1 hour at 4 °C to preclear the supernatant. Protein A–Sepharose was prepared by incubating the beads in swelling buffer (20 mM Hepes (pH 7.5), 0.15 M NaCl, and 0.1% NaN3) containing 2% BSA to block unspecific binding. Precleared supernatants were incubated overnight either with 80 µl of anti-FLAG-M2 gel-slurry  or anti-cofilin antibody (1∶100 dilution) (platelet lysates). The next day, 80 µl of 50% protein A–Sepharose slurry was added to the supernatant containing the anti-cofilin antibody followed by incubation at 4 °C for two hours. The beads were pelleted by centrifugation and washed twice with IP buffer containing 1 M GuHCI and 0.005% nonidet-40, followed by a wash with IP buffer lacking GuHCI. Finally, beads were resuspended in 100 µl 1x Laemmli buffer, boiled for 5 minutes at 95 °C, and subjected to SDS-PAGE and western blotting.Immunoprecipitation of cross-linked cofilin oligomers and cofilin-protein complexes was performed by using the method for immunoprecipitation of cross-linked proteins described previously The cell pellets after formaldehyde cross-linking were resuspended in 200 µl RIPA buffer  for one hour at 4°C. Cell lysates were homogenized with 22-gauge needle 30 minutes after resuspension in RIPA buffer. Cell lysates were then centrifuged at 16,000 x g for 30 minutes at 4°C, and the supernatant was used for further analysis. For cofilin immunoprecipitation, anti-cofilin antibody (1 100 dilution) was added to the supernatant and incubated overnight at 4 °C. Protein A magnetic beads  were added and incubated for one hour. The beads were magnetically removed, washed thrice with 500 µl IP wash buffer , and then resuspended in 50 µl PBS. For control, protein A magnetic beads (25 µl) was added to the supernatant, and it was proceeded as described above. Control and cofilin antibody bounds proteins were subjected to mass spectroscopy.Beads with control and cofilin antibody bound proteins were washed three times with 100 µl ammonium bicarbonate (20 mM) before proteolytic digestion with trypsin (300 ng) at room temperature under rotation for 16 h. The reaction was stopped with a final concentration of 1% acetic acid and the peptide mixtures were desalted on C-18 reverse phase material  and further processed as described earlier Proteins were identified via automated database search in a forward-reverse Uniprot database (rel. 01/2012) limited to human entries using the Sequest algorithm rel. 2.7 . Parent mass tolerance (MS) was set to 10 ppm and fragment mass tolerance 1 Da. Methionine oxidation was considered as optional modification. Proteins and peptides were considered as significantly identified when probability was >95%, respectively .6 cells using the HUVEC nucleofactor kit from Amaxa GmbH.In all experiments, HUVECs were transfected with 2 µg DNA per 1×10After 24h of transfection, cells were observed with a Nikon TE2000E-PFS fluorescence microscope with 37°C incubation chamber. The microscope was controlled through NIS-Elements software. The measurements were carried out in three independent experiments with 50 cells randomly selected in each experiment. The mean ± S.E. was calculated for each experiment.The experiments shown are representative of at least three others, which gave similar results. Values given are mean ± SEM or mean ± SD as indicated in the Figure legends. Significant difference was determined by the paired Student’s t-test or other tests as appropriate. A p-value of <0.05 was considered statistically significant.Figure S1Protein sequence alignment of human non-muscle cofilin (CFL1-Human) and human ADF (ADF-Human). Cysteine residues are highlighted in yellow. The positions of cysteine in human cofilin are indicated.(RTF)Click here for additional data file.Figure S2ADF does not form a distinct 65 kDa oligomer after cross-linking of endothelial cells. Endothelial cells (0.8–1×106 cells/20 µl) were incubated with DMSO (1 µl) or BMOE (1 mM). For formaldehyde cross-linking, endothelial cells (1×106 cells/ml) were treated with formaldehyde at a final concentration (1%). The cell lysates were subjected to SDS-PAGE on gradient gel (4–15%) and were then immunoblotted with an anti-ADF antibody. A smear of ADF cross-linked proteins was observed for both BMOE and formaldehyde cross-linked endothelial cells. Proteins were detected by fluorescence imaging of secondary antibodies labeled with infrared dyes.(PDF)Click here for additional data file.Figure S3Immunoblotting of cofilin-EGFP and cofilin-S3D-EGFPtransfected and cross-linked endothelial cells. Endothelial cells were transfected with EGFP, cofilin-EGFP or cofilin-S3D-EGFP plasmid. After 20 hours of transfection, cells (0.8–1×106 cells/20 µl) were treated with DMSO (1 µl) or BMOE at a final concentration of 1 mM. The cell lysates were subjected to SDS-PAGE on a gradient gel (4–15%) and were then immunoblotted with anti-cofilin or anti-EGFP antibody. A band of ∼100 kDa was apparent in both anti-EGFP and anti-cofilin immunoblots (arrow) of lysates of cofilin-EGFP and cofilin-S3D-EGFP transfected cells after BMOE cross-linking.(PDF)Click here for additional data file.Movie S1Live cell imaging of human endothelial cells transfected with DsRed2-cofilin. Endothelial cells transfected with cofilin-DsRed2 were stimulated with thrombin (1 U/ml) and then observed under the Nikon TE2000E PFS microscope with 63x magnification at 37°C. Pictures were taken every 30 seconds for 30 minutes using NIS elements software. Movie was edited with QuickTime Pro from Apple Inc.(MOV)Click here for additional data file.