M = 11 years, 10 months, SD = 8 months). Consistent with hypotheses, results from Structural Equation Modeling demonstrated that the Response Inhibition and Attentional Inhibition factors were empirically independent constructs that exhibited partial statistical dependence on the Working Memory Capacity factor. These findings have important implications for current theories and models of inhibitory control during development.Inhibitory control describes the suppression of goal-irrelevant stimuli and behavioral responses. Current developmental taxonomies distinguish between Response Inhibition – the ability to suppress a prepotent motor response, and Attentional Inhibition – the ability to resist interference from distracting stimuli. Response Inhibition and Attentional Inhibition have exhibited moderately strong positive correlations in previous studies, suggesting they are closely related cognitive abilities. These results may reflect the use of cognitive tasks combining Stimulus–Stimulus- and Stimulus–Response-conflict as indicators of both constructs, which may have conflated their empirical association. Additionally, previous statistical modeling studies have not controlled for individual differences in Working Memory Capacity, which may account for some of the empirical overlap between Response Inhibition and Attentional Inhibition. The aim of the current study was to test a hierarchical model of inhibitory control that specifies Working Memory Capacity as a higher-order cognitive construct. Response Inhibition and Attentional Inhibition were conceptualized as lower-order cognitive mechanisms that should be empirically independent constructs apart from their shared reliance on Working Memory Capacity for active maintenance of goal-relevant representations. Measures of performance on modified stimulus–response compatibility tasks, complex memory span, and non-selective stopping tasks were obtained from 136 preadolescent children ( See Tab studies . Respons studies . Attenti studies .interference effect’ and reflects the additional time required to resolve the conflict between competing stimulus representations and response tendencies  tasks, such as the Stroop, Eriksen Flanker (Flanker), and Simon tasks . These andencies . Studiesndencies .r = 0.67) and could be collapsed into a unitary construct called ‘Response-Distracter Inhibition.’ common inhibition ability,” reflecting a shared reliance on active goal maintenance or Working Memory Capacity (WMC). This theoretical perspective suggests that goal-relevant information is actively maintained in a readily accessible state in working memory through mechanisms of executive attention  and Attention Restraint (r = -0.40) factors and revealed a moderately strong positive correlation between these latter two constructs (r = 0.60), comparable in strength to that obtained by A study conducted by r = 0.71), indicating substantial empirical overlap. Furthermore, without inclusion of tasks measuring WMC, it is unclear whether this intercorrelation was attributable to a shared empirical overlap with WMC. Response Inhibition and Interference Suppression were also not yet differentiated in toddlers aged 24 – 32 months, suggesting increased functional segregation of component inhibitory control processes with ongoing development, similar to the divergent developmental trajectory and increased modularity observed for other executive functions  conflict and Stimulus–Response- (‘SR-’) conflict. SS-conflict reflects competition between the relevant and irrelevant stimuli or stimulus dimensions , whereas SR-conflict reflects competition between the correct, task-relevant response and the prepotent incorrect response   . The stuibition) . Howeveribition) . Conversibition) . The useA study conducted by Another limitation of previous studies is that they have not statistically controlled for the common empirical association of Response Inhibition and Attentional Inhibition with WMC . From thDue to a paucity of research, it is currently unclear how developmental changes affect the relationship between Response Inhibition and Attentional Inhibition. Response Inhibition exhibits a protracted developmental trajectory, with rapid maturation in early childhood followed by gradual improvements throughout adolescence that continue into early adulthood . SubstanThe aim of the current study was to test a model of inhibitory control in a developmental sample that differentiates between Response Inhibition and Attentional Inhibition as empirically independent constructs that are hierarchically organized, reflecting a shared dependence on WMC. Based on the theoretical and empirical framework introduced above it was expected that both the Response Inhibition and Attentional Inhibition factors would be significantly predicted by the WMC factor in a structural regression model, reflecting their shared dependence on active goal maintenance. It was also hypothesized that Response Inhibition and Attentional Inhibition would be empirically independent constructs after being regressed onto WMC, after which their shared variance with this factor would be accounted for. These hypotheses were tested in a pre-adolescent sample of children aged 11 and 12 years, given (a) the lack of prior research in young samples; (b) the importance of inhibitory control to cognitive development and developmental psychopathology; (c) the relative plateau in development of WMC after age 11 years. While the development of inhibitory control is a topic of interest, a restricted age range was employed here as a first step toward better understanding these constructs in children.M = 11 years, 10 months, SD = 8 months) that were tested as part of a larger study on cognition, self-regulation, and emotional and behavioral problems. Parents and caregivers nominated themselves and their child for participation in the study based on their child meeting the eligibility criteria requiring that they be aged between 11 years 0 months and 12 years 11 months; have normal, or corrected to normal, vision and hearing; speak English as his/her first language; have no history of a diagnosed learning disorder, brain injury, or psychiatric illness; and not currently be taking any psychoactive medications. Recruitment took place between December 2013 and April 2015 across year levels 5 (32.6%), 6 (37.9%), and 7 (29.5%) from 52 primary and secondary schools located in metropolitan Melbourne, Australia using advertisement flyers handed out to students in class and through school newsletters. Student participants largely attended Government schools (63.8%), with a smaller number recruited from Catholic (30.8%), and Independent (5.4%) schools. The majority (86%) of the student sample were of European descent. Sample size selection was based on a recommended minimum case-to-parameter ratio (N:q) of 10:1, required for obtaining parameter estimates with reasonable precision (The sample consisted of 136 (125 right-handed) typically developing, pre-adolescent children , aged 11 years, 0 months to 12 years, 11 months (recision . This stThe Automated Working Memory Assessment (AWMA) is a computerized test battery for assessing WMC in young persons aged 4–22 years in both verbal and visuospatial domains . The AWMz” key) or right-handed (“/” key) keyboard response, respectively. Following a response, or after the maximum reaction time had elapsed, there was an interval of 2000 ms prior to presentation of the subsequent trial stimulus.The stop signal task was administered using the Stop-It and Analyze-It program  installeZ< ± 1.96, p > 0.05), such that the subtraction method could be used to accurately calculate SSRT as the dependent variable  and represents a handicap for the inhibitory process in order to reduce the probability of successful inhibition . SSD is variable .H’ key on the computer keyboard. Trials on which participants failed to register their response during the interval of stimulus presentation were registered as omission errors. Conversely, participants were required to inhibit their key press on trials where an ‘X’ appeared. Responses on no-go trials were scored as commission errors. There were 5 practice trials followed by two blocks of 100 trials each, which consisted of 75% go and 25% no-go trials. Trials were also ordered such that there were no more than two consecutive no-go trials. The combination of a large number and high ratio of interspersed go to no-go trials, as well as minimal consecutive no-go trials, assisted in building up a strong prepotent response requiring the engagement of inhibitory processes . In the congruent condition, participants were presented with a stimulus in a spatial location that was compatible with the correctly mapped response . In the incongruent condition, participants were presented with a stimulus in a spatial location that was incompatible with the correctly mapped response . There were 6 practice trials followed by two experimental blocks containing 20 control, 60 congruent, and 60 incongruent trials in total. Each trial began with a fixation cross for 500 ms, followed by presentation of the stimulus until response, and a 500 ms inter-stimulus interval. The dependent variable was the difference in mean reaction times on incongruent compared to control trials (Simon SR-conflict). Behavioral selective stopping on the Simon task engages overlapping neurocognitive mechanisms to non-selective stopping on the stop-signal and go/no-go tasks . The Stroop task consisted of four blocks: word-reading and color naming blocks consisting of 4 practice and 20 experimental trials each, and two experimental blocks consisting of 8 practice and 174 experimental trials in total . The sequence for each trial began with presentation of the stimulus at central fixation until the participant responded. A 1000 ms inter-stimulus interval consisting of a blank screen then preceded the beginning of the next trial. The dependent variable was the differences in mean reaction time on incongruent response ineligible compared to control  trials (Stroop SS-conflict).The current study used a modified version of the Stroop Color-Word Interference Test, adapted from the one previously described by d naming . Responsd naming . Color sFigure 3). There was a practice block of 10 trials followed by two experimental blocks consisting of a total of 160 trials, including 15 control, 50 congruent, 48 incongruent response compatible, and 47 incongruent response incompatible trials. Each trial began with presentation of a fixation cross at the central target location for 500 ms, followed by presentation of the stimulus until response. After the participant responded there was a 500 ms inter-stimulus interval before the next trial began. The dependent variable was the difference in mean reaction time on incongruent response compatible compared to congruent trials (Flanker SS-conflict).The current study used a modified version of the Flanker task like that described by Figure 4). On each trial participants were required to indicate as quickly as possible using the appropriate manual response whether the target and comparison shapes were the same or different (the responses ‘Different’ and ‘Same’ were mapped onto the W and O keys). On control (no distractor) trials, the target and comparison shapes were presented on their own, whereas on distractor trials the target and comparison shapes were presented with the distractor shape, requiring participants to selectively attend to the green-colored target shape and ignore the red distractor shape to make the correct same/difference judgment. The sequence and timing of each trial is displayed in Figure 4. There were 14 practice trials, followed by two experimental blocks of 40 trials each. Conditions were balanced such that distractor trials were reproductions of control trials except for the presence of the underlying red distractor shape. The dependent variable was the difference in mean reaction time on distractor compared to control trials (shape matching SS-conflict).The shape matching task used in the current study was based on the task used by Participants were tested at varying locations, including their home, school, or at one of three research centers over a single 2-h session that was broken into 1-h blocks with additional short breaks in between tasks. Participants were seated comfortably in a location free from visual and auditory distractions. All stimuli were presented on a black background with viewing distance set at approximately 50 cm. An opaque cover was placed over the computer keyboard so that only the relevant response keys were visible for each task, with all the other keys disabled during the experiment. There was a 10-s break in between Blocks 1 and 2 for each task , after which participants pushed one of the response keys when they were ready to resume. Task order was fixed to control for any participant-by-order interaction effects, and was as follows, first block: Listening Recall, Counting Recall, Backward Digit Recall, Stroop, Flanker, and Simon tasks; second block: shape matching, and go/no-go tasks, and Stop-It. After completing Stop-It, participants were assessed using the Vocabulary, Matrix Reasoning, and Symbol Search subtests of the Wechsler Intelligence Scales for Children – Fourth Edition (WISC-IV)  and comp2(44) = 40.290, p = 0.631], indicating that the assumptions of MCAR were satisfied. For each participant the reaction time distributions for every SRC task condition were screened for outliers using a sequential fence procedure constructed using the upper and lower quartiles, defined as: fQ = n/4 + (1/4), and a 2.2 multiplicative of the interquartile range , and four participants (∼3%) were missing Stroop and shape matching task data; three due to color blindness and the fourth on each task due to separate incidences of equipment failure. Finally, one participant (0.7%) was missing SSRT data due to equipment failure. le range . This meZ statistics exceeding the critical value of Z ± 1.96, p < 0.05 were considered to violate the assumption of univariate normality and these distributions were normalized using the appropriate transformation (see Table 4) (2(10) = 8.561, p = 0.574], however, Small’s test of multivariate kurtosis was significant . An omnibus test of multivariate normality, based on Small’s statistics was also significant , indicating that the data set violated the assumption of multivariate normality (2(10) = 29.588, p < 0.001]  . After rormality . The var< 0.001] .Interference effects for each participant were calculated by subtracting mean reaction time trials in the baseline condition from mean reaction time on incongruent trials. Control trials were used as the baseline in the Stroop and Simon tasks because congruity effects often result in response facilitation and faster reaction times on congruent trials . The con2 statistic toward significance (Type II error), as well as attenuating standard errors for the model parameters estimates biasing them toward significance (Type I error)  . Therefostimates . Congenestimates . Latent stimates .N:q) of less than 10:1 can be biased and untrustworthy (Parameter estimates for models with a case-to-parameter ratio (stworthy . A two-sstworthy . Factor stworthy .2) test statistic; the Root Mean Square Error of Approximation (RMSEA); the Comparative Fit Index (CFI); and the Standardized Root Mean Square Residual (SRMR) (2 test statistic has been found to be the most sensitive fit index for identifying model misspecification and was therefore referred to first in order to adjudge model fit (df) and small sample sizes are underpowered to detect model misspecification using the χ2 test statistic and RMSEA (2) was used for comparing the relative fit of competing nested models  . The χ2 odel fit . Generalodel fit . Howevernd RMSEA . Therefond RMSEA . A cut-ond RMSEA  was usednd RMSEA . Values nd RMSEA . The chid models .Table 2 and were indicative of a participant sample with slightly higher than average WMC compared with the age-related population, along with a slight truncation in score ranges with no individuals scoring below a standard score of 70 (Extremely Low). Results from the various conditions of the SRC tasks are displayed in Table 3 and reveal good to excellent internal consistency reliability. In general, the expected pattern of differences across task conditions were observed with slower mean reaction times on incongruent compared to congruent and control trials, and slower mean reaction times on response eligible and response incompatible trials compared to response ineligible and response compatible trials of the Stroop and Flanker tasks. As displayed in Figure 5A, 84% of the standard Stroop interference effect of 181 ms obtained between control and incongruent response eligible trials was attributable to SS-conflict (152 ms), whereas SR-conflict contributed only 16% of the total interference effect (29 ms). In contrast, Figure 5B shows that 86% of the 116 ms total interference effect in the Flanker task was attributable to SR-conflict (99 ms), whereas only 14% was attributable to SS-conflict (16 ms).Results from the AWMA subtests are displayed in F = 138.608, p < 0.001, F = 70.754, p < 0.001, F = 1.334, p = 0.263]. Tests of within-subjects contrasts on the Stroop task indicated that mean reaction time was not significantly different between control and congruent trials ; but was significantly slower on incongruent response ineligible compared to congruent trials , suggesting no statistically significant differences between male and female participants across the dependent variables in the study, although observed power was low.Repeated measures ANOVA with Greenhouse-Geisser correction for within-subject comparisons was significant for the Stroop  ] . SSRT, nTable 6. The initial correlated three-factor model (Model 1) provided a good fit to the data. In particular, the χ2 test statistic and RMSEA point estimate indicated that the null hypotheses of exact and close approximate fit to the observed covariance matrix could not be rejected (p = 0.233). This result was not surprising given the very small proportion of variance in overall interference effect on the Flanker task attributable to SS-conflict. However, dropping this item from the model caused estimation problems due to local under-identification of the Attentional Inhibition latent variable (Fit statistics for the competing measurement models are displayed in rejected . The onlvariable . Neithervariable . Therefovariable .2(9) = 77.303, p < 0.05], suggesting that there was sufficient covariance in the data to identify the hypothesized Response Inhibition and Attentional Inhibition factors, along with WMC. However, the H index was computed for the Response Inhibition (H = 0.56) and Attentional Inhibition (H = 0.62) factors and indicated less than adequate construct reliability (H < 0.70)  .χ2(1) = 2.725, p > 0.05], suggesting a more parsimonious representation of the data.Constraining factor intercorrelations to zero or one provides a powerful statistical test of comparative fit for the theoretical measurement model relative to more constrained nested models . Constra2(1) = 3.246, p > 0.05], but resulted in local under-identification of the Attentional Inhibition factor . Therefore, this model was not retained. Constraining the intercorrelation between WMC and Response Inhibition to zero (Model 6) resulted in a significantly poorer model fit . Therefore, the model with Response Inhibition and Attentional Inhibition specified as uncorrelated factors (Model 4) provided the best representation of the data. This model is displayed in Figure 7.Applying a parameter constraint of zero on the factor intercorrelation between WMC and Attentional Inhibition (Model 5) resulted in a poorer overall model fit that was not significantly worse , as well as nested models with the factor intercorrelation constrained to one  and zero . Thus, Response Inhibition and Attentional Inhibition could not be represented as independent constructs when WMC was not included in the model.The Response Inhibition and Attentional Inhibition factors were also modeled without the WMC factor included (Model 7). The 95% confidence interval of the standardized factor intercorrelation between Response Inhibition and Attentional Inhibition did not contain one or zero  and Attentional Inhibition  were both statistically significant and indicated that the efficiency of inhibitory control increased concomitantly with higher levels of WMC. The residual correlation between Response Inhibition and Attentional Inhibition was not statistically significant , suggesting that their previous marginally significant association , was explained by their shared empirical association with WMC. Constraining this residual correlation to zero provided a more parsimonious model  that did not lead to a significant decrement in model fit , and was thus retained as the final model. In this final model, WMC explained 41%  and 11%  of the variance in the Response Inhibition and Attentional Inhibition factors, respectively.The Response Inhibition and Attentional Inhibition factors were regressed onto the single-indicator WMC latent variable to test the first hypothesis that these two constructs would exhibit a shared statistical dependence on WMC as a higher-order factor. The residual correlation between Response Inhibition and Attentional Inhibition was freely estimated to determine if there was a decrease in the empirical association between these constructs after the variance shared with WMC was accounted for. The structural regression model is displayed in p = 0.014) explaining 9.3% of the variance in WMC. However, constraining the non-significant correlation between Attentional Inhibition and Response Inhibition increased the regression coefficient , explaining 11% of the variance in WMC.A theoretically plausible equivalent model was generated using Lee-Herschberger replacing rules, which assists in determining whether the theoretical structural regression model provides the optimal representation of the data compared to an alternative model with equivalent overall fit . Equivalp > 0.05; κ = 0.421, p > 0.05; W(136) = 0.992, p = 0.684] and Attentional Inhibition  latent variables did not deviate from normality as revealed by skewness and kurtosis coefficients and Shapiro–Wilk tests. Similarly, neither the residuals of WMC factors score estimates regressed onto the Attentional Inhibition factor score estimates , nor the reverse  were significantly skewed. Direction of dependence cannot be determined from third- and fourth-order moments when the distributions of factor scores estimates are univariate normal  compared to the factor intercorrelation in the full latent model   . Thus, edictions .The proposed hierarchical model of inhibitory control was partially supported by the results of structural equation modeling on task data obtained from a sample of 11- and 12-year old children. Consistent with hypotheses, variance in both the Response Inhibition and Attentional Inhibition factors was significantly explained by individual differences in WMC. As further predicted, the Response Inhibition and Attentional Inhibition factors were empirically independent constructs after their shared statistical dependence on WMC was accounted for in the structural regression model. However, results obtained from the initial measurement model revealed that the correlation between the Response Inhibition and Attentional Inhibition factors was modest and only marginally significant. Furthermore, the empirical fit of a more constrained nested model demonstrated that the Response Inhibition and Attentional Inhibition factors could be represented as empirically independent constructs prior to being regressed onto the WMC factor. We chose to focus on a narrow age range in the current study to reduce the impact of developmental changes on the results . The undNotwithstanding these limitations, the current study is the first to demonstrate that Response Inhibition and Attentional Inhibition are empirically independent cognitive constructs in a developmental sample. A lack of dissociation between these two constructs in previous studies may be attributable to the use of cognitive tasks with low reliability and specificity. Previous studies have tended to conceptualize and use variants of the Flanker task as a measure of Attentional Inhibition . HoweverTo our knowledge, ours is the first study to test and empirically support a hierarchical model of inhibitory control. The structural organization of the proposed model is consistent with the unified model of inhibitory control proposed by Specification of WMC as a higher-order factor supporting inhibitory control is also consistent with experimental research highlighting the dependence of response inhibition and visual selective attention on WMC . ResponsWorking memory capacity corresponds with the Central Executive in the multi-component model of working memory, originally described by Taxonomies of inhibitory control previously recognized a distinction between the ability to ignore irrelevant stimuli and the ability to withhold a prepotent motor response . FriedmaTheoretical and empirical distinctions between these two inhibitory control abilities and WMC may assist in providing conceptual clarity to studies of cognitive development and developmental psychopathology. For example, the developmental trajectory of Attentional Inhibition has not yet been investigated, because it has not been traditionally distinguished from WMC and Response Inhibition. Researchers have suggested that WMC and Attentional Inhibition are largely overlapping constructs, and that greater WMC may thus support more efficient visual selective attention . The curA potentially important practical implication of the hierarchical model of inhibitory control is in relation to the neuropsychological profile and clinical presentation of children with low WMC. The current model predicts that children with deficits in WMC may also exhibit concomitant difficulties in selectively attending to task-relevant stimuli and inhibiting behavioral responses. Such predictions are in keeping with the cognitive and behavioral profile of children with poor WMC, including those with Attention Deficit Hyperactivity Disorder (ADHD) . ConversThe hierarchical model of inhibitory control predicts that response inhibition is a distinct neurocognitive mechanism from active goal maintenance in working memory. This perspective challenges the prominent Unity/Diversity model of executive function in which variance associated with inhibiting prepotent behavioral responses is completely subsumed by a general goal maintenance factor . Thus, aFrom a research and clinical perspective, the current study indicates the need for independent assessment of Attentional Inhibition, Response Inhibition, and WMC, using multiple tasks where possible to surmount the task impurity problem . We recoIdentification of the three cognitive constructs in the current study was based upon a synthesis of previous factor analytic studies, which have demonstrated patterns of intercorrelations in task performance largely consistent with the separate factors identified in the current model . Neverthexecutive function,’ a multidimensional construct encompassing several interrelated, higher-order processes involved in the goal-directed control of cognition and behavior (N = 137) in first establishing the Unity/Diversity model of executive function. However, the use of a larger sample is needed for sufficient power to compare competing nested models, validate the precision of the parameter estimates, as well as assess the adequacy of model fit to the broader population (Response Inhibition and Attentional Inhibition have both been categorized under the broader rubric of ‘behavior . Severalbehavior . A limitbehavior . Additiopulation . Use of pulation . The hieThe current study introduced and tested a hierarchical model of inhibitory control. The results challenge existing models and theories of inhibitory control in two respects. First, it was demonstrated that Response Inhibition and Attentional Inhibition both exhibit a statistical dependence on WMC, consistent with cognitive and neurobiological models of top-down control. Second, the Response Inhibition and Attentional Inhibition factors could be modeled as empirically independent constructs, suggesting that they are distinct, and perhaps functionally unrelated, cognitive abilities. The hierarchical model of inhibitory control may provide a useful conceptual framework for future theorizing and empirical studies examining the relationship between these processes and other aspects of cognitive, affective, and behavioral functioning, as well as how they change across the course of development.https://osf.io/u2w79/].The dataset analyzed for this study can be found in PsyArXiv Open Science Framework [JT was responsible for conceptualizing and designing the study, data collection, analyses, and write-up. RT, MB, CP, and SW advised on study design, data collection, interpretation of results, and write-up of the manuscript. All authors contributed to manuscript revision, as well as read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The recent finding that PAG can function as a positive regulator of the high-affinity IgE receptor (FcεRI)-mediated mast cell signaling suggested that PAG and CSK have some non-overlapping regulatory functions in mast cell activation. To determine the regulatory roles of CSK in FcεRI signaling, we derived bone marrow-derived mast cells (BMMCs) with reduced or enhanced expression of CSK from wild-type (WT) or PAG knockout (KO) mice and analyzed their FcεRI-mediated activation events. We found that in contrast to PAG-KO cells, antigen-activated BMMCs with CSK knockdown (KD) exhibited significantly higher degranulation, calcium response, and tyrosine phosphorylation of FcεRI, SYK, and phospholipase C. Interestingly, FcεRI-mediated events in BMMCs with PAG-KO were restored upon CSK silencing. BMMCs with CSK-KD/PAG-KO resembled BMMCs with CSK-KD alone. Unexpectedly, cells with CSK-KD showed reduced kinase activity of LYN and decreased phosphorylation of transcription factor STAT5. This was accompanied by impaired production of proinflammatory cytokines and chemokines in antigen-activated cells. In line with this, BMMCs with CSK-KD exhibited enhanced phosphorylation of protein phosphatase SHP-1, which provides a negative feedback loop for regulating phosphorylation of STAT5 and LYN kinase activity. Furthermore, we found that in WT BMMCs SHP-1 forms complexes containing LYN, CSK, and STAT5. Altogether, our data demonstrate that in FcεRI-activated mast cells CSK is a negative regulator of degranulation and chemotaxis, but a positive regulator of adhesion to fibronectin and production of proinflammatory cytokines. Some of these pathways are not dependent on the presence of PAG.C-terminal Src kinase (CSK) is a major negative regulator of Src family tyrosine kinases (SFKs) that play critical roles in immunoreceptor signaling. CSK is brought in contiguity to the plasma membrane-bound SFKs  The first biochemically well-defined step in FcɛRI-mediated cell activation is tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of FcɛRI β and γ subunits by Src family kinase (SFK) LYN, followed by recruitment of protein tyrosine kinase (PTK) SYK to FcεRI γ and its activation. LYN and SYK, together with FYN and some other PTKs, phosphorylate the tyrosine motifs of transmembrane adaptor proteins (TRAP) such as linker for activation of T cells , non-T ame LAT1 ], and phame LAT1 –6], whicame LAT1 , lipid rame LAT1 , and PTKame LAT1 , 10.397) and 508 (Y508)  domain with phosphorylated Y508 stabilizes the inactive conformation of the catalytic domain. Y397 of LYN is autophosphorylated in trans, whereas Y508 phosphorylation is catalyzed by the CSK. The critical role of CSK in cell physiology was demonstrated by the phenotype of mice lacking CSK. These mice die in early embryonic stages and their tissues possess Src family tyrosine kinases (SFKs) with increased enzymatic activity  –13. Phosactivity –16. Whenactivity . Furtheractivity .via their N-terminal myristoyl and/or palmitate moieties, CSK lacks the transmembrane domain and fatty acyl modifications and is predominantly localized in the cytosol  leads to rapid PAG dephosphorylation, which results in the release of CSK from PAG and relief of CSK-mediated inhibition of SFKs  with PAG knockout (KO) or PAG knockdown (KD) exhibited impaired antigen-induced degranulation, calcium response, tyrosine phosphorylation of FcεRI, SYK, and some other signal-transduction molecules, production of several cytokines and chemokines, and chemotaxis. At the same time, the enzymatic activities of LYN and FYN kinases were increased in nonactivated cells, suggesting involvement of a LYN and/or FYN-dependent negative regulatory loop . These dTo better understand the contribution of CSK and PAG in FcεRI-mediated signaling, in this study we examined the antigen-induced activation events in BMMCs from WT and PAG-KO mice with reduced or enhanced expression of CSK and corresponding controls. We also analyzed the involvement of CSK and PAG in adhesion to fibronectin and chemotaxis toward antigen and SCF. Our data indicate that CSK and PAG in mast cells are, respectively, negative and positive regulators of events leading to degranulation and chemotaxis toward antigen and SCF, whereas they are both positive regulators of FcɛRI-induced production of cytokines and chemokines. The combined data indicate that CSK regulates production of proinflammatory cytokines and chemokines through the LYN/SHP-1/STAT5 axis.Y783 (Cat. No. sc-12943), and GRB2 (Cat. No. sc-255), as well as horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cat. No. sc-2005) and goat anti-rabbit IgG (Cat. No. sc-2004) were obtained from Santa Cruz Biotechnology Inc. Antibodies specific for phospho-LynY507 , phospho-SFKsY416 , phospho-SYKY525/Y526 (Cat. No. 2711), phospho-STAT5Y694 (Cat. No. 9351), and phospho-SHP-1Y564 (Cat. No. 8849) were obtained from Cell Signaling. Phospho-LATY191 (Cat. No. 07-278) and Protein G HRP conjugate (Cat. No. 18-161) were obtained from Millipore. Anti-mouse β1-integrin-specific antibody HM β1-1 (Cat. No. 553837) was purchased from BD Biosciences. Alexa Fluor (AF)488-conjugated goat Ab specific for hamster IgG (Cat. No. A-21110) or mouse IgG (Cat. No. A-11001) were obtained from Life Technologies. HRP-conjugated anti-phosphotyrosine mAb  and V450-conjugated rat anti-mouse CD107a (Cat. No. 560648) were obtained from BD Biosciences. Anti-mouse FcεRI-fluorescein isothiocyanate  and anti-mouse c-KIT (CD117)-allophycocyanin  conjugates were obtained from eBiosciences. Polyclonal antibodies specific for mouse tumor necrosis factor (TNF)-α (Cat. No. 500-P64), interleukin (IL)-13 (Cat. No. 500-P178), recombinant mouse TNF-α (Cat. No. 315-01A), recombinant IL-13 (Cat. No. 210-13), and recombinant IL-6 (Cat. No. 216-16) were obtained from PeproTech. Murine anti-IL-6 (Cat. No. 554400) was purchased from Becton Dickinson. Colloidal gold nanoparticles , consisting of approximately 2 × 1011 Au-NPs/ml, were obtained from BB International. TNP-bovine serum albumin (BSA) conjugate (15–25 mol TNP/mol BSA) was produced as described previously  and [γ-32P] ATP  were purchased from the Institute of Isotopes Co., Ltd. . All other reagents were from Sigma-Aldrich.The following monoclonal antibodies (mAbs) were used: mouse IgE mAb specific for 2,4,6-trinitrophenol (TNP) clone IGEL b4.1 , anti-Fcl-glutamine, 12 mM d-glucose, recombinant mouse SCF , mouse recombinant IL-3 , and 10% fetal calf serum (FCS). Stable mast cell lines derived from mouse WT BMMCs (Lyn+/+) or mouse LYN-KO BMMCs (Lyn−/−), were obtained from Hibbs et al.  and the reverse primer 3′-TTTGCGGCCGCCTACTTGTACAGCTCGTCCAT-5′ (the NotI restriction site is underlined) were used. The construct was verified by sequencing and denoted hCSK-mCherry. In all experiments, empty pLKO.1 and/or non-target (NTG) vectors or empty pCDH vectors were used as controls. In some experiments related to CD107a and β-glucuronidase detection, we also used the pLKO.1 vector containing NTG shRNA (Sigma-Aldrich) as a control.Lentiviral transductions were done as described previously using HEK 293 T/17 packaging cells for virus preparation . A set og; 3 min), cells were fixed with 4% paraformaldehyde for 15 min and washed with PBS. IgE was visualized using AF488-conjugated anti-mouse IgG (H + L) antibody, which cross-reacts with IgE, as previously describe  and analyzed with an LSRII flow cytometer (BD Biosciences) and further processed using FlowJo software . For analysis of the CD107a presence on the cell surface, BMMCs were activated or not with antigen (10–100 ng/ml) for 10 min at 37°C. Activation was stopped by centrifugation at 4°C. The cells were then resuspended in 50 µl of PBS-1% BSA containing 200-fold-diluted V450-conjugated rat anti-mouse CD107a and stained on ice for 30 min. After washing with PBS, the cells were examined as described above. For analysis of IgE internalization, IgE-sensitized cells were activated with antigen (500 ng/ml) or not for various time intervals  at 37°C. Activation was stopped by centrifugation at 4°C (300 × describe . To quandescribe .2+ were determined by spectrofluorometry using the Infinite 200M plate reader with excitation wavelengths at 340 and 380 nm and with constant emission at 510 nm.Bone marrow-derived mast cells were cultured for 4 h in SCF- and IL-3-free medium supplemented with TNP-specific IgE (1 µg/ml). The IgE-sensitized cells were washed, and degranulation assay, based on evaluation of β-glucuronidase release, was performed and evaluated as previously described . FluoresmRNA was extracted from IgE-sensitized and antigen-activated (100 ng/ml) BMMCs using a TurboCapture 96 mRNA kit or RNeasy miniKit (Qiagen). Single-stranded cDNA was synthesized with M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (RT-qPCR) amplifications of cDNAs and oligonucleotide primers used were described previously . Actin, 5 cells/well). After activation with antigen  or SCF , unbound cells were washed out using a microplate washer  and the bound cells were determined using an Infinite 200M Fluorometer with excitation and emission filters at 485 and 538 nm, respectively.Chemotaxis responses were assayed in 24-well transwell chamber (Corning) with 8 µm-pore-size polycarbonate filters in the upper wells. Chemoattractants, antigen (250 ng/ml) or SCF (50 ng/ml), were added to the lower compartments, and cell migration was assessed as previously described  supplemented with 0.2% Triton X-100 (for FcεRI immunoprecipitation), 1% n-dodecyl-β-d-maltoside and 1% Nonidet P-40 (for LYN and FYN immmunoprecipitations), or 0.5% Triton X-100 . After incubation on ice for 30 min, the lysates were centrifuged  and postnuclear supernatants were immunoprecipitated with the corresponding antibodies prebound to UltraLink-immobilized protein A . The immunoprecipitated proteins were size-fractionated by SDS-PAGE as described above, transferred to nitrocellulose membrane and immunoblotted with PY20-HRP or with protein-specific antibodies, followed by HRP-conjugated anti-mouse or anti-rabbit IgG antibody. The HRP signal was detected by a Luminescent Image Analyzer LAS 3000 (Fuji Photo Film Co.). Aida software (Raytest GmbH) was used for signal quantification. The amount of phosphorylated proteins was normalized to the amount of immunoprecipitated proteins after stripping of the membranes, followed by development with the corresponding antibodies.Whole-cell extracts from nonactivated or antigen (250 ng/ml) activated cells were prepared by solubilizing cell pellets in sodium dodecyl sulfate (SDS) sample buffer, followed by sonication and boiling of samples for 5 min. Proteins were size-fractionated by 10% SDS-polyacrylamide gel electrophoresis, electrophoretically transferred onto nitrocellulose membrane, and analyzed by immunoblotting with protein- or phosphoprotein-specific antibodies, followed by HRP-conjugated anti-mouse or anti-rabbit IgG antibodies. Phosphorylation levels were normalized to the corresponding loading proteins. In immunoprecipitation experiments, cells were solubilized in ice-cold immunoprecipitation buffer  and then resuspended in 25 µl kinase buffer supplemented with 2.5 μCi (92.5 kBq) of [γ-32P]ATP, 100 µM ATP, and 0.5 µg/µl of acid-denatured enolase as the exogenous substrate. Immunoprecipitates were eluted with reducing SDS-PAGE sample buffer. The 32P-labeled proteins were size-fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and visualized by autoradiography. Films were quantified with Aida image analyzer software (Raytest).The escribed , 36, witt-test for comparison between two groups. In experiments where more than two groups were compared, the significance of intergroup differences was determined by one-way ANOVA with Tukey’s post-test. Comparison of changes over time between different groups was performed using two-way ANOVA with Bonferroni post-test. P values of less than 0.05 were considered significant. GraphPad Prism 5  or Microsoft Excel 2010 were used for statistical analyses. Statistical method used and number of replicates are indicated in the corresponding figure legends.Results are expressed as means ± SEM. Statistical significance was evaluated by unpaired two-tailed Student’s To determine the role of CSK in mast cell signaling, we prepared BMMCs from C57BL/6 mice with reduced or enhanced expression of CSK. For CSK silencing we used four CSK-specific shRNAs  in lentiviral pLKO.1 vector. As controls, BMMCs transduced with empty pLKO.1 vector or pLKO.1 vector containing an NTG sequence were used. Because there were no significant differences between the results obtained with these different control vectors, the data were pooled and are presented together as control pLKO.1 vector. Lentiviral-mediated delivery of individual CSK-specific shRNAs resulted in reduction of CSK protein to 26 ± 4% , 18 ± 6% (shRNA 36), 26 ± 5% (shRNA 64), and 36 ± 4% (shRNA 90) when compared to cells transduced with control pLKO.1  or pooled shRNAs were sensitized with IgE and activated with various concentrations of antigen. Degranulation was determined by quantification of β-glucuronidase released from the cells upon antigen exposure or spontaneously. In nonactivated cells, there were no significant changes in spontaneous β-glucuronidase released from the cells transduced with various individual shRNA vectors or control pLKO.1  for 1 or 5 min, solubilized, and tyrosine phosphorylation of the immunoprecipitated FcɛRI chains was examined by immunoblotting. Data show that after antigen-mediated activation, FcεRI β and γ chains were rapidly tyrosine phosphorylated in both cell types  in the course of FcεRI-mediated activation by immunoblotting. In nonactivated cells, there was only weak tyrosine phosphorylation of this phosphatase, and CSK had no significant effect on it. Interestingly, phosphorylation of SHP-1Y564 was significantly enhanced in antigen-stimulated CSK-KD cells when compared to control pLKO.1 cells  with CSK-OE (Lyn−/−/CSK-OE) and compared it with WT BMMC line with CSK-OE (Lyn+/+/CSK-OE) and the corresponding control cells . As shown in Figures Y564 was observed in non-activated control Lyn+/+ cells and after antigen triggering the phosphorylation was increased. In Lyn+/+/CSK-OE cells phosphorylation of pSHP-1Y564 was significantly reduced in antigen-activated cells. In Lyn−/− cells reduced phosphorylation of SHP-1Y564 was observed in both cell types, with CSK-OE or control pCDH vector. This result is consistent with enhanced tyrosine phosphorylation of SHP-1Y564 observed in BMMCs with CSK-KD , control PAG-KO cells transduced with empty pLKO.1 vector (pLKO.1/PAG-KO), and control WT cells transduced with empty pLKO.1 vector (pLKO.1/WT).Our previous study showed that BMMCs isolated from mice with PAG-KO exhibited significantly reduced tyrosine phosphorylation of several molecules involved in early FcεRI-mediated signaling, including FcɛRI β and γ chains, SYK, LAT, and pPLCγ. This caused reduced degranulation and CaKO BMMCs . On the  Figures B,E. To b2+ response. The cells were sensitized with IgE, loaded with Fura-2, and activated with antigen. We found that in the BMMCs with CSK-KD/PAG-KO, the Ca2+ response was significantly higher when compared to the Ca2+ response in control pLKO.1/PAG-KO and pLKO.1/WT cells , LAT (pLATY191), and NTAL (pNTAL) in IgE-sensitized and antigen-activated or nonactivated CSK-KD/PAG-KO BMMCs and control pLKO.1/PAG-KO cells  and national guidelines (law 409/2008), and was in compliance with the EU Directive 2010/63/EU for animal experiments.LP and PD designed the study and wrote the manuscript. LP performed most of the experiments. LD performed chemotactic and kinase assays, immunoprecipitations, and immunoblotting experiments. IH performed flow cytometry analysis of β1-integrin, FcεRI internalization, and RT-qPCR analyses of cytokines and chemokines. TP performed calcium response analysis and immunoblotting experiments. All authors analyzed the data and read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chemical inhibition of ATM in rat cardiomyoblast cells (H9c2) significantly decreased the excited-state autofluorescence lifetime of enzyme-bound reduced NADH and its phosphorylated form, NADPH (NAD(P)H; 2.77 ± 0.26 ns compared to 2.57 ± 0.14 ns in KU60019-treated cells). This suggests an interaction between ATM and the electron transfer chain in the mitochondria, and hence may have an important role in oxidative phosphorylation in terminally differentiated cells such as cardiomyocytes.The absence of Ataxia-Telangiectasia mutated protein kinase (ATM) is associated with neurological, metabolic and cardiovascular defects. The protein has been associated with mitochondria and its absence results in mitochondrial dysfunction. Furthermore, it can be activated in the cytosol by mitochondrial oxidative stress and mediates a cellular anti-oxidant response through the pentose phosphate pathway (PPP). However, the precise location and function of ATM within mitochondria and its role in oxidative phosphorylation is still unknown. We show that ATM is found endogenously within cardiac myocyte mitochondria under normoxic conditions and is consistently associated with the inner mitochondrial membrane. Acute  A recent meta-study revealed that heterozygous A-T carriers, that make up as much as 1.4–2% of the general population3, have a significantly increased risk of developing cancer and ischaemic heart disease4. Mouse studies have also shown that heterozygous ATM mice suffer from accelerated atherosclerosis development5, and that the absence of ATM can lead to structural and functional changes in the heart6. Although ATM protein kinase is best known for its role in the signalling and repair of double strand breaks in DNA7, available data suggests that the absence of ATM protein kinase can also result in a plethora of metabolic and cardiovascular abnormalities. Its potential importance with regards to the development of insulin resistance and vascular dysfunction has also been highlighted8. Studies have shown that anti-oxidative treatment specifically targeting mitochondria can decrease metabolic syndrome in ATM-null mice5, and supports the notion that A-T might be a mitochondrial disease9. Currently, an emerging role for ATM is being investigated independently from the DNA damage response pathway with regards to its activation in response to mitochondrial oxidative stress11 and contribution towards oxidative homeostasis in the cytosol13.Ataxia-Telangiectasia (A-T) is a rare, recessive disease characterised by the absence of Ataxia-Telangiectasia Mutated (ATM) protein kinase. The disease results in progressive neurodegeneration, increased incidence of cancer, radio -sensitivity, insulin resistance and cardiovascular disease14. Mitochondria comprise approximately 30% of the cardiomyocyte volume15 and are essential for the maintenance of healthy cardiomyocytes16. Mitochondrial dysfunction leads to increased reactive oxygen species (ROS) production and consequently, oxidative stress. Moreover, dysfunctional mitochondria have been identified as a contributing factor to both insulin resistance and coronary artery disease17, as well as various cardiac pathologies including the progression of heart failure18.Cardiomyocytes make up approximately 70–85% of all heart cells10, but the underlying mechanisms are still largely unclear. ATM is known to regulate mitochondrial biogenesis and DNA content19 and can lead to mitochondrial dysfunction when absent20. ATM has also been detected in mitochondrial fractions of normal human fibroblasts, and can be activated in response to mitochondrial uncoupling21. Atm-deficient cells (ATM−/−) show decreased respiratory rates compared to their wild-type counterparts, which can be partially repaired with anti-oxidant treatment20. Earlier reports of ATM localisation showed that mitochondrial subcellular fractions of human fibroblast21 and hepatocyte cell-cultures are enriched with phosphorylated ATM in response to cytosolic oxidative stress, whilst the fraction containing peroxisomes only contained traces of ATM22. Most recently however, ATM was shown to be imported into the peroxisome via the PEX5 peroxisome import receptor in response to oxidative stress23 and reacts to peroxisomal ROS to induce autophagy of the peroxisome (pexophagy)24. Furthermore, in HeLa cells, ATM localises in the nucleus in response to endogenous mitochondrial oxidative stress, independently of DNA damage response, whilst promoting glucose flux through the pentose phosphate pathway (PPP) and increasing cellular anti-oxidant capacity by increasing glucose-6-phosphate dehydrogenase (G6PD) in bone osteosarcoma cells (U2OS)11. Impaired ROS sensing by ATM results in increased glutathione synthesis, possibly due to a decreased flux through the PPP. This reduces NADPH availability for glutathione recycling and places ATM, as a redox sensor, at a critical junction of cytosolic carbohydrate metabolism11. The redox pairs, NAD+/NADH and phosphorylated NADP+/NADPH, play critical roles respectively in cellular redox states during ATP production and ROS production, or lipid, amino acid and nucleotide biosynthesis through glycolysis or the PPP25.ATM can act as an important sensor of oxidative stress in cells and regulate redox stress defences26, the protein is found mainly in the cytoplasm of differentiated neuronal cells where it maintains basal metabolic flux27. This study sought to establish where ATM is located within terminally differentiated cardiac cells, and if so, whether there are any mitochondrial respiratory or metabolic implications.Whilst it is well known that ATM resides predominantly in the nucleus of mitotic cellsWe investigated endogenous ATM protein kinase in cardiac mitochondria isolated from male Wistar rats as well as its role in oxidative phosphorylation and effect on NAD(P)H/NADH ratios. Our results show that endogenous ATM is located in cardiac mitochondria from rats under basal conditions, and that direct chemical inhibition of ATM results in decreased oxidative phosphorylation in cardiac mitochondria. We also established that the ATM inhibitor, KU60019, significantly reduced enzyme binding of NAD(P)H/NADH (2.77 ± 0.26 ns compared to 2.57 ± 0.14 ns in KU60019-treated cells), using fluorescence lifetime imaging microscopy (FLIM) in H9c2 rat cardiomyoblast cells.28. The efficacy of digitonin-based removal of the outer mitochondrial membrane (OMM) was evaluated with transmission electron microscopy  as shown in Fig. 20. In light of the observed consistent association between ATM and the IMM of cardiac mitochondria, we investigated the effect of acute ATM inhibition on oxidative phosphorylation in isolated hearts obtained from 12-week old male Wistar rats . The hearts were stabilised and subjected to retrograde Langendorff perfusion with either DMSO  or 3 µM KU60019  for 20 minutes prior to mitochondrial isolation in KE buffer. This concentration of the inhibitor does not influence isolated cardiomyocyte cell viability  for the complex I-mediated reduction of the NADH-linked substrates, GM, in KU60019-perfused hearts compared to controls. Together with a reduction in complex I respiratory rate, this resulted in a significant reduction (p = 0.0024) in the oxidative phosphorylation rate  in GM, but not PCM respiratory buffer.Mitochondrial oxygen uptake was measured with a Clarke-type electrode (HansaTech.) and State 2 , State 3 (active respiration in presence of ADP), and State 4 (resting respiration after ADP usage) were recorded respectively in glutamate (5 mM) + malate  and palmitoyl-L-carnitine (0.45 mM) + malate  respiratory buffers. Respiratory control ratio  reflects the control of oxygen consumption by phosphorylation and did not differ significantly between control and KU60019 treated hearts. The substrates glutamate and malate, investigate the mitochondrial aspartate shuttle that produce NADH which is oxidized by complex I (NADH dehydrogenase) of the electron transfer chain (ETC) to NAD2 values = 1.041 ± 0.05; 1.045 ± 0.044; mean values ± s.d.). Fluorescence decay parameters were recorded for τ-bound, α-bound  to 2.57 ± 0.14 ns (n = 3) compared to DMSO-treated cells , whereas α-bound increased, albeit not significantly (p = 0.14). Similarly, inhibition of ATM with KU60019 in HEK293 cells, significantly reduced τ-bound mean values (±s.d.) to 2.66 ± 0.11 ns compared to the control treated HEK293 cells   were treated with either DMSO  or 3 μM KU60019 dissolved in DMSO (n = 3) for 3–6 hours prior to FLIM. Fluorescence decays obtained at each pixel were fitted with a two-component model that yielded good fits  that consequently decreased the mitochondrial coupling efficiency, and resulted in reduced oxidative phosphorylation and ATP production. Moreover, the inhibition of ATM resulted in a significant decrease in NAD(P)H fluorescence decay lifetime which suggests an increase in NADH and supports the notion that complex I-mediated NADH oxidation is impaired by the inhibition of ATM. Likewise, inhibition of complex I-mediated oxidative phosphorylation with rotenone results in a decrease in NAD(P)H fluorescence and have been associated with an increase in NADH relative to NADPH, potentially suggesting metabolic disturbanceet al.22, we made use of detergent-based subcellular fractionation of rat cardiac mitochondria to obtain mitoplasts. ATM was found to be consistently associated with the IMM and expands on previously published work that shows that the absence of ATM is associated with mitochondrial dysfunction in A-T patient derived fibroblasts20 and ATM−/− mice thymocytes21. However, this is, to the best of our knowledge, the first evidence to show that ATM is located on the inner mitochondrial membrane of rat cardiac mitochondria. The removal of the OMM and integrity of the mitoplasts were confirmed with TEM  remained intact. At 20 minutes the majority of mitoplasts remained intact  by significantly lowering the oxidative phosphorylation efficiency . NADH transfers electrons to the ETC at complex I, whilst the FADH2 transfers electrons to the ETC at complex II, and is the main source of ATP production in a healthy heart18. The glutamate-malate substrate assesses the aspartate-malate shuttle that produces NADH which provides electrons to complex I, and suggests that the inhibition of ATM has a larger effect on complex I mediated NADH oxidation than FADH2 oxidation at complex II. Atm-deficient mice thymocytes also exhibit a decrease in complex I enzyme activity and reduced ATP synthesis, but increased oxygen respiration which has been suggested to be context specific21. For example, heterozygous ATM+/− mice shows reduced complex I activity in the liver, but not in the heart5. However, the latter study suggests that accumulating nuclear double strand breaks in the heterozygous model could lead to mitochondrial dysfunction5. The same research group found significantly increased markers of oxidative stress in ATM+/− mouse hearts compared to wild-type litter mates, which was significantly reduced when fed a mitochondrial specific anti-oxidant40. Consequently, the underlying mechanism for ATM in this context still remains unknown, and warrants further research to disseminate between acute  and chronic responses where DNA damage could lead to mitochondrial dysfunction in response to the absence or inhibition of ATM.The IMM consists of mitochondrial cristae and contains the electron transport complexes as well as ATP synthase which is responsible for energy conversion and ATP production41. ATM promotes glucose flux through the PPP by increasing both the abundance as well as activity of G6PD, consequently increasing NADPH production which is required for the anti-oxidant response in cells11. Moreover, the inhibition of COX increases ROS production and electron leak at complex I or III by reducing the redox centres of these complexes42. A reduction in the redox centre of Complex I should thus lead to decreased NADH oxidation if ATM inhibition impacts complex I.The acute inhibition of ATM decrease Cytochrome C oxidase  activity in permeabilised muscle tissue in a post-transcriptional manner (within an hour of KU55933 treatment), and is correlated with an increase in G6PD, which suggests a shift towards glycolysis32. Reduced NADH, and its phosphorylated form, NADPH are autofluorescent electron donors with identical fluorescent spectra, and is referred to as NAD(P)H due to the uncertain origin of the signal33. Changes in the lifetime of NAD(P)H are determined by fitting an exponential function to the observed time-resolved fluorescence decay, and have been used to distinguish between mitochondrial protein localisation44, redox ratio45 and glucose carbon diversion32. It has been suggested that the protein-bound form of NADH localizes mainly within the mitochondria whilst free NADH is primarily located within the cytosol46. We observed a bound lifetime (τ-bound) of 2.76 ± 0.26 ns H fluorescence lifetime decay to 2.57 ± 0.14 ns H τ2 can effectively and non-invasively quantify diversions of carbon away from the TCA cycle or the ETC32  which is sensitive to microenvironment changes at the molecular level and can distinguish between free (τ1) and enzyme-bound NAD(P)H (τ2) based on fluorescence lifetime (where τ1 > τ2), which makes it a useful tool for probing metabolism in living cells ns Fig. . Inhibit50 and can be imported in response to stress. Our results indicate all are possible.We showed that ATM is located within cardiac mitochondria under normoxic conditions, and our results contribute towards a better understanding of the potential role of ATM in mitochondrial function through ETC complex I, as well as its potential contribution towards the development of cardiovascular dysfunction by decreasing oxidative phosphorylation efficiency. The results presented here stress the need to further establish the role of ATM and determine whether ATM directly phosphorylates mitochondrial proteins, is activated in response to mitochondrial damageAll the procedures involving animals were in compliance with the South African National Standard for the care and use of animals for scientific purposes (SANS 10386:2008); the Medical Research Council Guidelines on Ethics for Medical Research; Book 3: Use of Animals in Research and Training and the National Institutes of Health Office of Laboratory Welfare: The Guide for the Care and Use of Laboratory Animals, 8th Edition; USA, and was approved by the Research Ethics Committee: Animal care and Use, Stellenbosch University, South Africa .Male Wistar rats were injected with Eutha-naze , and euthanised through exsanguination after rapid excision of the heart.51. For oxidative active mitochondria, all isolations were performed in ice-old mitochondrial isolation buffer . Briefly, the ventricular tissue was homogenised with a Heidolph SilentCrusher M, followed by a slow centrifugation step (755 × g for 10 minutes at 4° Celsius) in order to obtain a pellet containing the nuclei, cell membranes and remaining tissue. The supernatant was transferred to a clean centrifuge tube and centrifuged at 18 800 × g for 10 minutes at 4 °C to obtain a mitochondrial pellet. The pellet was re-suspended in ice-cold mitochondrial isolation buffer with a 2 cm3 Potter-Elvehjem glass-teflon homogenizer, and stored on ice until use.Mitochondrial isolation was performed with a standard differential centrifugation protocol52 and53 , finely diced, and subjected to homogenization (Heidolph SilentCrusher M). Mitochondria were isolated as described above and resuspended in ice cold M/S buffer. The mitochondrial fraction was washed with centrifugation at 10 000 × g for 10 min at 4 °C, and resuspended in ice-cold M/S buffer. Protein concentration was determined with a Bradford protein assay54 prior to 1.2% digitonin treatment . A volume equal to 100 μg mitochondria was aliquoted into a clean Eppendorf tube, and an equal volume of 1.2% digitonin (prepared by boiling for 1 minute in M/S buffer) was added. An aliquot was taken at the moment of addition (0 minute time point), and the reaction was quenched in a 10x dilution with ice-cold M/S buffer containing bovine serum albumin . The master digitonin-permeabilisation reaction was allowed to continue for 30 min whilst gently stirring at 0° Celsius, and aliquots were taken at 15 min, 20 min and 30 min time points, and quenched as previously described. The quenched 10x dilution time-point reactions were subsequently centrifuged at 10 000 × g for 10 min at 4 °C (Sigma 1–14 K) to obtain the mitoplasts (sediment), and the supernatant was carefully aspirated. The supernatant was centrifuged at 100 000 × g for 1 hour at 4° Celsius to obtain the OMM fraction.For subfractionation, the atria were removed directly after excision, and the remaining ventricular tissue was placed in ice-cold D-mannitol/sucrose buffer adapted fromIn brief, samples were processed with a Leica EM tissue processer and were scoped with a JEOL 10–11 transmission microscope  and a SIS imaging according to standard protocol .56) and VDAC , or ATM and TOM20 , washed and probed with the appropriate fluorescently Goat Anti-Rabbit IgG H&L  and Goat Anti-Mouse IgG H&L  secondary antibody.Super-Resolution Structured Illumination Microscopy (SR-SIM) was performed at the Stellenbosch University Central Analytical facility (CAF) Fluorescence Microscopy unit. Mitochondria and mitoplasts were prepared as described previously and fixed with 2.5% glutaraldehyde. The samples were incubated overnight with either ATM  z- stacks of high-resolution image frames were collected with 5 rotations by using an alpha Plan-Apochromat 1006/1.46 oil DIC M27 ELYRA objective and an ELYRA PS.1 (Carl Zeiss Microimaging) microscope equipped with a 488 nm laser (100 mW), 561 nm laser (100 mW) and Andor EM-CCD camera (iXon DU 885). Micrographs were reconstructed using ZEN software  using a structured illumination algorithm3, 4.7 mM KCl, 1.2 mM KH2PO4, 0.6 mM MgSO4·7H2O, 0.59 mM Na2SO4, 1.25 mM CaCl2·12H2O and 10 mM glucose; pH 7.4) at a constant pressure of 100 cm H2O and gassed with 95% oxygen and 5% carbon dioxide. Hearts were stabilized for 20 minutes, after which they were perfused in a recirculating manner with KHB containing 3 μM KU60019 (n = 5) or an equal volume DMSO . Protein determination for the mitochondrial data was performed with a Lowry assay58.Excised hearts (n = 10) were cannulated via the aorta and perfused with Krebs-Henseleit buffer , equilibrated to ambient oxygen levels at 25 °C, were analysed in parallel. Data was captured polarographically with HansaTechPlus software. Oxygen consumption prior to the addition of ADP (State 2), in the presence of ADP (State 3), and after complete utilization of ADP (State 4) was measured as well as State 3 oxygen consumption after 20 minutes anoxia followed by reoxygenation was noted. These values were used to calculate the oxygen consumption , oxidative phosphorylation rate , respiratory control rate , and the ADP/O ratio.Mitochondrial oxygen consumption was analysed with a Clarke-type oxygraph  immediately following isolation. Briefly, two chambers containing a glutamate or palmitoyl-L-carnitine assay buffer , 8.5 mM K4, 0.5 mM NaF, 2% SDS). The samples were boiled for four minutes and stored at −80° Celsius until use. Samples were boiled again for 4 minutes prior to being loaded on 4–15% Mini-PROTEAN® TGX™ Stain free Precast Protein Gels (Bio-Rad Laboratories), and subjected to electrophoresis for 1 hour. Due to major size differences in the proteins investigated 59; VDAC #4866, 32 kDa, CST; and ANT 1/2/3/4 #H-188, 34 kDa Santa Crus Biotechnology60), a modified transfer buffer was used , and the gels were allowed to rest for 30 minutes in the buffer prior to activation in the ChemiDoc MP system (Bio-Rad Laboratories), and transfer. Transfer was performed at 200 V and 200 mA for 1 hour 30 minutes at 4° Celsius onto an Immobilon®-P PVDF membrane (EMD Millipore). The membranes were briefly placed in MeOH, and allowed to dry before blocking in a 5% liquid fat-free milk solution. The membranes were cut below the 170 kDa molecular mark for ATM, and probed from 170 kDa to- 450 kDA for ATM. For VDAC and ANT1/2/3/4 the membranes were cut just below the 55 kDa and probed from approximately 20 kDA to just above 43 kDA. The antibodies were diluted 1:1000 as follows: SignalBoost™ Immunoreaction Enhancer Kit (EMD Millipore) for ATM protein kinase antibody (CST); TBS-T for VDAC antibody and secondary antibody, and TBS-T (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20) with 2.5% fat free milk solution for ANT 1/2/3/4 (SCBT) and its secondary antibody. Donkey anti-rabbit secondary antibody  was diluted 1:4000 in either SignalBoost™ Immunoreaction Enhancer Kit (EMD Millipore) for ATM detection, TBS-T for VDAC and TBS-T + 2.5% milk for ANT1/2/3/4. After primary and secondary antibody incubation the membranes were treated with Clarity™ Western ECL Blotting Substrate and visualised on the ChemiDoc MP system on the Chemi Hi sensitivity setting, which was stopped prior to overexposure (which appears red on program settings).Briefly, the mitochondrial, mitoplast, OMM, IMM and matrix fraction lysates were prepared for western blotting with the addition of one volume M/S buffer and one volume 1x SDS buffer  or 3 μM KU60019 (n = 3), 3–6 hours prior to imaging. HEK293 cells were previously obtained from ATCC (CRL-1573™), and were maintained in MEM (Gibco) with 2 mM-L-Glutamine, 10% FBS and 1% pencillin/streptomycin at 37 °C and 5% CO2. Cells were seeded at a concentration of 1.5 × 105 cells/ml on 35 mm MaTIK plates for 48 hours prioris to treatment. The HEK293 cell cultures were treated identical to the H9c2-cell cultures .H9c2 (2-1) cells were obtained from ATCC®  and maintained in DMEM (Gibco) with 4 mM L-Glutamine, 4.5 g/L glucose, 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO62. Multiphoton excitation was generated from a titanium: sapphire laser, tuned to 760 nm, operating at 76 MHz with 200 fs pulse width . A BG39 filter was used to block the near infra-red light before non-descan detection using a hybrid detector, HPM-100 . A pixel dwell time of 5.04 μs was used for 256 × 256 pixel images over a 212 μm × 212 μm field of view. TCSPC electronics  were used to acquire fluorescence decay curves with 256 time bins. Following a 3 × 3 binning of photons and a threshold of 25, two-component fluorescence decay curves were used to fit the decay curves for FLIM images using iterative reconvolution in SPCImage 33. NAD(P)H fluorescence lifetime parameters were obtained from the FLIM data and reported as an average of 10 pixels over at least three to five regions of three separate cultures imaged on different days.These experiments were performed at the Rutherford Appleton Laboratory, the Science and Technology Facility Council, Oxfordshire, UK. Fluorescence intensity and lifetime images were acquired using a custom-built multiphoton fluorescence lifetime system around a modified inverted microscope . Samples were imaged using a 60× , NA 1.2 water-immersion objectiveThe data were analysed using GraphPad Prism 6.0 software . Statistical comparisons were made using a two-tailed Student’s t-test or 2-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test where applicable. All values and error bars are presented as mean ± s.d, and the level of significance was set at p < 0.05.Supplementary Information: Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation


The results showed that these compounds exhibited tyrosinase inhibitory activity. Compound 3--6,8-dihydroxycoumarin (8) is the most potent compound (0.27 mM), more so than umbelliferone (0.42 mM), used as reference compound. The kinetic studies revealed that compound 8 caused non-competitive tyrosinase inhibition.In the present work we report on the contribution of the coumarin moiety to tyrosinase inhibition. Coumarin-resveratrol hybrids  The active centre of tyrosinases , formed by dinuclear coppers, is flexible during catalysis.Tyrosinase EC 1.14.18.1) is an enzyme with a dinuclear copper centre, widely distributed in Nature and mainly involved in the formation of pigments such as melanins and other polyphenolic compounds .14.18.1  establisWithin different taxa, the sequence homology of tyrosinases is high and conserved domains can be identified . The enzThe study of tyrosinase inhibitory activity became of interest in recent years because of the significant industrial and economic impact of the inhibitors of this protein. Recently, different inhibitory compounds derived from natural sources or partly/fully synthetic have been tested . StudiesOur research aimed to study compounds that hypothetically would be good tyrosinase inhibitors, by using all available structural information. Our experience on both coumarin and resveratrol structures, it has encouraged us to examine coumarin-resveratrol hybrids in this context.trans-stilbene) is a polyphenolic phytoalexin produced in plants via a metabolic sequence induced in response to biotic or abiotic stress factors. It has been found in a multitude of dietary plants and derived products, such grapes, peanuts, legumes and in red wine. It has been shown to be an antimutagen, antioxidant, and inducer of quinone reductase. In addition, many biological effects have been attributed to resveratrol, such as modulation of hepatic apolipoprotein, activation of the estrogens’ receptor, inhibition of platelet aggregation and in vitro eicosanoid synthesis, inhibition of transcription and enzyme activity of cyclooxygenase-2 [Resveratrol  oxidizing diphenolase activity of tyrosinase of the hydroxyphenyl coumarin derivates and umbelliferone are summarized in 1 is an umbelliferone analogue with one phenyl group at C3. The phenyl substituent led to a dramatic loss of potency, and compound 1 did not inhibit any tyrosinase activity. This result indicates that 3-phenyl substituent of umbelliferone may be an important factor for the absence of tyrosinase inhibition activity.Inhibitory effects on 2. Furthermore, we decided to examine the effect of adding hydroxyl groups at the 4’ position of compound 1 (thus obtaining compound 3). As shown in 4), suggesting that a different position of hydroxyl group substituent of the A ring might be advantageous.Having identified the importance of the 4’-hydroxyl group of flavonoid compounds in the pharmacological activity, we studied the role of hydroxyl group at position 4’ of the coumarin-resveratrol hybrid 5-8 with hydroxyl groups in positions 3’ and 5’ were found to exhibit different inhibitory potencies, depending on the position and number of hydroxyl groups in the A and B rings. The activities of the compounds 5-7 did not provide interesting results as none of them showed any significant inhibitory activity (8 showed the highest tyrosinase inhibitory activity (IC50=0.27 mM). This could be due to a synergistic effect of the hydroxyl groups on the aromatic rings. This implies that the number of free OH groups plays an important role in determining the activity. It is unclear, however, why the additive effect of the 4’-hydroxyl is different from that of the other hydroxyl substitution patterns, although Gao and co-workers have reported the inhibitory activity of 3’,4’,5,5’,7-pentahydroxyflavone that has a similar structure to compound 8 [The coumarin-resveratrol hybrids activity , but commpound 8 . Further8, we studied its effect on the diphenolase activities of the enzyme. A plot of 1/V vs inhibitor concentration  A, and the corresponding arylacetic acids B, using dicyclohexylcarbodiimide (DCC) as dehydrating agent  between ng agent  [25,26,2ent  was added and the reaction was monitored at 475 nm for 5 min. The detected absorbance increases accompanying the oxidation of the substrate (L-DOPA). The final concentration of dopaquinone was measured using the molar absorption coefficient of 3,700 M-1 cm-1 [L-DOPA per minute. The percentage of inhibition of tyrosinase activity was calculated as inhibition (%) = (A-B)/A×100, where A represents the difference in the absorbance of control sample between an incubation time of 0.5 and 1.0 min, and B represents the difference in absorbance of the test sample between an incubation time of 0.5 and 1.0 min. The activity of mushroom tyrosinase was determinate by spectrophotometric techniques (Varian Cary 50). DMSO inhibitory activity, in the same experimental conditions, was evaluated as 10.7%.Tyrosinase activity assays were performed with escribed  with sliM-1 cm-1 . One uni1-8 on mushroom tyrosinase activity have been evaluated. The experimental data showed that compound 8 possesses the highest activity among the tested hybrids, and it was a non-competitive inhibition with respect to L-DOPA. This result indicates that the number and the position of free OH groups play an important role in determining the activity. Further experiments are in progress in order to know how these compounds interact with the enzyme on molecular basis. Moreover, since the initial step in melanin synthesis is also the same in catecholamine synthesis, we are beginning to study a possible close relationship of inhibitory tyrosinase activity and vasorelaxant activity.Inhibitory effects of hydroxylated coumarin-resveratrol hybrids


The methylbenzenesulphonate derivative 1f (IC50 = 0.38 ± 0.01 μM) was found to be the most active h-TNAP inhibitor. Another 4-fluorobenzenesulphonate derivative 1i (IC50 = 0.45 ± 0.02 μM) was found as the strongest inhibitor of h-IAP. Some of the derivatives were also identified as highly selective inhibitors of APs. Detailed structure-activity relationship (SAR) was investigated to identify the functional groups responsible for the effective inhibition of AP isozymes. The study was also supported by the docking studies to rationalise the most possible binding site interactions of the identified inhibitors with the targeted enzymes.Tissue-nonspecific alkaline phosphatase (TNAP) is an important isozyme of alkaline phosphatases, which plays different pivotal roles within the human body. Most importantly, it is responsible for maintaining the balanced ratio of phosphate and inorganic pyrophosphate, thus regulates the extracellular matrix calcification during bone formation and growth. The elevated level of TNAP has been linked to vascular calcification and end-stage renal diseases. Consequently, there is a need to search for highly potent and selective inhibitors of alkaline phosphatases (APs) for treatment of disorders associated with the over-expression of APs. Herein, a series of tricyclic coumarin sulphonate  Further, the most effective inhibitors of either h-TNAP or h-IAP were selected for the molecular docking studies to rationalise the possible binding interactions with the respective targeted enzymes.Recently, we have reported coumarinyl sulphonates derivatives as novel and potent inhibitors of supplementary file.The synthetic procedures, purification methods, and the spectral analysis data have been reportedh-TNAP (46 ng/well) or of h-IAP (57 ng/well). The plates were incubated at 37 °C for 5–10 min, and luminescence signals were recorded by microplate reader . Then, 20 µl of CDP-star® substrate was added to each reaction mixture and signals were again measured as after-read, after 15–20 min of incubation. The data were scrutinised by PRISM 5.0  software and the inhibitory concentration values  were attained as discussed earlier.The inhibition assay was performed by following the already reported method1f and 1i, potent inhibitors of h-TNAP and h-IAP, respectively, were conducted to investigate the probable binding interactions of these compounds within the active site of the respective target enzyme. Our previously reported models of h-TNAP and h-IAP were used for docking calculationMolecular docking studies of compounds 1a-za were prepared. Cycloheptanone (2a) or cyclooctanone (2b) was reacted with diethyl carbonate in the presence of NaH in benzene at the reflux temperature to get the ester intermediates 3a,b that exist in keto-enol tautomers4a-f1a-za1a-za were tested for their potential to inhibit h-TNAP and h-IAP. Except two compounds (1w and 1 y), all compounds were found to be active against both isozymes of AP with IC50 values in the lower micro-molar range. Compounds showed their h-TNAP inhibitory potential in the range of IC50 = 0.38 ± 0.01 to 8.57 ± 0.73 μM as compared to standard levamisole (IC50 = 20.21 ± 1.9 μM), and h-IAP inhibitory activity in the range of IC50 = 0.45 ± 0.09 to 11.8 ± 1.91 μM as compared to standard L-phenylalanine (IC50 = 100.1 ± 3.15 μM) (All synthesised tricyclic coumarin sulphonate derivatives 3.15 μM) .1a-za were divided into two categories, on the basis of fused cycloheptane and cyclooctane ring. Moreover, the detailed structure-selectivity relationship was also rationalised on the basis of effect of unsubstituted, and chloro- and methoxy-substituted derivatives on the inhibition of both APs. Compounds 1q and 1z were found as selective inhibitors of h-TNAP while none of the derivative was found as a selective inhibitor of IAP.In order to find out the comprehensive structure-activity relationship for AP inhibition, all tricyclic-fused coumarin sulphonates 1f expressed maximum inhibitory effects on h-TNAP and compound 1i depicted the strongest potency against h-IAP. From the unsubstituted derivatives, except compound 1c, 1g, and 1m, the other target derivatives expressed varying inhibitory effects on h-TNAP. Compounds 1f, 1a, and 1d possessing p-toluene sulphonate, methane sulphonate, and cyclopropane substitution, respectively, depicted higher inhibition of h-TNAP than compounds 1c, 1g, and 1j-m. Among the cyclooctane-fused derivatives, compound 1l with benzenesulphonate substitution demonstrated the strongest inhibitory potential on h-TNAP (IC50 ± SEM = 1.13 ± 0.21 µM). Compounds 1c with the fused cycloheptane ring and compound 1j with the fused cyclooctane ring were found as stronger inhibitors of h-IAP with the inhibitory values of IC50 ± SEM = 0.91 ± 0.06 and 0.84 ± 0.09 µM, respectively. Among the cyclooctane-fused derivatives, compound 1j with ethanesulphonate substitution demonstrated the strongest inhibitory effect on h-IAP (IC50 ± SEM = 0.84 ± 0.09 µM). From the cycloheptane-fused derivatives, compound 1c with propanesulphonate substitution demonstrated the strongest inhibitory potential on h-IAP (IC50 ± SEM = 0.91 ± 0.06 µM). Although more lipophilic side chain was substituted in 1c, the inhibitory effect of this derivative was found less because of the presence of seven-membered fused ring. Moreover, from the obtained results this may be rationalised that the presence of more lipophilic side chain in compound 1c may form stronger hydrophobic interactions with the target enzyme.Among the tested derivatives, compound 1h and 1n with tert-butylbenzene suphonate moiety expressed the dual inhibition of both isozymes of APs. The derivative 1n depicted more inhibitory potential because of the presence of fused eight membered ring . In comparison to 1h, the replacement of tert-butylbenzene with flouorobenzenesulphonate results in the most potent inhibitor of h-IAP i.e. 1i. Compound 1i was identified as the strongest inhibitor of h-IAP with and inhibitory value of IC50±SEM = 0.45 ± 0.09 µM. The increased potency might be due to the presence of most electronegative halogen.The inhibitory effect by other derivatives; h-TNAP, compound 1o (IC50±SEM = 1.21 ± 0.11 µM) was found more potent as compared to compound 1q (IC50±SEM = 4.31 ± 0.99 µM), 1p (IC50±SEM = 1.49 ± 0.13 µM), and 1r (IC50±SEM = 2.60 ± 0.49 µM) but with reference to h-IAP, 1p (IC50±SEM = 1.51 ± 0.17 µM) was found more better than 1o (IC50±SEM = 11.8 ± 1.91 µM) and 1r (IC50±SEM = 0.75 ± 0.07 µM) was found better than 1q (IC50 ± SEM = >100 µM). It means that the presence of functional group is playing more important role on the inhibitory effects. The derivatives with less carbon number were found more potent that the propane derivative. But, with detailed SAR it was found that the heptane ring was favourable for TNAP, while octanone derivative was found potent towards IAP. When the activity of 1o was compared with 1r and 1p, it was observed that the reduced inhibitory effect of both the derivatives on TNAP was due to the presence of n-propane substitution in case of 1r, and the presence of fused cyclooctane ring in case of 1p.On the other hand, the chloro- and methoxy-substituted derivatives were found more active against both APs than the unsubstituted analogues. With reference to 1o, compound 1s having benzenesulphonate substitution was found almost as an equipotent inhibitor of TNAP with the inhibitory value of 1.26 ± 0.07 µM. The other derivative i.e. 1t having the same substitution of benzenesulphonate as in compound 1s, but with the cyclooctane ring, a reduced inhibitory effect on TNAP was observed with an increased inhibitory effect on IAP isozymes . The derivatives 1w and 1x having the same substitution at R1 and R2 positions but with cycloheptane and cyclooctane ring in 1w and 1x, respectively. When the SAR of these two derivatives was considered, it was observed that cycloheptane derivative 1w was found less reactive than that of the cyclooctane derivative 1x which inhibited both isozymes effectively. This inhibitory effect confirms that like 1p, the presence of more carbon number in the derivative is responsible for the more inhibition.In comparison to 1u and 1za processing cycloheptane ring along with p-tosylate substitution at R2 position but different substitution at R1 position. The derivative with electron-donating group i.e. OMe in case of 1u (IC50±SEM = 0.79 ± 0.05 µM) was found more active inhibitor as compared to the derivative 1za (IC50±SEM = 1.11 ± 0.01 µM) with electron-withdrawing substitution. These two derivative also exert some inhibitory potential on h-TNAP, but less  in comparison to h-IAP. The other derivatives 1 y and 1z with methanesulphonate and benzenesulphonate substitution were found inactive towards h-IAP. Moreover, it was found that the aromatic sulphonate derivatives 1s, 1u, and 1za depicted stronger inhibitory effects on h-IAP than h-TNAP.An interesting inhibition of IAP was also exhibited by the aromatic derivative h-TNAP than the corresponding analogues having fused cyclooctane ring. Similarly, an inverse effect was observed towards h-IAP. It can be suggested from the obtained data that the derivatives with more lipophilic side chains led to the more inhibition of h-IAP and vice versa in case of h-TNAP. This lipophilic side chains might be involved in some hydrophobic interactions within the pocket of the receptor site and increase the affinity of such derivatives for the targeted enzyme and ultimately accounts for the enhanced h-IAP inhibition.The results obtained here are in consistent with our previously reported work. The 2-methoxy- or 2-chloro- substituted derivatives possessing the fused cycloheptane ring were found stronger inhibitors of 1f and 1i were carried out within the respective target enzyme and their most probable binding interactions within the modelled enzymes are depicted in 1f within h-TNAP revealed that oxygen atom in both sulphonate group and carbonyl group are responsible for making three strong hydrogen bonds with amino acid residues. Two hydrogen bonds were formed by oxygen of sulphonate group with amino groups of Arg167 while the third hydrogen bond was formed by carbonyl oxygen in coumarin ring with amino acid Thr436. Additional to hydrogen bonding, second oxygen atom of the sulphonate group also formed direct interaction with the Zn+2 ion within the active site of h-TNAP. The excellent h-TNAP inhibition activity of compound 1f can be related with this direct binding interaction of sulphonate oxygen with the Zn+2 ion of the active site. Furthermore, two π–π interactions were formed by benzene ring adjacent to sulphonate group with amino acid residues His154 and His321. One π-alkyl interaction was also exhibited by the seven-membered ring fused with the coumarin ring and His434 as shown in 1i with h-IAP is shown in 1f with h-TNAP, compound 1i formed four hydrogen bonds with different amino acids of h-IAP. Oxygen atom in sulphonate group of compound 1i also formed two hydrogen and one metal interaction. These two hydrogen bonds were formed by one oxygen atom in sulphonate group with amino acid residue of Arg166 and His143. While other oxygen of sulphonate group formed interaction with Zn+2 ion. Carbonyl oxygen in coumarin ring and fluorine atom in compound 1i also formed hydrogen bond with amino acid residues Arg150 and Gln108 as shown in 1i. Docking of the inactive derivatives 1q and 1w showed weaker affinity and fewer bonds formed, which supports the SAR discussion. The figures are given in the supplementary file.Molecular docking studies of the most potent derivatives i.e. 1a-za were evaluated for their effective inhibitory potential against h-TNAP and h-IAP. Except 1w and 1 y derivatives, all the other compounds exhibited excellent inhibitory potential against both h-TNAP and h-IAP. Methylbenzenesulphonate derivative 1f (IC50 = 0.38 ± 0.01 μM) was found to be the most active h-TNAP inhibitor. Another 4-fluorobenzenesulphonate derivative 1i (IC50 = 0.45 ± 0.02 μM) was found as the strongest inhibitor of h-IAP. Detailed SAR studies and molecular docking studies were performed to identify the importance of functional groups within the structures and to explore the possible binding site interactions, respectively. Moreover, previously these derivatives have been reported for their antiproliferative effects against different cell lines including, leukaemia, CNS, prostate, renal, colon, lung cancer, melanoma, ovarian, and breast cancers. From that data we got 1i and 1 m as the strongest antiproliferative derivatives against colon cancer cell line along with the maximum anti-inflammatory effects. Here in this study, we have identified these compounds as stronger inhibitors of h-IAP isozymes. This can be rationalised that the IAP, responsible for the maintenance of intestinal homeostasis, might be over-expressed in the colon cancer cell lines. Moreover, the over-expression of h-TNAP can be associated with the lung cancer, melanoma, ovarian, breast cancers, and renal cancer. Therefore, the obtained results synergise with the previously reported data. It can be suggested that the potent inhibitors of both h-TNAP and h-IAP can be selected for further exploration at molecular level.Tricyclic-fused coumarin sulphonates