A ninth challenge is accurate verification of biomarkers as therapy final result predictors calls for randomized phase III clinical trials during which the biomarker predicts progression-free survival or general survival during the therapy arm but not the management arm, a attribute in only two in the 22 trials in Tables one? Otherwise, in case the biomarker predicts survival in each groups, it is actually a prognostic marker, not a treatment-specific biomarker. Demonstration of the predictive biomarker calls for a statistical check of interaction in between the therapy plus the biomarker, which traditionally increases the sample size properly beyond that expected to demonstrate clinical efficacy on the drug, that means that definitive research verifying biomarkers of anti-angiogenic treatment is going to be a expensive endeavor.
Sample dimension is important even in non-randomized trials through which biomarkers are investigated, as evidenced smoothened antagonist by a preliminary report on the initial 16 glioblastoma patients handled with RTKI cediranib within a phase II trial, in which basic fibroblast growth issue elevated when tumors escaped treatment method , whilst during the total 31 patient cohort reported later, bFGF did not predict progression . Despite these issues, a few candidate biomarkers have emerged for anti-angiogenic treatment, which we summarize under. Biomarkers predicting response to anti-angiogenic treatment . Variety I?physiologic Hypertension can be a physiologic side impact of anti-angiogenic drugs, taking place, as an example, in 6% of bevacizumab-treated sufferers . VEGF lowers blood strain by activating protein kinase B, which phosphorylates endothelial nitric oxide synthase , cutting down plasminogen activator inhibitor-1 expression . Consequently, anti-VEGF treatments maximize PAI-1 activity, which promotes hypertension.
Angiotensin converting enzyme inhibitors have been proposed to deal with hypertension induced by anti-angiogenic treatment considering that they inhibit PAI-1 expression . Though some studies have discovered that drug-induced hypertension increases OS just after RTKI or bevacizumab treatment method , other research identified no correlation . Even further perform ought to define if a background of hypertension has an effect on its predictive this content worth; the cutoff worth for blood pressure rise past which hypertension is predictive; plus the value of speed of hypertension onset and duration in its predictive value. Validating hypertension as being a biomarker of anti-angiogenic treatment efficacy is essential due to the fact producers may well endorse discontinuing drug just after treatment-associated hypertension and many trials exclude individuals with pre-existing hypertension .
As a result, right up until the predictive worth of hypertension as a biomarker is established or until finally newer medicines without having this side impact are produced, individuals might possibly be denied therapy that, if hypertension is really a biomarker of anti-angiogenic therapy efficacy, is most likely to get useful, dependant on a treatable side impact.
Monthly Archives: June 2013
Alternatively, methylation of intermediate followed by deprotecti
Alternatively, methylation of intermediate followed by deprotection of the phenylsulfonamide group gave the N methylpyrazole derivative . Furthermore, we prepared the truncated versions of GSK in which either the arylamine group or even the phenylurea group was removed. Compound was ready as X see front matter Elsevier Ltd. All rights reserved. doi j.bmcl . Corresponding writer. Tel . E mail deal with: jesus.r.medina gsk.com . Bioorganic Medicinal Chemistry Letters Contents lists obtainable at ScienceDirect Bioorganic Medicinal Chemistry Letters journal homepage: www.elsevier.com find bmcl illustrated in Scheme . Suzuki coupling of N ethylpyrazolyl boronic ester with bromoazaindole followed by reduction in the nitro group gave the corresponding intermediate . Reaction of aniline with dimethylcarbamoyl chloride afforded compound . The syntheses of compound and its respective isomer had been initiated by sequential Suzuki couplings of azaindole with all the suitable boronic acid or ester followed by N methylpyrazolyl boronic ester.
Reductive amination of intermediate a followed by phenylsulfonamide deprotection afforded . Compound was obtained from your direct deprotection of b . Compounds and showed rather weak activity while in the Aurora B enzyme assay but fantastic binding efficiency , which defined selleck chemical custom peptide synthesis the baseline amount of action of your azaindole pyrazole template . Compound , which represents the truncated edition of GSK lacking the arylamine group, exhibited a fold expand in enzyme potency relative towards the pyrazoloazaindoles and , but that has a substantial reduce in binding efficiency . Furthermore, the enzyme potency didn’t correspond using a very similar degree of exercise during the cell proliferation assay. Interestingly, compounds and , which lack the phenylurea but contained an arylamine group, the two exhibited beneficial activity while in the Aurora B enzyme assay , superior binding efficiency , and wonderful exercise inside the A proliferation assay .
Compound does display oral publicity and bioavailability in rats, even though it has large clearance . To investigate the mechanism with the antiproliferative activity, we evaluated A lung tumor cell lines treated with and by using fluorescence activated cell sorting . Offered that compounds had diverse potencies towards tumor Rosiglitazone cell growth , in an effort to evaluate them inside the FACS evaluation they have been all tested at their EC values inside the cell proliferation assay. A lung tumor cell lines taken care of with showed an increase in sub N DNA and tiny or no enhance in N N DNA in contrast for the DMSO control . Considering inhibition of Aurora B really should result in a rise in N N DNA this end result strongly recommended that exerts its antiproliferative results by a non Aurora B mechanism of action.
Asymmetric reduction implementing catalytic Rh DuPhos underneath
Asymmetric reduction working with catalytic Rh DuPhos underneath psi hydrogen, followed by subsequent ester hydrolysis offered the desired a amino acid in fantastic yield and ee, as determined by chiral HPLC. The b amino acids originated from readily on the market phenyl acetate esters . Hydroxymethylation of the ester by treatment with paraformaldehyde and NaOMe, followed by activation to the mesylate and concomitant b elimination gave acrylate . Introduction in the amine by Michael addition followed by successive amine safety since the t butyl carbamate and ester hydrolysis gave the racemic b amino acid in excellent overall yield. The chiral amino acid was obtained from separation from the racemate through SFC. With all the requisite amino acid and core in hand, coupling was carried out utilizing the circumstances illustrated in Scheme . Treatment method within the amine with H?nig?s base, HBTU as well as the proper amino acid gave in outstanding yield.
Subsequent hydrolysis within the phenyl sulfonamide and Boc deprotection afforded the sought after compound selleck reversible microtubule inhibitor since the bis HCl salt. All through our lead optimization efforts we have been capable of get an X ray framework of bound to Akt. The crystal structure confirmed the pyrrolopyrimidine core interacts through a pair of Hbonds to the hinge area of Akt at Ala and Glu . The longer amine side chain interacts during the carbonyl wealthy region with all the carboxylate side chain of Glu as well as the backbone carbonyl of Glu . The side chain carbonyl oxygen of Asn is ca. ? in the amine nitrogen, as well as carboxylate oxygen of Asp is ?. The Cl phenyl group occupies a compact hydrophobic pocket under the P loop that may be formed when Phe is displaced towards the C helix.
The Cl group appears almost suitable, provided its reasonably large C log P and little size. Tyrphostin AG 879 In vitro ADME evaluation of quite a few analogs revealed as being a candidate for further evaluation. Despite the fact that exhibited reasonable cell potency , it also displayed very low predicted clearance , and evaluation of plasma protein binding in the two human and mouse models suggested the prospective for high free of charge fraction . The Caco permeability was measured for at . cm s, and . cm s for your reverse path The modest asymmetry recommended some kind of lively efflux, probably mediated by P glycoprotein transporters. The solubility of was exceptional at higher than mg mL . The predicted low clearance, large free of charge fraction, and great solubility properties of suggested it might be ideal for use in evidence of idea studies in human tumor mouse xenograft experiments.
The U human glioblastoma tumor cell line is PTEN negative and displays a high level of Akt phosphorylation. U tumor Boc N Br PhOS HN HCl PhOS Scheme .
This prospects to dacomitinib and afatinib for being irreversible
This leads to dacomitinib and afatinib to get irreversible inhibitors of EGFR. Extra especially, they covalently bind to Cys of EGFR through the sulfur atom . Thanks to their covalent binding mode, irreversible inhibitors have action in the direction of EGFRTM, EGFRTM LR too as wild type EGFR. Consequently, dose limitation resulting from the concomitant inhibition of wild kind EGFR is often a likely concern and, in clinical settings, this accompanying inhibition translates into skin rash and diarrhea. The outcomes of various phase III clinical trials for dacomitinib and afatinib are awaited in . More just lately, ?third generation? inhibitors which have an anilinopyrimidine core happen to be recognized by means of large throughput screening of a library of irreversible kinase inhibitors. Notably, anilinopyrimidinebased irreversible inhibitors are to fold even more potent towards EGFRTM than other primary and second generation inhibitors and therefore are as much as fold much less potent against wild form EGFR.
An additional third generation, mutant selective inhibitor entered clinical trials in Mutant selective inhibitors, from this source such as WZ and CO , could probably supply greater therapeutic windows and greater toxicity profiles. It stays to be witnessed how these third generation inhibitors will behave from the clinic. A different mechanism by which sufferers produce acquired resistance to EGFR inhibition happens by means of the amplification from the MET oncogene MET amplification happens in roughly on the NSCLC individuals. In delicate cells, the phosphorylation of ERBB by EGFR is inhibited by tyrosine kinase inhibitors. Nonetheless, when MET is amplified, the proto oncogene MET compensates, and phosphorylates ERBB.
As soon as ERBB is phosphorylated, MK-4827 it binds for the regulatory subunit of PIK which benefits while in the activation of pa and its downstream target, AKT . Thus, upon MET amplification, the activation of your PIK AKT pathway leads to cell survival, even beneath sustained inhibition of EGFR. The concomitant inhibition of MET and EGFR employing the monoclonal antiboby MetMab and erlotinib proved to be efficacious in the phase II trial and early clinical trials of small molecule inhibitors of MET given with EGFR inhibitors are now now underway . These combinations may demonstrate enhanced total survival and progression cost-free survival in specified individuals, subject to their genotypes. The combination of crizotinib and dacomitinib, although in early phase, is notably fascinating considering the fact that a single of its components, dacomitinib, was built to handle the acquired resistance by the TM secondary mutation in EGFR.
This blend could probably be efficient in individuals with acquired resistance by means of TM and or MET amplification. Another probable approach to overcome acquired resistance could be the utilization of combination therapies which include heat shock protein inhibitors.
Two mechanisms govern pkip inactivation while in human carcinogen
Two mechanisms govern pkip inactivation all through human carcinogenesis: reduction of protein expression and exclusion from your nuclear compartment The level of pkip is lowered in of human cancers Cytoplasmic sequestration of pkip is often a mechanism whereby cancer cells conquer pkip imposed development inhibition and has become reported for colon, esophagus, thyroid, ovarian, and breast carcinomas. Importantly, the reduction of pkip expression and its presence within the cytoplasm of cancer cells are markers that predict shorter ailment cost-free and or overall survival in sufferers impacted by different types of cancer Reduction of pkip expression in cancer generally occurs by means of sustained protein degradation, a 4 step practice that calls for phosphorylation of pkip at threonine by cyclin E cdk recognition of T phosphorylated pkip by the ubiquitin ligase SCFSkp, ubiquitylation, and degradation from the S proteasome of T phosphorylated pkip. Cytoplasmic retention of pkip may possibly come about by way of greater export or lowered import. Interaction of pkip with JAB CNS or phosphorylation of serine through the hKIS kinase promotes pkip export in the nucleus whereas phosphorylation of threonine through the protein kinase B AKT impairs its import.
Whereas reduction of pkip and its cytoplasmic relocalization in human cancer is very well established, the signaling pathways that regulate these processes are mainly obscure. In an attempt to cast light to the signaling pathways that govern loss of pkip and its cytoplasmic relocalization in human MLN8237 cancer, we studied thyroid follicular cell neoplasms due to the fact in these tumors pkip is inactivated by both loss of expression and cytoplasmic sequestration. In addition, thyroid cancer is different in that distinct histological features, malignant prospective, and degree of differentiation can arise from a single cell and therefore are connected to certain oncogenic lesions Particularly, papillary thyroid carcinomas are characterized by chromosomal rearrangements that result in the activation in the RET PTC tyrosine kinase receptor , by activating mutations while in the gene encoding the serine threonine kinase BRAF or by overexpression with the MET oncogene.
Follicular thyroid carcinomas are as an alternative characterized by activating point mutations in 1 from the three RAS genes . Alteration in the PIK PTEN AKT pathway, by diminished expression within the dual specificity phosphatase PTEN or by hyperexpression of AKT, occurs in each FTCs and PTCs Proliferative signaling elicited by activated tyrosine kinase receptors TKI-258 or by RAS proteins is funneled as a result of a network regulated through the phosphatidylinositol kinase and by the mitogen activated protein kinase . Activation of PIK and generation of phosphatidylinositol triphosphate are needed for activation of AKT and pSK.
These gene goods may possibly get the job done in a coordinated m
These gene products may well get the job done in a coordinated manner to block apoptosis at many steps along the apoptotic signaling cascade . Apart from, NF ?B also protects the cells from various apoptotic agents, which can be accountable for your drug resistance . Dependant on the above information that NF ?B requires in apoptosis modulation and NF ?B activity is linked with thioredoxin, we are able to infer that the thioredoxin program may possibly regulate apoptosis by means of handle of NF ?B exercise. In a earlier analysis, our lab effectively synthesized a novel organoselenium compound , ethane targeting thioredoxin reductase, which has become shown to inhibit thioredoxin reductase action in vitro , and to repress the growth of the assortment of human cancer cells from several organs, such as lung, gastric, hepatic, cervix, prostate, blood etc Also, our previous work discovered that BBSKE induced apoptosis in a variety of tumor cell lines, and that this apoptotic effect contributes to the anticancer action of BBSKE .
The current review investigates the molecular pathway of apoptosis induced by BBSKE in the cells. We try out to elucidate whether NF ?B pathway is responsible for BBSKE ATP-competitive p38 MAPK inhibitor induced A cells apoptosis, and so investigate the molecular mechanism of BBSKE anticancer potential. Our preceding get the job done has shown that BBSKE can inhibit the growth of a variety of human cancer cells . In order to research the selectivity of BBSKE on cancer cells, development inhibition for a human lung cancer cells and CCC HPF human embryonic lung diploid cells was measured via MTT assay. A cells viability was sharply decreased by BBSKE in the dose dependent method at diverse time factors . The ICs of BBSKE for h, h and h time factors had been about M, M and M respectively. In contrast, BBSKE suppressed CCC HPF cells development far more mildly . The ICs of BBSKE on CCC HPF cells for h, h time factors exceeds M, whilst the IC for time stage was approximate to M. Then we further confirmed the apoptosis inducer role of BBSKE on a cells.
The apoptotic price was quantified making use of movement cytometry evaluation with Annexin V Aprepitant FITC and propidium iodide staining. The apoptotic charge enhanced dependently of BBSKE concentration, and approximated on the . M dose level for h . DNA fragmentation within the form of the laddering pattern was also analyzed. The DNA fragmentation was detecInhibitors immediately after exposing cells to many different concentrations of BBSKE for h, specifically with the dose factors of M and . M BBSKE induced A cells apoptosis as a result of mitochondria dependent way As we know, you can find two big pathways for apoptosis which were elucidated so far: the intrinsic as well as extrinsic pathways , and cellular pressure typically triggers mitochondria dependent apoptotic way .
For this function, HeLa cells blocked at M phase with nM nocodazo
For this purpose, HeLa cells blocked at M phase with nM nocodazole had been handled for h with MG before hour incubation with eupatorin from the continued presence of nocodazole and MG. The cells have been subsequently fixed and immunostained for BubR, Aurora B, Survivin, INCENP, pCenpA and CREST auto immune serum. Manage cells blocked at mitosis with nocodazole had been incubated for h with MG ahead of fixation, immunostainings, and measurement within the kinetochore fluorescence intensities of your immunostained marker proteins. BubR kinetochore accumulation was considerably lowered after eupatorin therapy , probably explaining the impaired SAC in these cells. Importantly, CREST signals on the kinetochores of eupatorin handled cells were comparable to these in manage cells , indicating that the common architecture of the kinetochores is simply not affected from the flavonoid. Aurora B is needed for correcting improper kinetochore MT interactions and sustaining SAC signaling when sister kinetochores are connected toMTs but aren’t under tension . In accordance with previous research , Aurora B accumulated at inner centromeres in cells exposed to nocodazole andMG .
The localization was confirmed by co immunostaining with INCENP, a regarded inner centromeric protein . Interestingly, addition of eupatorin on these cells led to mislocalization of Aurora B on the chromosome selleck chemicals special info arms. To investigate if eupatorin interfered with Aurora B function, cellswere stained with an antibody recognizing Cenp A phosphorylated at S , a specific target epitope for Aurora B . A extremely important reduction in kinetochore accumulation of pCenp A was observed in the eupatorin treated cells , which is comparable for the reduction in pCenp A staining induced from the Aurora inhibitor ZM . Together these outcomes demonstrate that eupatorin inhibits Aurora B exercise in cells. As Aurora B is part of the chromosomal passenger complicated we even more studied regardless of whether eupatorin has an effect on the localization from the other CPC subunits survivin and INCENP.We observed that during the presence of eupatorin also survivin and INCENP were mislocalized for the chromosome arms in the majority of cells as evaluated by immunofluorescence staining .
This suggests that eupatorin abolishes the correct localization on the CPC in the inner centromeres. To review regardless of whether the dramatic lessen in Aurora B action attributable to eupatorin in cells is because of direct inhibition of Aurora B or modulation of its upstream regulators, we performed an in vitro kinase assay, in which ? P ATP incorporation to myelin primary protein by recombinant Aurora B was established from the presence of different concentrations of eupatorin. The flavonoid inhibited Aurora supplier Veliparib B in vitro at a concentration that causes escape from mitosis with an IC worth of about M .
Sections had been incubated with blocking serum in PBS containing
Sections were incubated with blocking serum in PBS containing bovine serum albumin, followed by incubation with rabbit anti human Bcl xl polyclonal antibody or with rabbit anti phosphorylated human c Met polyclonal antibody for hour, followed by incubation having a biotinylated goat secondary anti rabbit antibody . Immunoreactive signals have been detected using a streptavidin biotin peroxidase complex from Vector Laboratories, according to the manufacturer?s advisable procedures. All of the slides were counterstained with hematoxylin . For that adverse control, slides had been subjected to your same procedures, together with antigen retrieval, except for treatment of samples with management rabbit IgG. This detrimental manage clearly demonstrated the specificity of the immunostaining that we observed. Subcellular localization of ETS proteins was detected by indirect immunofluorescence. In brief, ETS , PU. or Tel transfected I cells had been plated on coverslips in RMPI medium containing fetal bovine serum. The cells were then serum starved or grown in fetal bovine serum for hrs.
The serum starved cells were exposed to ng ml HGF for minutes, fixed, after which stained with ETS , PU or selleck wnt signaling inhibitors Tel antibodies. Beneficial immunostaining was detected by incubation that has a fluorescein isothiocyanate conjugated secondary antibody plus a ng ml concentration of Hoechst dye and visualized by using epifluorescence microscopy . Quantitative Measurements of Bcl xl mRNA Bcl xl mRNA amounts in both patient samples and cell lines had been measured applying serious time PCR. Total RNAs have been extracted working with TRIzol from Sigma Aldrich, and g aliquots of complete RNA from each sample had been reversetranscribed utilizing a TaqMan reverse transcription kit . Primers and probes to detect Bcl xl and glyceraldehyde phosphate dehydrogenase have been obtained from Utilized Biosystems. Human total RNA was utilised as being a associated common and human glyceraldehyde phosphate dehydrogenase was utilized as the internal PCR management. Real time PCR was performed making use of an MX Multiplex quantitative PCR program . All reactions had been performed in triplicate.
Bcl xl expression amounts in mesothelioma cell lines and in normal lung and pleural tissue have been evaluated by Western blotting with an anti human Bcl xl polyclonal antibody. The robust expression of Bcl xl Irinotecan was evident in all mesothelioma cell lines in contrast with all the two regular tissues examined . Differential Bcl xl expression in human tumor samples was demonstrated by immunohistochemical analysis during which a strong Bcl xl signal was detected inside the tumor spot, whereas the adjacent usual tissue showed no expression of this protein . The differences inside the Bcl xl RNA levels concerning the mesotheliomas and typical tissue have been even further confirmed employing real time PCR evaluation from the exact same human samples made use of for immunohistochemical staining .
Lately, a FRET based conformational sensor for FAK was reported
A short while ago, a FRET primarily based conformational sensor for FAK was reported. Nevertheless, the utility of this construct to quantify small molecule inhibition of FAK stays to get established . Typically, split enzyme complementation tactics are already utilised to the detection of protein protein interactions . Much more not too long ago, a luciferase primarily based phosphorylation sensor was built for AKT . This AKT sensor has split luciferase fragments with the distal ends, a phospho Thr binding FHA domain, and an AKT substrate peptide . On the whole, luciferase based sensors are greater suited for higher throughput screening purposes than are FRET based mostly sensors, if due only on the higher sensitivity with the enzyme amplified signal plus the greater robustness toward compound interference. Having said that, phosphorylation sensors counting on promiscuous peptide substrates are unlikely for being very discriminatory for almost any offered target kinase inside a cellular context.
Also, current phosphorylation sensors detect conformational improvements while in the substrate constructs but not while in the target kinase itself. Although distinctive conformational dynamics are linked to kinase activation, this characteristic hasn’t been immediately exploited for the development of HTS compatible kinase assays and modest molecule screening. Right here we current the generation and validation of split luciferase based intracellular kinase erk inhibitors conformational sensors for Abl. Mutagenesis studies confirmed that these Abl conformational sensors particularly detect the two aggressive and allosteric Abl inhibitors. Furthermore, our data strongly suggest that inhibitor induced stimulation of luciferase exercise is without a doubt the direct outcome in the compound induced conformational improvements in Abl and not triggered indirectly by adjustments in intracellular protein protein interaction events. The Abl assays are hassle-free, robust, and HTS friendly, particularly in the case of the TI Abl mutant .
In principle, this cellular assay format may very well be adopted alot more broadly to other kinases too as to unrelated enzymes displaying sizeable conformational alterations on their activation. Structure analysis shows that, in the smoothened agonist inactive state, c Abl adopts a compact and tightly packed conformation with all the CAP SH SH domain docked onto the back of your catalytic domain. In its energetic state, within the other hand, Abl is likely to adopt an extended conformation with the SH domain contacting the N lobe on the catalytic domain . Provided the large conformational alter in between the inactive and lively states of Abl, we reasoned that a split enzyme complementation approach or possibly a FRET based mostly technique may possibly let us to detect these various Abl conformations in cells.
Dysregulation of Aurora kinase functions success in aneuploidy an
Dysregulation of Aurora kinase functions outcomes in aneuploidy and tumorigenesis, making this class of kinases as interesting oncology therapeutic targets . The preclinical information on VX compound, a pan Aurora inhibitor, showed tumor regression in different animal versions of cancer thus validating Aurora kinase as bonafide oncology targets. A variety of Aurora inhibitors patents have emerged from the current many years and ongoing current publications from many providers highlight the higher level of interest in Aurora as an anticancer biological targets . There can be three mammalian members within the Aurora protein loved ones, Aurora A, B and C. The 2 big Aurora proteins, Aurora A and Aurora B, share higher sequence conservation during the kinase domain . The residues involved in binding of the adenine ring in Aurora A and B ATP binding pocket are identical. Despite the higher sequence conservation inside the catalytic areas, the two proteins have distinct subcellular localization and biological functions. Aurora A is implicated in centrosome maturation and separation, when Aurora B plays a important purpose in cytokinesis, furthermore to its position in mitosis.
Activation of Aurora A is triggered allosterically by binding of an activator TPX . Current crystal structure determination with the Aurora A: TPX complex provided a structural basis for comprehending the activation of Aurora A by TPX recommended site . The N terminal segment of TPX was proven to bind towards the tiny lobe of Aurora A. During the presence in the activator, the Aurora A protein demonstrated an extended energetic conformation on the activation loop that harbors Thr, a webpage that should be autophosphorylated for rendering the Aurora A protein totally energetic . Similar to Aurora A, the activation of Aurora B takes place by binding of an activator, INCENP . The tremendously conserved IN box region of INCENP binds and activates Aurora B. Current biochemical and structural scientific studies have highlighted the differences during the activation mechanism of Aurora A and B .
INCENP was shown to activate Aurora B by a two stage mechanism wherein INCENP only partially activated Aurora B kinase, as well as the total activation was contingent on phosphorylation of a conserved Thr Ser Ser motif on the C terminus of the protein. The Xenopus Aurora B: IN box segment structure that was recently solved corroborated the biochemical information that chlorpheniramine advised distinctions during the activation mechanisms within the Aurora A and Aurora B proteins . INCENP bound Aurora B, inside a binding mode that was distinct from TPX binding to Aurora A. INCENP was proven to not make any direct contacts using the activation loop of Aurora B which makes it probably that INCENP promotes the extended conformation of your Aurora B activation loop by means of an allosteric mechanism .