PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals  Howe in vivo . Our rec in vivo . We furtsruption . We hypoin vivo in multiple settings including myocardial ischemia, skin inflammation, and T cell senescence . Activation of p38α by TAB1 binding has been shown to function nescence , 10. We ammation , 5. HoweIn contrast to canonical p38 activation, noncanonical activation of p38 by endothelial GPCRs occurs primarily from endosomes. We showed that ubiquitination of PAR1 by activated NEDD4-2 E3 ubiquitin ligase initiates recruitment of TAB2, a structural homolog of TAB1, that binds to TAB1 and triggers p38 activation . While aTo elucidate the pathways and proteins that engender thrombin-activated PAR1-stimulated p38 signaling in human endothelial cells, we used a quantitative phosphoproteomic approach. Here, we report the global thrombin phosphoproteome regulated by p38 using tandem mass tag (TMT) mass spectrometry (MS). Our study unveils four unique dynamic phosphoproteome profiles of thrombin signaling regulated by p38 and the identification of multiple enriched biological functions associated with microtubules, focal adhesions, stress fiber, endocytosis, Rho, small GTPases, and cell–cell adherens junctions, which function as key modulators of endothelial dysfunction. We also identified distinct sets of kinases and subsets of candidate proteins predicated to mediate thrombin-stimulated p38-induced cellular responses. In validation studies, we show that p38 negatively regulates basal phosphorylation of extracellular signal–regulated protein kinase 1/2 (ERK1/2) in endothelial cells resulting in amplified ERK1/2 phosphorylation after thrombin stimulation. We further show that p38 uniquely mediates phosphorylation of α-catenin, a key component of adherens junctions, which has not been previously reported. Together, these studies identify an important array of proteins and pathways that specify thrombin-stimulated p38 signaling in endothelial cells.A). To identify p38 MAPK targets that mediate thrombin-induced proinflammatory responses, human cultured endothelial EA.hy296 cells were pretreated with SB203580, a p38α and p38β selective inhibitor, or dimethylsulfoxide (DMSO) vehicle control and then stimulated with thrombin to determine changes in protein phosphorylation linked to p38 signaling. Cell lysates were collected from the nonagonist treated, 0 min pretreated DMSO and SB203580 controls, performed with two biological replicates, and three thrombin-stimulated replicates for each condition and phosphopeptides were enriched by TiO2, processed for TMT 10-plex labeling and analyzed by LC-MS2/MS3 to quantify the proteome and phosphoproteome  . The phosphosite distribution was 84.6% phosphoserine, 13.5% phosphothreonine, and 1.9% phosphotyrosine . The majority of the peptides were phosphorylated at a single site (78.5%) with 19.9% peptides phosphorylated at two sites and considerably fewer (1.6%) peptides phosphorylated at three sites . Cell lysates from endothelial cells treated with or without SB203580 stimulated with thrombin showed agonist-induced p38 phosphorylation in DMSO control cells that was markedly reduced in cells pretreated with the p38 inhibitor SB203580 , confirming thrombin-stimulated p38 signaling is inhibited by SB203580 pretreatment in EA.hy926 as we previously reported -41 residue and activation of PAR1 resulting in ubiquitin-driven noncanonical stimulation of p38α MAPK signaling mediated by TAB1 and TAB2 on endosomes  Fig. 1AAFig. 1A.Fig. 1B) , 13. We Fig. 1B)  (https:/A and B) (C), indicating minimal variance of the 0 min control replicates. The thrombin-induced phosphoproteome with and without the p38 inhibitor SB203580 pretreatment separated into five temporally distinct clusters (C) of phosphopeptides. C1 peptides displayed a significant increase in phosphorylation after thrombin stimulation that was reduced by SB203580, whereas C2 peptides showed a more modest significant increase in phosphorylation following thrombin incubation that was markedly enhanced in cells pretreated with the SB203580 p38 inhibitor (A and B). Phosphorylation of C3, C4, and C5 peptides was significantly decreased after thrombin stimulation (A and B). Inhibition of p38 by SB203580 increased C3 peptide basal phosphorylation at 0 min compared to vehicle control, whereas C5 peptides showed a decrease in basal phosphorylation at 0 min in SB203580 treated cells (A and B). In either case, thrombin reduced C3 and C4 peptide phosphorylation, which appeared greater in C3 compared to C5 (A and B). No change in phosphorylation was detected in C4 peptides in SB203580-treated cells following thrombin stimulation compared to DMSO control (A and B). These data indicate that thrombin stimulates four distinct temporal dynamic changes in protein phosphorylation that are differentially modified by the inhibition of p38 MAPK signaling.Temporal changes in the thrombin phosphoproteome in the presence or absence of the SB203580 was evaluated using κ-means clustering of significantly altered phosphopeptides , A and BA and B) . SpearmaA and B) C, indicanhibitor , A and Bmulation , A and Bed cells , A and Bed to C5 , A and B control , A and BD). Focal adhesion, integrin signaling, and stress fiber were enriched in C2, whereas the actin-binding calponin homology domain was represented in both C1 and C2 clusters . Thus, C1 and C2 were enriched in important mediators of endothelial inflammatory responses. C3 was also significantly enriched in the mediators of endothelial proinflammatory responses such as cell junction, PDZ, and ANK domain-containing proteins, autophosphorylation and calcium . Interestingly, thrombin-modulated C4 phosphopeptides appeared to be independent of p38 activity and were significantly associated with endocytosis, clathrin-coated pit, and modulators of endothelial barrier disruption such as cell–cell adherens junction, PDZ, and Src homology-3 domain . The C5 cluster having the lowest phosphopeptide representation was enriched in gene expression and nucleus  and may be attributed to thrombin-stimulated p38-mediated regulation of gene expression , 1514, 1Fig. 2pression . Overallpression , 18, conpression .A). Several other kinases including protein kinase D (PKD) and MAPKAPK  were also enriched in C1 . Cluster 2 showed high enrichment of pyruvate dehydrogenase kinase 1 and cyclin-dependent kinase (CDK) . Kinase enrichment was less evident in C3 and C4 within the experimental time-frame examined . Using the motif-X algorithm, we found the strongest enrichment was for the RRxS motif in Cluster 1 and for the PxSP motif in Cluster 2 (B and C). The motif logos show the amino acid distributions around the RRxS and PxSP consensus sites of the significant phosphopeptides (B and C). Other ACG kinases and CMGC kinases were also predicted in Cluster 1 and 2 based on GPS analysis . The RRxS motif is a PKA consensus phosphorylation site and the PxSP motif is a consensus phosphorylation site for CDKs. Thus, two independent prediction analysis are largely in agreement and consistent with the literature implicating a role for many of these kinases particularly for PKA and CDK in thrombin signaling.To identify the kinases known or predicted to target proteins for phosphorylation in the specific clusters of the thrombin phosphoproteome regulated by p38, we used group-based prediction system (GPS) algorithm  to prediFig. 3A). Cells pretreated with SB203580 and stimulated with thrombin resulted in significant changes in 342 total phosphopeptides, of which 223 phosphopeptides from 172 unique proteins displayed increases in phosphorylation, and 119 phosphopeptides from 88 unique proteins showed decreases in phosphorylation . The corresponding heat maps of the individual TMT-MS replicates are highly similar (A and B), verifying reproducibility of the data.The effect of thrombin on specific subsets of phosphopeptides was examined using pi-score analysis, which combines statistical significance with agonist-induced fold change in phosphorylation into a single, comparable value . In contFig. 4C and D). The six phosphopeptides with the greatest significant decreases in phosphorylation represent six unique proteins including ankyrin repeat domain 50 (ANKRD50) a regulator of membrane trafficking and recycling, casein kinase 1 delta (CSNK1D), α-catenin-1 or α-E-catenin (CTNNA1), an adapter that binds to cadherins and facilitates interaction with the actin cytoskeleton, the scaffold neuroblast differentiation-associated protein AHNAK and the transcriptional regulators tripartite motif containing 28 (TRIM28) and transforming growth factor-β-stimulated clone 22 domain family member 4 (TSC22D4) (C and D). Heat maps of the corresponding individual TMT-MS replicates are similar indicating high reproducibility of the data (D). Together, these findings reveal the identity of several key proteins displaying divergent changes in phosphorylation in response to thrombin and p38 activity. These candidate proteins have not been previously linked to thrombin-induced p38-regulated signaling.Next, we examined the effect of p38 inhibition by SB203580 on thrombin-induced changes in a subset of phosphopeptides using pi-score analysis. Eight phosphopeptides with the most significant increases in phosphorylation encode six unique proteins. The six proteins include nuclear factor kappa B subunit 1 (NF-κB1), a key inflammatory mediator; son of sevenless homolog 1 (SOS1), a guanine nucleotide exchange factor for Ras; MAPK1 , the key effector of the MAPK/ERK cascade; the transporter solute carrier family 12 member 2 (SLC12A2), and zinc finger protein 326 (ZNF326) that facilitates transcript elongation and alternative splicing , C and DTSC22D4) , C and Dthe data D. TogethA). These findings suggest that p38 MAPK may function in a negative feedback loop to attenuate NF-κB1 S907 phosphorylation following thrombin stimulation.We evaluated thrombin-induced p38-regulated changes in specific phosphosites of key proteins predicted or shown to associate with endothelial dysfunction. The NF-κB transcription factor, formed by the NF-κB1-105 kDa protein in complex with Rel-like domain-containing proteins, is activated by thrombin in endothelial cells and promotes inflammatory responses . PhosphoFig. 5B, Inset). In DMSO control cells, thrombin caused a marked increase in endothelial barrier permeability that was significantly reduced in cells pretreated with U0126, at 40 and 60 min (B). These findings indicate that ERK1/2 functions as an effector of thrombin signaling to regulate endothelial barrier disruption. Interestingly, analysis of the thrombin phosphoproteome indicates that inhibition of p38 with SB203580 alone enhanced basal phosphorylation of ERK1 and ERK2 peptides at multiple sites compared to DMSO control cells (C and D). Despite increased basal ERK1/2 phosphorylation in SB203580-treated cells, thrombin modestly increased ERK1/2 phosphorylation, at multiple sites including ERK1 T202, Y204, ERK2 T185, Y187, T190, ERK2 T185, Y187, and ERK2 T181, T185 peptides (C and D). These findings suggest that p38 may function in multiple negative feedback loops to control changes in NF-κB and ERK1/2 protein phosphorylation, key proteins associated with endothelial dysfunction.Next, we first established ERK1/2 function in endothelial dysfunction using the U0126 MEK inhibitor, which effectively blocked thrombin-stimulated ERK1/2 phosphorylation in human cultured endothelial cells B, Inset.d 60 min B. These ol cells , C and Dpeptides , C and DA). However, S1352 phosphorylation was significantly decreased by thrombin in SB203580-treated cells compared to control cells . The α-catenin protein, a key component of adherens junction that links cadherins to the actin cytoskeleton, showed a marked decrease in phosphorylation of S641 and S652 sites in SB203580-treated cells compared to DMSO control cells . However, thrombin caused a further modest decrease in α-catenin phosphorylation in control and SB203580-treated cells . While a role for casein kinase-2 in cadherin phosphorylation has been reported . Taken together, these results indicate that p38 activity functions to control both increases and decreases in basal phosphorylation of several key proteins implicated in endothelial dysfunction and in some cases, the p38-dependent changes in protein phosphorylation were further altered by thrombin stimulation.In addition to regulating increases in protein phosphorylation, we examined whether p38 regulated decreases in phosphorylation at specific sites of other key proteins. ANKRD50, a large protein involved in membrane trafficking and recycling , is exprFig. 6reported , 26, mucin vitro . Our resA, lanes 1–4, top panels, and B), indicating that PAR1 is required for thrombin-stimulated p38 phosphorylation. Similarly, thrombin induced a significant increase in ERK2 phosphorylation with a modest increase in ERK1 phosphorylation detected in control cells that was virtually ablated in cells pretreated with vorapaxar , consistent with a role for PAR1. In contrast to ERK1/2, a high basal level of α-catenin S652 phosphorylation was detected even in the absence of thrombin stimulation and was not altered by vorapaxar , suggesting that basal S652 phosphorylation of α-catenin occurs independent of thrombin and activation of PAR1.To validate thrombin and p38 regulation of ERK1/2 and α-catenin phosphorylation, we used a PAR1-selective antagonist vorapaxar and commercially available antibodies that specifically detect phosphorylated ERK1, ERK2, or α-catenin at sites identified in our phosphoproteomic analyses. In these studies, endothelial cells were pretreated with or without the PAR1 antagonist vorapaxar or the p38 inhibitor SB203580 and then stimulated with thrombin. Cell lysates were immunoblotted using a phospho-ERK1/2 antibody that detects ERK1 phosphorylation at T202 and Y204 sites and ERK2 phosphorylation at T185 and Y187 sites and a phospho-α-catenin antibody that detects the S652 phosphorylated site. In control cells, thrombin induced a significant increase in p38 phosphorylation that was significantly reduced in endothelial cells pretreated with vorapaxar , confirming that thrombin/PAR1-stimulated p38 phosphorylation is attributed primarily to autophosphorylation rather than upstream MAPKs in endothelial cells , consistent with our phosphoproteomic analysis (C and D). Regardless, thrombin significantly amplified ERK1 and ERK2 phosphorylation over basal in SB203580-treated cells . SB203580 inhibition of p38 activity also caused a significant decrease in basal α-catenin S652 phosphorylation in nonagonist-treated cells compared to control cells and was not altered by thrombin stimulation , similar to that observed in the phosphoproteomic analysis (B). These findings indicate that thrombin-induced PAR1-stimulated p38 signaling negatively regulates ERK1/2 phosphorylation, while α-catenin phosphorylation at the S652 site is regulated by p38 activity but not by thrombin-stimulated PAR1 signaling.Next, we evaluated the effect of p38 inhibition on thrombin-stimulated phosphorylation of ERK1/2 and α-catenin. As expected, inhibition of p38 catalytic activity with SB203580 significantly decreased thrombin-induced p38 phosphorylation compared to control cells , suggesting that p38α function as the major effector for thrombin signaling in endothelial cells. In p38α siRNA–depleted cells, basal ERK1/2 phosphorylation was elevated , confirming that p38α functions to negatively regulate ERK1/2 phosphorylation as demonstrated in our phosphoproteomic analysis (C and D). However, thrombin significantly amplified ERK1/2 phosphorylation in p38α-depleted cells compared to siRNA-transfected control cells . These results indicate that the p38α isoform contributes to a negative feedback mechanism resulting in amplified ERK1/2 phosphorylation induced by thrombin (A). In contrast, depletion of the p38α isoform failed to perturb α-catenin phosphorylation either basally or in thrombin-treated cells compared to nonspecific siRNA-transfected control cells , suggesting that p38α expression is dispensable for basal α-catenin phosphorylation at the S652 site. These data suggest a complex regulation of α-catenin phosphorylation that requires p38α catalytic activity for basal phosphorylation but is not entirely dependent on p38α expression (B), suggesting that another kinase may function redundantly in the absence of p38α to phosphorylate α-catenin at the S652 site.To examine the function of the p38α isoform in thrombin-stimulated ERK1/2 phosphorylation, siRNA was used to specifically knockdown p38α isoform protein expression in endothelial cells. In nonspecific siRNA-transfected control cells, thrombin induced a significant increase in p38 phosphorylation that was virtually abolished in p38α siRNA–transfected cells depleted of p38 protein expression . In validation studies using a commercially available antibody that detects the α-catenin S652 phosphorylation, we found that inhibition of p38 activity with SB203580 virtually ablated basal α-catenin phosphorylation at the S652 site. Unexpectedly, however, we found that depletion of p38α failed to block basal S652 phosphorylation of α-catenin detected in endothelial cells, suggesting that p38α and α-catenin may physically associate and upon depletion of p38α, the S652 site is exposed and phosphorylated by a different kinase (B). Since, SB203580 targets both p38α and p38β, it is also possible that p38β is functionally redundant and phosphorylates α-catenin in the absence of p38α, but this remains to be determined.Cell–cell contacts are formed by adherens junctions and are important for maintaining endothelial barrier integrity. Adherens junctions are dynamic structures composed of transmembrane cadherin proteins that are linked to the actin cytoskeleton through interactions with α- and β-catenins. Interestingly, α-catenin has been shown to function in actin dynamics , 35, sugFig. 6t kinase B. Since,C and D) and in cell lysates using immunoblot analysis of ERK1/2 phosphorylation (B), consistent with its role in endothelial inflammation (A) . A. AC and tion (A) , suggestIn summary, the current study provides a global analysis of the thrombin phosphoproteome regulated by the p38 MAPK signaling and critical information regarding the proteins and pathways that mediate endothelial dysfunction. Clearly, future studies are needed to interrogate the function of p38 and ERK1/2 crosstalk in GPCR-induced endothelial dysfunction and the role of the p38-regulated α-catenin S652 phosphorylation in endothelial barrier disruption. In addition, the mechanism by which p38 negatively regulates thrombin-induced ERK1/2 phosphorylation is not known and important to determine. Finally, this study has identified numerous other proteins likely to have important functions in GPCR- and p38-induced endothelial dysfunction that may serve as targets for therapeutic intervention.Human α-Thrombin was from Enzyme Research Laboratories (#HT1002a). The following antibodies were from Cell Signaling Technologies including p44/42 MAPK ERK1/2 (#9102), phospho-p44/42 MAPK ERK1/2 (#9106), p38 MAPK (#8690), phospho-p38 MAPK (#4511), α-E-catenin (#3236), and phospho-α-E (Ser652)-catenin antibody (#13061). Horseradish peroxidase-conjugated goat-anti rabbit (#1706515) and goat-anti mouse antibodies (#1706516) were from BioRad. Vorapaxar or SCH 530348 (#1755) was from Axon MedChem, SB203580 (#S8307) was from Sigma-Aldrich, and U0126 (#U-6770) was from LC Laboratories.Human umbilical vein endothelial cell–derived EA.hy926 cells were  were obt5 cells per well and grown for 72 h and then pretreated with or without 100 nM U0126 or DMSO vehicle control for 30 min at 37 °C prior to 10 nM thrombin treatment. Diffusion of Evans Blue bound to bovine serum albumin was used to monitor barrier disruption and quantified over a 1 h time-course by measuring the A605 using a Molecular Devices SpectraMax ABS Plus 384 Microplate Readers as previously described  per the manufacturer’s instructions. The siRNAs used in these studies. AllStars negative control nonspecific siRNA (#1027281) and p38α-specific siRNA 5′-AACTGCGGTTACTTAAACATA-3′ were purchased from Qiagen.Endothelial EA.hy926 cells were seeded on 24-well plates at 1.7 ×105 cells per well and transfected. Serum-starved cells were then either left untreated or treated as described above, lysed, and protein concentrations determined using a bicinchoninic acid protein assay (ThermoFisher Scientific). Equivalent amounts of cell lysates were resolved by SDS-PAGE, transferred to PVDF membranes, and incubated with antibodies as indicated. Membranes were developed by chemiluminescence and quantified by densitometry using NIH ImageJ software or analyzed using a ChemiDoc Imaging System (BioRad).Endothelial EA.hy926 cells were seeded into 24-well plates at 1.7 ×102 dishes, serum starved overnight, pretreated with DMS0 vehicle control or 5 μM SB203580 for 30 min, and then stimulated with or without 10 nM Thrombin for 10 min at 37 °C. Cells were washed with 1X PBS and lysed in a buffer containing 75 mM NaCl, 3% SDS, 1 mM NaF, 1 mM β-glycerophosphate, 1 mM NaVO4, 1 mM NaPP, 1 mM PMSF, 1X complete EDTA-free protease inhibitor cocktail and 50 mM Hepes EA.hy926 cells were grown in 150 cmmM Hepes . Cells wProteins were denatured by addition of urea , then reduced and alkylated with DTT and iodoacetamide, respectively . The den2PO4, pH 10. TiO2 beads were washed . Peptides were resuspended in binding buffer, mixed with beads at a ratio of 1:4 (peptides to beads), and vortexed at room temperature for 1 h. Beads were then washed three times with binding buffer, followed by three times with wash buffer. Phosphopeptides were eluted from the beads with elution buffer (two 5 min incubation while vortexing). Enriched peptides were desalted with C18 Sep-Paks, then lyophilized and stored at −80 °C until they were labeled for quantitation.Phosphopeptides were enriched by TiO2 beads as previously described . BrieflySamples were labeled with TMT 10-plex reagents , 50 for Fractionation was carried out by basic pH reverse-phase liquid chromatography. BrieflyLabeled peptides were resuspended in 5% acetonitrile/5% formic acid and analyzed on an Orbitrap Fusion Tribrid mass spectrometer with an in-line Easy-nLC 1000 System. All data acquired were centrioded. Samples were loaded onto a 30 cm in-house pulled and packed glass capillary column . The column was packed with 0.5 cm of 5 μm C4 resin followed by 0.5 cm of 3 μm C18 resin, with the remainder of the column packed with 1.8 μm of C18 resin. Following sample loading, peptides were eluted using a gradient ranging from 11 to 30% acetonitrile in 0.125% formic acid over 85 min at a flow rate of 300 nl/minute and heating the column to 60 °C. Electrospray ionization was assisted by the application of 2000 V of electricity through a T-junction connecting the column to the nLC.5 with a maximum ion inject time of 100 ms and a lower threshold for ion intensity of 5 × 104. Ions selected for MS2 analysis were isolated in the quadrupole at 0.5 Th. Ions were fragmented using collision-induced dissociation, with a normalized collision energy of 30% and were detected in the linear ion trap with a rapid scan rate. AGC was set to 1 × 104 and a maximum inject time of 35 ms. MS3 analysis was conducted using the synchronous precursor selection option to maximize TMT quantitation sensitivity  was set to 2 × 10sitivity . Up to 1Resultant data files were processed using Proteome Discoverer 2.1. MS2 data were queried against the Uniprot human database  using the Sequest algorithm . A decoyPTMphinder R package was used to localize phosphorylated residues in the context of their corresponding full-length proteins and extract flanking sequences prior to motif analysis . Statistical analysis methods are indicated in the Figure legends.Data were analyzed using Prism 7.0 statistical software, Microsoft Excel, R and Morpheus (The mass spectrometry data have been deposited on MassIVE (MSV000085235) and on the ProteomeXchange (PDX018406).This article contains The authors declare that they have no conflicts of interest with the contents of this article.


To compare the biomechanical stability of transsacral-transiliac screw fixation and lumbopelvic fixation for “H”- and “U”-type sacrum fractures with traumatic spondylopelvic dissociation.Finite element models of “H”- and “U”-type sacrum fractures with traumatic spondylopelvic dissociation were created in this study. The models mimicked the standing position of a human. Fixation with transsacral-transiliac screw fixation, lumbopelvic fixation, and bilateral triangular fixation were simulated. Biomechanical tests of instability were performed, and the fracture gap displacement, anteflexion, rotation, and stress distribution after fixation were assessed.For H-type fractures, the three kinds of fixation ranked by stability were bilateral triangular fixation > lumbopelvic fixation > transsacral-transiliac screw fixation in the vertical and anteflexion directions, bilateral triangular fixation > transsacral-transiliac S1 and S2 screw fixation > lumbopelvic fixation in rotation. The largest displacements in the vertical, anteflexion, and rotational directions were 0.57234 mm, 0.37923 mm, and 0.13076 mm, respectively. For U-type fractures, these kinds of fixation ranked by stability were bilateral triangular fixation > lumbopelvic fixation > transsacral-transiliac S1 and S2 screw fixation > transsacral-transiliac S1 screw fixation in the vertical, anteflexion, and rotational directions. The largest displacements in the vertical, anteflexion, and rotational directions were 0.38296 mm, 0.33976 mm, and 0.05064 mm, respectively.All these kinds of fixation met the mechanical criteria for clinical applications. The biomechanical analysis showed better bilateral balance with transsacral-transiliac screw fixation. The maximal displacement for these types of fixation was less than 1 mm. Percutaneous transsacral-transiliac screw fixation can be considered the best option among these kinds of fracture fixation. Traumatic sacrum fractures and spondylopelvic dissociation are two severe injuries that are caused by falling and high-energy vehicle accidents. “U”-type and “H”-type sacrum fractures are the common subtypes of sacrum fractures , 2. The In recent years, with the development of Starr-frame closed reduction tools for pelvic fractures, most pelvic fractures can be reduced without open surgery, and the range of indications for percutaneous fixation has expanded , 8. PercSome biomechanical studies have focused on lumbopelvic fixation and transsacral-transiliac screw fixation –14. HoweThe geometric properties of the bony tissue of the pelvis and lumbar spine of a healthy 30-year-old female were assessed by a CT scan with a slice thickness of 1 mm, and the imaging data were then converted into a coarse geometric model by using Mimics 17.0 . The lumbar intervertebral disc cartilage, cortical bones, cancellous bones, articular cartilage, fiber rings, and ligaments of the FE model were created based on the geometrical model in Hypermesh 14.0 . The geometric model of the lumbar, ilium, and sacrum was then refined with reverse engineering software  and meshed into a finite element (FE) model with FE preprocessing software . Based on the structure of the bone, an FE model of the cartilage and ligaments was created according to the anatomical studies , 16 (TabThe following ligaments were constructed as 3D tension truss elements: iliolumbar ligament (IL), anterior sacroiliac ligament (ASL), interspinous ligament (ISL), long posterior sacroiliac ligament (LPSL), short posterior sacroiliac ligament (SPSL), sacrospinous ligament (SS), sacrotuberous ligament (ST), superior pubic (SP), arcuate pubic (AP), ligamentum flavum (LF), anterior longitudinal ligament (ALL), posterior longitudinal ligament (PLL), interspinous ligament (IL2), supraspinous ligament (SL), ligamenta intertransversaria (LI), and capsular ligament (CL). The regions were attached according to the methods described in previous studies , 16. TheThe FE model was built based on the methods described in a previous study . The resThe model mimicked the standing position of a human. The model was loaded with a 500-N force, and the forces were directed straight downward and toward the center of the L4 superior endplate. Both sides of the ischial tuberosity were restricted to six degrees of freedom.The H-type sacrum fracture involved a fracture line between the two S1 foramina and a vertical fracture line crossing both sides of the sacral foramina Fig. . The U-t1 to D1.Four points, A to D, were marked on the model of the H-type sacrum fracture. A was located at the top of the fracture line on the left. B was located at the top of the fracture line on the right. C was located at the bottom of the fracture line on the left. D was located at the bottom of the fracture line on the right. After loading, the displacement of the four points was recorded. The displacement values after loading were recorded as A1 to C1.Three points, A to C, were marked on the model of the U-type sacrum fracture. A was located at the top of the fracture line on the left. B was located at the top of the fracture line on the right. C was located in the middle of the S2 horizon fracture line. After loading, the displacement of the three points was recorded. The displacement values before and after loading were recorded as AWe chose the following 4 typical methods of fixation that many clinical studies and biomechanical studies have used: single S1 transsacral-transiliac screw fixation, S1 and S2 transsacral-transiliac screw fixation , L4-L5 pedicle screw and iliac screw lumbopelvic fixation (lumbopelvic fixation), and S1 transsacral-transiliac screw combinations of L4-L5 pedicle screw and iliac screw lumbopelvic fixation. We used single S1 transsacral-transiliac screw fixation for the U-type fractures but not for the H-type fractures.The stress distribution of internal fixation and displacement of the points on the model were recorded to determine the biomechanical stability and loading situations.The vertical displacement of the four points of the H-type fracture gap under load ranged from 0.40162 mm to 0.57234 mm with transsacral-transiliac S1 and S2 screw fixation, 0.36754 mm to 0.55696 mm with lumbopelvic fixation (L4–L5 pedicle screw and iliac screw fixation), and 0.09874 mm to 0.55761 mm with bilateral triangular fixation   and at the bar above the iliac screw with both lumbopelvic fixation (2.250e+02) and bilateral triangular fixation (1.695e+02) Fig. The vertical displacement of the three points for the U-type fracture gap under load ranged from 0.20655 mm to 0.38296 mm with single S1 transsacral-transiliac screw fixation, 0.17605 mm to 0.33581 mm with S1 and S2 transsacral-transiliac screw fixation, 0.14006 mm to 0.27315 mm with lumbopelvic fixation, and 0.02739 mm to 0.06335 mm with bilateral triangular fixation  and at the bar above the iliac screw with lumbopelvic fixation (2.307e+02), bilateral triangular fixation (1.620e+02), and single S1 fixation (8.911e+01) Fig. This FEA study aimed to study the biomechanics of internal fixation for “H”- and “U”-type sacrum fractures with traumatic spondylopelvic dissociation. Since the Starr frame is popular, closed reduction with percutaneous fixation can be used to treat an increasing number of displaced pelvic fractures. Routt et al.  was the For H-type fractures, our data showed that all three kinds of internal fixation yield great biomechanical stability in the standing position, and none of the fractures displacement values exceeded 1 mm. In transsacral-transiliac S1 and S2 screw fixation, there was great symmetry of the four points at the fracture sites . The maximal displacement was at AA1 at the top of the fracture line on the left. In lumbopelvic fixation, there was less displacement at the top of the fracture site (0.36754 mm and 0.39059 mm) and slightly more displacement at the bottom (0.50983 mm and 0.55696 mm), which means that L4–L5 pedicle screw and iliac screw lumbopelvic fixation yields more stability at the top of fractures and great bilateral symmetry. The maximal displacement was at DD1 at the bottom of the fracture line on the right. In the bilateral triangular fixation, there was less displacement (less than 0.2 mm) at points A, B, and C, which means that this kind of fixation is the most stable. The maximal displacement was at DD1 at the bottom of the fracture line on the right. Adding S1 transsacral-transiliac screw fixation can increase the stability at the top of the fracture site. Regarding anteflexion displacement, the results showed that maximal displacement at AA1 with transsacral-transiliac S1 and S2 screw fixation was 0.37923 mm, which is less than 1 mm. This means that lumbopelvic fixation yields more stability than does transsacral-transiliac fixation and that bilateral triangular fixation is the most stable. For rotation displacement, the results showed that the maximal displacement was 0.13076 mm with lumbopelvic fixation. Additionally, the displacement with transsacral-transiliac S1 and S2 screw fixation was, on average, less than that with bilateral triangular fixation, which means that lumbopelvic fixation was more rotationally stable than was transsacral-transiliac screw fixation. The displacement values for all three kinds of fixation were less than 1 mm, which meets the clinical requirements. In summary, for H-type fractures, these three kinds of fixation ranked by stability were bilateral triangular fixation > lumbopelvic fixation > transsacral-transiliac S1 and S2 screw fixation > transsacral-transiliac S1 screw fixation in the vertical and anteflexion directions, bilateral triangular fixation > transsacral-transiliac S1 and S2 screw fixation > lumbopelvic fixation in rotation.For U-type fractures, our displacement data showed that the three kinds of internal fixation yielded great biomechanical stability in the standing position, and none of the methods had displacement values exceeding 1 mm. In transsacral-transiliac S1 and S2 screw fixation, there was great symmetry for the three points on the fracture sites . The maximal displacement was at AA1 at the top of the fracture line on the left. In the lumbopelvic fixation, there was less displacement at the top of the fracture site (0.36754 mm and 0.39059 mm) and slightly more displacement at the bottom (0.50983 mm and 0.55696 mm), which means that L4–L5 pedicle screw and iliac screw lumbopelvic fixation yields more stability at the top of fractures and great bilateral symmetry. The maximal displacement was at DD1 at the bottom of the fracture line on the right. In the bilateral triangular fixation, there was less displacement (less than 0.2 mm) at points A, B, and C, which means that this kind of fixation is the most stable. The maximal displacement was at DD1 at the bottom of the fracture line on the right. Adding S1 transsacral-transiliac screw fixation can increase the stability at the top of the fracture site. The maximal stress distribution location of internal fixation for the 4 types internal fixation were the fracture site with S1 screw in transsacral-transiliac S1 and S2 screw fixation (8.364e+01) and at the bar above the iliac screw with lumbopelvic fixation (2.307e+02), bilateral triangular fixation (1.620e+02), and single S1 fixation (8.911e+01). Therefore, single S1 and transsacral-transiliac S1 and S2 screw fixation have more biomechanical symmetry in internal fixations and slightly higher stress concentrations than does lumbopelvic fixation. Lumbopelvic fixation leads to stress being concentrated on the lower part of the bar, which can be decreased slightly by adding transsacral-transiliac S1 screws. In summary, for U-type fractures, these 4 kinds of fixation methods ranked by stability were bilateral triangular fixation > lumbopelvic fixation > transsacral-transiliac S1 and S2 screw fixation > transsacral-transiliac S1 screw fixation in the vertical, anteflexion, and rotation directions.For U-type and H-type fractures, percutaneous transsacral-transiliac fixations, L4–L5 pedicle screw and iliac screw lumbopelvic fixation and bilateral triangular fixation can offer sufficient biomechanical stability. However, percutaneous transsacral-transiliac fixation yields better symmetry and a slightly higher stress concentration than does lumbopelvic fixation in U-type and H-type fractures. In the clinic, percutaneous transsacral-transiliac fixation leads to fewer complications and greater range of motion in L4–L5–S1 than does lumbopelvic fixation or bilateral triangular fixation. Therefore, we considered that percutaneous screw fixation  are the optimal choices for clinical applications.There were some limitations of our study. First, it was based on an FE model of the pelvis after surgical fixation, which may not exactly reflect how a biological pelvis would behave. Second, the pelvic model we generated may not be completely representative of all pelvic morphologies and kinematics/dynamics seen in the general population and thus may be an oversimplification. Additionally, this FE model considers the behavior of the pelvis in a static environment, and our results may differ in more dynamic settings. Additionally, some muscle and ligaments of pelvic and lumbar constructs exist that were not considered and may yield differing results. However, we feel that our study offers a good representation of the available constructs utilized to typically address H- and U-type fractures, and the FE model utilized accurately represents the clinical dilemma at present. Nevertheless, additional studies are warranted to evaluate the applicability of our findings in clinical practice.For orthopedics, more and more studies can be done through translational medical , 27. In


R parameter in frequency domain. The value of R allows one to determine if an ice sheet had been pebbled and if the shape of pebbles is suitable for the game of curling. The presented system was tested in various ice conditions—on both freshly prepared and used ice. Ice technicians and players may use the proposed system to decide whether the ice surface is suitable for play or if it should be remade.Despite the significant influence of ice conditions on results in the sport of curling, players and ice technicians lack a measurement device that would objectively measure ice quality during a curling competition. This paper presents such a new measurement method by using a device consisting an inertial measurement unit (IMU) attached to the handle of the curling stone and data processing software. IMU is used to measure the vibration of curling stone during its movement on the surface of the ice. The acceleration signal is recorded, and then the software calculates the value of so-called  Curling is a worldwide popular winter Olympic team sport, in which athletes slide 20-kilogram granite stones over the flat surface of ice. The field of play (sheet) is 45-meters long and 5-meters wide. On both ends of the sheet, colored rings called the house are the targets for players. A game consists of 8 or 10 ends and features two four-person teams . After each end (which features 16 stones and 8 per team), a point is awarded for each stone that is closer to the center of the house  than any stone of the opposition [During delivery, players set the stone in direct and rotational motion. Direct speed may be up to 4–5 m/s, and imposed angular speed is usually between 50 and 150 °/s. Rotation makes the stone follow a curved trajectory, and the lateral displacement in the stone’s path is called the curl. Curl direction depends on the direction of rotation—clockwise rotation makes the stone turn right and vice versa. Kameda has shown that the amount of curl mostly depends on the roughness of the curling stone’s running band . This faThe physics behind curling stone movement is still not fully described, and new models are being proposed. Recently proposed models include front-back asymmetry models (assuming an asymmetry of friction forces at the front and the back of a running curling stone) ,9,10,11 Curling ice must be level (any unevenness would cause stones to drift to one side), but its surface must not be flat. Droplets of water must be sprayed on the ice to make it possible to play the game. After freezing, they form so-called pebbles that reduce the contact area between the ice and the bottom of the stone. However, these droplets form sharp cones on the ice, which is not desirable either because curling stones would crush these sharp cones. This is why pebble tops must be cut off with a knife (ice must be “scraped”), and only that makes it possible for the stones to slide in the field of play with ease.The three ice structures are presented in Despite the important influence of ice conditions on game result, curlers and ice technicians lack a measurement device that would objectively measure the quality of ice to ensure a fair game. This research aims to investigate whether an accelerometer attached to the stone handle and data processing software can be used to differentiate between the three abovementioned structures .A MEMS  inertial measurement unit (IMU) with a 3-axis accelerometer was attached to the stone’s handle . The stoIn this experiment, the sampling frequency of IMU was set to 400 Hz. The sampling frequency was chosen based on IMU bandwidth (550 Hz). Collected acceleration samples were analyzed in the time and frequency domain (which is described in the next paragraph).X-axis was defined as parallel to the curling sheet length, pointing from the start point to the target. The Z-axis was defined as pointing downward, and the Y-axis was defined accordingly to obtain a left-handed coordinate system .The most critical metrological parameters of the developed measurement device are presented in The firmware used in the microcontroller was developed by the authors in C programming language. The main task of the firmware is to record the data. After interfaces and modules initialization, the main loop of the program is started. Data from sensors are read every 2.5 ms (to achieve 400 Hz sampling frequency) and buffered in RAM. Every second, buffered data are written to a file on an SD card. The file can be read on a PC using a standard socket. Data processing was performed on a PC using MATLAB software.Measurements were taken at Curling Łódź, a state-of-the-art four-sheet curling rink located in Łódź, Poland. The stone used during the experiment came from one of the Olympic sets that are used in all top-level competitions around the world. During a training session, a competitive curler was asked to deliver the stone with IMU. No mechanical device was used for stone delivery.Results obtained with the accelerometer during the movement of a curling stone are presented in Three periods mentioned above are clearly visible in gyroscope data, presented in x, y, z are defined in the measuring device coordinate system, and X, Y, and Z are defined in the ice sheet coordinate system. α is the angle between X and x.The measuring device was rotating with the stone itself; thus, to obtain acceleration parallel to the length of a curling sheet, gyroscope data was used according to Equations (1)–(3):Xa was analyzed in the frequency domain after Fourier transform  was performed on it. Xa. The obtained spectrum does not change rapidly during the middle part of curling stone movement. One can observe fading of the signal during the last 5 s both in the frequency domain and in the time domain. In further analysis, we will use a signal portion from the middle of a shot. Signal acquired during a curling shot must be compared to the sensor’s noise. In order to compare spectra and types of ice objectively, parameter R was introduced. It is defined as follows.R was calculated for 30 curling shots on each type of ice, and each obtained sample set was tested for normality using the Lilliefors test in MATLAB. In order to perform the test, data were converted back to a linear scale (instead of using results expressed in dB). The significance level was set to 0.05, and the null hypothesis stated that the dataset was normal. The Lilliefors test proved that obtained datasets might be treated as normal distributions  in the stone ,19,23,24Inertial measurement units are used in sports measurements ,29,30. IR was proposed to characterize the ice state . Changes in R-value can be used to monitor the quality of ice during curling competitions continuously. It was also confirmed that the value of R is stable for the all stone movements during a draw shot and does not vary significantly between repeated attempts on the same ice conditions.In this manuscript, a new, objective acceleration-based method was proposed to assess curling field of play (ice) quality during the curling competition. The procedure is based on signal analysis from the accelerometer attached to the handle of a moving curling stone. In the studies, it was found that the sensor signal acquired during a curling shot is about 40–50 dB higher than the sensor’s noise. It confirms that IMU range and resolution was correctly selected, and results of short-time analysis of signal frequency spectra could be used to characterize curling stone vibration during movement. Parameter R varies from one ice rink to another. If confirmed, a correction formula on R-value should be elaborated .The experiments were conducted only on one rink. Further research is necessary to determine whether the absolute value of The method proposed in this manuscript may be easily applied by ice technicians and players to evaluate the state of curing ice during competition and practice. It may indicate whether the ice surface is suitable for the game and when it should be remade to assure fair play. The method is fast, as curling stone delivery usually takes about 20–30 s and is easy to apply. It could be used in between end breaks during a curling game (which lasts for 1 to 5 min). The electronic recording device is small and light and does not change the trajectory of a granite curling stone.It is also worth considering extending the device functionality with wireless communication with PC , improving sample transfer.However, the proposed method also has some limitations. Most likely, it would not be usable in an ice rink with high air humidity . In such a case, water is condensed on the ceiling and then falls onto the ice as huge droplets, changing the shape of the ice surface. It is also unknown how frost deposition on the ice would influence results.


A similar inhibition pattern, but with comparatively higher values, was observed with EGCG. Of the flavonols, quercetagetin was the strongest inhibitor of α-glucosidases, with inhibition constants approaching those of acarbose, followed by galangin and kaempferol, while the weakest were quercetin and EGCG. The varied inhibitory effects of flavonols against human α-glucosidases depend on their structures, the enzyme source and substrates employed. The flavonols were more effective than EGCG, but less so than acarbose, and so may be useful in regulating sugar digestion and postprandial glycaemia without the side effects associated with acarbose treatment.Certain flavonoids can influence glucose metabolism by inhibiting enzymes involved in carbohydrate digestion and suppressing intestinal glucose absorption. In this study, four structurally-related flavonols  were evaluated individually for their ability to inhibit human α-glucosidases , and were compared with the antidiabetic drug acarbose and the flavan-3-ol(−)-epigallocatechin-3-gallate (EGCG). Cell-free extracts from human intestinal Caco-2/TC7 cells were used as the enzyme source and products were quantified chromatographically with high accuracy, precision and sensitivity. Acarbose inhibited sucrase, maltase and isomaltase with IC One of the earliest signs of type 2 diabetes (T2D) is elevated and erratic postprandial glycaemia that promotes oxidative stress at various sites within the body . ControlThere are four relevant types of digestive α-glucosidases in humans, maltase , glucoamylase , sucrase  and isomaltase . Maltase and glucoamylase have a unique, high α-1,4 hydrolytic activity for longer chain maltooligosaccharides to produce glucose ,6 and fuActinoplanes species [The most commonly used FDA-approved pharmaceutical α-glucosidase inhibitor is acarbose, a fermented product from  species  that is  species ,9, but i species , and dru species . Several species ,13. Amon species ,15. Food species ,17,18.Flavonoids are found ubiquitously in plants and represent ~60% of all dietary (poly)phenolic compounds ,20. FlavUnfortunately, many enzyme inhibition studies have been conducted using α-glucosidases from yeast or bacteria, with fewer studies using human intestinal enzymes. The inhibition of yeast and human α-glucosidases is very different, specific to the type of substrate, as reported for maltose . Here weBuffer components, sugar substrates and standards, and most inhibitors  were purchased from Sigma-Aldrich Corp., Merck , with purity >98%. Quercetagetin was purchased from EMD Millipore, Merck  and quercetin was purchased from Extrasynthese . Maltose monohydrate, sucrose and isomaltose were used as substrates for the enzyme assay, and together with fructose and glucose, were used as sugar standards for the chromatographic analyses. All other chemicals were of analytical grade and also purchased from Sigma-Aldrich, unless specified otherwise.2O supplied by a MilliQ system (Millipore) was used throughout.The Dionex™ Integrion™ HPIC™ (High Performance Ion Chromatography) system was used for High-Performance Anion-Exchange chromatography with Pulsed Amperometric Detection (HPAE-PAD)  for the separation and analysis of sugars. A PHERAstar FS plate reader  was used for measuring absorbance in the total protein assay. High-purity (18.2 mΩ/cm) H2O while following the same multistep eluent gradient was performed at the end of every batch (12–14 samples). All injections were performed in duplicate and peak identification was achieved by comparing retention times to the standards. Dionex™ Chromeleon™ 7 Chromatography Data System, version 7.2.9 , was used to process the chromatograms, ensuring peaks were suitably integrated before recording peak area. Sugars were quantified from peak areas using standard curves, with the standards prepared in the same buffer as the samples.Disaccharides and monosaccharides were analyzed by HPAE-PAD on the Dionex™ Integrion™ HPIC™ system (Thermo Fisher Scientific). Separation of the carbohydrates was achieved on a CarboPac PA210 column (2 × 150 mm), preceded by a CarboPac PA210 Guard column (2 × 30 mm), with the column and compartment temperatures maintained at 30 °C and 20 °C, respectively. Eluent was generated using a Dionex™ EGC 500 KOH eluent generator cartridge with Dionex™ continuously regenerated-anion trap column 600, with eluent concentration following a multistep gradient: 12 mM for 12 min, 100 mM for 8 min and 12 mM for 12 min, at a flow rate of 0.2 mL/min and with a sample injection volume of 2.5 μL. Detection was performed using a gold working electrode and AgCl reference electrode at pH ~ 12.0, with a collection rate of 2.00 Hz using the “Gold, Carbo, Quad” waveform. The total run time per sample was 32 min, with 3 min allowed between analyses for sample injection by autosampler. A wash injection of only H2O, quercetin and quercetagetin were prepared in DMSO, while kaempferol and galangin were dissolved in absolute ethanol. The maximum working concentrations for each compound was pre-determined before enzymatic reaction assay to ensure no precipitation occurred in the system. Compared to acarbose and EGCG, lower solubility of tested flavonols was expected due to their structural differences, where compounds with less than 100 µg/mL solubility were considered poorly soluble, as reported previously [v/v) of DMSO were ≤2% and ≤0.5% for quercetin and quercetagetin, respectively, and ethanol was ≤0.5% for kaempferol and galangin. The solvents did not affect enzyme activity, as demonstrated by vehicle controls. Cell-free extracts (CFE) were prepared as described in Stock solutions (250 mM) of sucrose, maltose and isomaltose (substrates), plus glucose and fructose (standards only), were prepared in sodium phosphate buffer . Stock solutions of acarbose (1 mM) and all flavonoids (10 mM) were prepared by dissolving in their respective solvents, stored at −20°C and used within 2 weeks. Acarbose and EGCG were prepared in Heviously ,29. Inhig, 15 min at 4 °C. The resulting supernatants were then diluted at least 10× in H2O ). Additionally, all standards and samples containing enzyme and substrates were filtered through 0.2 µM polyether sulfone (PES) filters . All standards, samples and blanks were kept at 4–8 °C until analysis by HPAE-PAD, as described in All prepared standards and assay samples, for both method validation and post-assay quantification, underwent the same treatment prior to injection on the HPAE-PAD system. All were deproteinated by mixing with an equal volume of acetonitrile, vortexed for 30 s and centrifuged at 17,000× The HPAE-PAD method for the quantification of glucose, sucrose, fructose, isomaltose and maltose was set up based on our previously published method , but witv/v <2%)). Blank samples spiked with a known amount of maltose, sucrose and isomaltose served as reference. The matrix effect was evaluated by comparing the analytical response of sugar spikes in SPB to those in H2O, to ensure accurate calibration plots were constructed.Specificity was determined by evaluating any endogenous interferences from the CFE. A comparison study was conducted on chromatograms of a blank incubated CFE matrix sample (CFE/enzyme only), CFE incubation sample with inhibitors (without substrate), CFE incubation sample with a substrate (without inhibitor), individual inhibitors only and blank assay solvent . Six different concentrations of maltose, sucrose and isomaltose  were prepared in assay incubation buffer or distilled water and measured in triplicate on four different days, giving a total of twelve replicates to construct the curve. Linearity was evaluated by calculating a regression line by the least-squares method, determining a linear equation (Equation (1)), where y = peak area, x = concentration, a = slope, b = intercept, and Rstandard .(1)y=axThe sensitivity of the method was evaluated by determining the LOD and LOQ using data generated from the calibration curve. LOD and LOQ were measured using the SD of the Precision (repeatability and reproducibility) was determined through the analysis of intra- and inter-day assay using standards in SPB quantification buffer. Intra-assay precision was assessed by measuring six concentrations of each standard measured in triplicate on the same day, in one laboratory by one person. Inter-assay precision was carried out by measuring the same concentrations of standards measured (in triplicate) over four different days by two analysts in the same laboratory. Precision was expressed as percent coefficient of variance (%CV), according to Equation (4), calculated as:As standard reference material was not used, accuracy was determined using the extraction recovery calculated by comparing the analytical response of two different concentrations of substrates spiked pre-assay to the values recovered post-assay in triplicates. The accuracy was calculated following Equation (5) as below:6 cells/T75 culture flask, maintained at 37 °C in an atmosphere of 10% CO2/90% air at a relative humidity in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4.5 g/L) . The medium was supplemented with 2% (v/v) Glutamax , 1% (v/v) nonessential amino acids, 1% (v/v) penicillin-streptomycin (100 IU/mL penicillin and 100 ug/mL streptomycin) and 20% (v/v) heat-inactivated foetal bovine serum  . Cells were passaged with 0.25% trypsin-EDTA before reaching ~70% confluence. Cells were used for experiments during passages 31–34 and were maintained until 21 days post-confluence, with medium routinely changed every 2–3 days, to differentiate. On day 21, cells were thoroughly washed twice with ice-cold PBS and harvested into ice-cold PBS containing 1% protease inhibitor cocktail . Following centrifugation at 200× g, 10 min at 4 °C, the supernatant was discarded and cell pellets were frozen immediately at −80 °C until required for the enzyme assay.Caco-2/TC7 cells, originating from human colon adenocarcinoma, were a kind donation from Dr Rousset, INSERM U505, Paris, France. Cells were seeded at ~1 × 10g, 10 min at 4 °C and the supernatant containing cell-free extract (CFE) collected. Total protein concentration in the CFEs was determined by Bradford assay [To mimic intestinal digestion, an in vitro assay using Caco-2/TC7 cell extracts containing sucrase, maltase and isomaltase was conducted. Frozen cells were thawed, 1 mL ice-cold SPB added and then passed through a 21-G needle 15–20 times. The lysate was centrifuged at 14,000× rd assay ,35, usinAssay mixtures, total volume 250 µL, containing CFE , with or without various concentrations of inhibitors/flavonoids, were prepared and kept on ice. The enzyme reaction was initiated by the addition of ≥20 mM sucrose, maltose or isomaltose and immediately incubating in a 37 °C water bath for 10 min . Following incubation, the enzyme activity was terminated by incubating in a 96 °C water bath for a further 10 min. A positive control without any added inhibitor/flavonoid was simultaneously tested in each batch, and negative controls without enzymes or substrates were also assayed to evaluate the stability of the inhibitors/compounds. Samples were prepared for HPAE-PAD analysis as described in Preliminary assays to optimize the substrate and enzyme concentrations and incubation time were performed to ensure enzyme kinetic experiments were carried out under initial linear velocity conditions (substrate depletion <10%). CFE protein concentration was tested, and specific activities found to be linear, at 0.10–0.35 mg/mL , with sumax)  . Sucrase15, IC25 and IC50 value determination, respectively, or the maximum percentage of inhibition expressed. Estimation of inhibition values were determined in GraphPad Prism and the percentage of inhibition of the sample was calculated following Equation (7) below, where SA = specific activity:Using the optimal assay conditions, various concentrations of acarbose and flavonoids were tested. Controls (CFE and substrate only) were prepared by replacing the volume of inhibitor with SPB. Activities of sucrase, maltase and isomaltase were considered as 100% (or 0% inhibition) in the absence of an inhibitor. Compounds that exhibited enzyme inhibition of at least 15%, 25% and 50% were subjected to IC15, IC25 and IC50 values were calculated based on the plots created using GraphPad, using the dose-response inhibition (log (inhibitor) vs. normalized response—variable slope) model. The same software was used to determine the apparent Km and Vmax values under the enzyme–kinetic inhibition model, and for statistical analyses using non-parametric multiple comparisons tests. A difference was considered significant at p < 0.05 for all comparisons. All data are expressed as the mean ± SD or SEM, specified accordingly.Enzyme assays were performed at least once for CFEs from biological triplicates, with duplicate injections of each analyzed by HPAE-PAD. MS Excel  was used for data processing and analysis. Final inhibition values were expressed as the percentage of control activity (%). The IC2O or in sodium phosphate assay buffer (SPB) were almost identical, with the same retention times and peak areas, demonstrating the absence of matrix effects (p = 0.225). LOD and LOQ for all five sugars were determined as a signal to noise ratio of 3:1 and 10:1, respectively. All sugar standards showed low LOD  and LOQ   .Intra- and inter-run precision was determined by analyzing the standards in triplicate in a single run on the same day and repeated on four different days within four months. Samples were kept at 4 °C at all times to avoid repeated freeze/thaw cycles or deterioration at RT. The mean peak areas and coefficients of variation (%CV) were calculated to determine the precision, as per ICH guidelines,  as preseaccuracy and the %CVprecision were <10% for all concentrations , and without the presence of any additional glucose or fructose peaks in the chromatograms. High precision values were obtained for all tested sugars with inhibitors (%CVprecision < 15%), indicating no interference between sugars and tested substances. No peaks were observed in blank/control samples, where  inhibitors, or assay buffer, or water, acetonitrile and eluent alone were tested.50) could not be determined, IC25 and IC15 were calculated instead. All flavonols, acarbose and EGCG inhibited α-glucosidase activity to some extent  (Based on the IC < 0.05) . Only ac < 0.05) .15 values, the decreasing order of the isomaltase inhibitory activity of the studied inhibitors was concluded to be quercetagetin ≥ acarbose ≥ galangin ≥ kaempferol > quercetin ≥ EGCG. This pattern is almost identical to sucrase inhibition. The inhibition potentials of acarbose and EGCG were generally lower towards isomaltase than sucrase and maltase, where a few fold higher concentrations were required to exhibit half-maximal inhibition (On the basis of the IChibition . The inhhibition .Saccharomyces cerevisiae) with limited reports on enzymes of mammalian or human origin [Flavonoids and other (poly)phenols potently inhibit α-amylase and α-glucosidase activities  without n origin . Multipln origin ,40. We en origin . Four flQuercetagetin was first identified as part of spinacetin  in spinach , and a f50 values against human and yeast α-glucosidases [Kaempferol has one less hydroxyl group in the B ring than quercetin. It has consistently shown lower inhibition than quercetin against rat maltase ,51,52, rosidases . These cGalangin has an unsubstituted B ring and is rich in many root plants, and possesses antiviral and anti-inflammatory properties with no toxic effects observed even at high doses in rats ,62,63. T50 = 281.2 µM for maltase, IC50 > 400 µM for sucrase) [Quercetin is a widely distributed flavonoid and therefore most researched in human and animal models. With a half-life of ~24 h , a few fsucrase) , similarAmong all tested flavonoids, while EGCG was most soluble with the highest inhibition reached for human sucrase (100%), maltase (68%) and isomaltase (71%) at 1500 µM, this is a supra-physiological concentration and EGCG generally exhibited the weakest inhibitory potential when compared by concentration alone, as indicated previously . Convers50 values closest to acarbose. This suggests that stronger enzyme inhibition is observed with increasing hydroxyls on the A ring since quercetagetin is a stronger inhibitor than quercetin. Increasing hydroxylation of the B ring  improves the solubility of compounds but lowers inhibition. The lower aqueous solubility of flavonols, observed in quercetagetin, kaempferol and galangin, is a shortcoming of this study, and is the reason why some IC50 values could not be determined.The most active flavonol was quercetagetin, with ICAt the molecular level, the binding between hydroxyls in ring A, B or C of flavonoids to the active sites of α-glucosidases leads to structural changes in the enzyme evidenced by several docking studies with yeast α-glucosidase ,55,72,79The hydroxylation patterns, particularly 3-OH at a B ring catechol moiety, are among the major determinants of various biological effects of flavonoids . The hydAlthough mild α-amylase inhibition is beneficial for blunting glucose spikes, excessive inhibition may induce starch indigestion and abnormal bacterial fermentation, causing abdominal pain, bloating or cramping . AcarbosWe have elucidated the inhibitory effects of flavonoids against human α-glucosidases compared with acarbose, influenced by structure, enzyme origin and substrates. A higher concentration of acarbose is required to inhibit maltase than sucrase, while all flavonoids showed similar inhibition of sucrase and maltase, in agreement with our previous findings using olive leaf extracts . Isomalt50 of 20 µM for EGCG was determined for human maltase expressed in yeast [Enzyme inhibition by plant extracts was consistently superior to acarbose when tested using yeast α-glucosidase ,73, in cin yeast , which iin yeast , using h50 values and necessitating the use of IC25 or IC15 values instead.Our study highlights the potential of selected flavonoids to inhibit human intestinal α-glucosidases, hence slowing carbohydrate digestion and reducing postprandial glycaemia. A sensitive and accurate method to determine sugar hydrolysis by sucrase, maltase and isomaltase has been successfully developed and validated. The use of HPAE-PAD to detect subtle changes in the concentrations of five sugars simultaneously, with minimal sample preparation and high precision within 32 min, has been central to this study. Acarbose and flavonoids exhibit different inhibition of human enzymes to those reported for yeast or mammalian α-glucosidases, emphasizing the need for a more pragmatic screening approach on individual human enzymes to elucidate their actual inhibitory potentials in vivo. Flavonoids from various sources are more effective against α-glucosidase than α-amylase . The lowQuercetagetin, similar to acarbose, followed by kaempferol and galangin, exhibited greater inhibitory action against sucrase, maltase and isomaltase than EGCG and quercetin, although the latter compounds were more soluble in aqueous buffer. Two key structural elements of flavonoids for enhanced α-glucosidase inhibition in humans are the C6-OH A ring hydroxylation and reduced B ring hydroxylation. Improving understanding of how flavonoids bind to human α-glucosidases should provide a rational basis for exploiting antidiabetic compounds from dietary sources.


We also report a targeted siRNA-based screen which suggests a possible role for mTOR and Akt in DNA repair inhibition by this class of drugs. Taken together, these data reveal a new class of mibefradil-based DNA repair inhibitors which can be further advanced into pre-clinical testing and eventually clinical trials, as potential GBM radiosensitizers.Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system with a dismal prognosis. Locoregional failure is common despite high doses of radiation therapy, which has prompted great interest in developing novel strategies to radiosensitize these cancers. Our group previously identified a calcium channel blocker (CCB), mibefradil, as a potential GBM radiosensitizer. We discovered that mibefradil selectively inhibits a key DNA repair pathway, alternative non-homologous end joining. We then initiated a phase I clinical trial that revealed promising initial efficacy of mibefradil, but further development was hampered by dose-limiting toxicities, including CCB-related cardiotoxicity, off-target hERG channel and cytochrome P450 enzymes (CYPs) interactions. Here, we show that mibefradil inhibits DNA repair independent of its CCB activity, and report a series of mibefradil analogues which lack CCB activity and demonstrate reduced hERG and CYP activity while retaining potency as DNA repair inhibitors. We present  Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system (CNS). The current standard of care involves maximal surgical resection followed by radiation therapy (RT) with adjuvant temozolomide (TMZ) chemotherapy. Despite high doses of RT and TMZ, most patients have a recurrence within 1–2 years, and the overall prognosis is dismal .DNA repair inhibitors have shown great promise as tumor cell radiosensitizers , 5. Cellin vitro [in vivo GBM models [We previously published a microplate-based assay to measure both HR and alt-NHEJ simultaneously in live cells, termed EJ-DR . The EJ-in vitro , which wM models , 11. A PM models . These iM models , 14.in vivo models and eventually clinical trials for improving RT efficacy in GBMs.Based on these findings, we sought to create a new class of radiosensitizers which retained mibefradil’s activity as a DNA repair inhibitor, but showed reduced hERG and CYP450 enzyme inhibition. We found that the CCB ability of mibefradil is non-essential for its ability to inhibit alt-NHEJ. Through structure activity relationship (SAR) analysis, we created and synthesized a series of 140 analogues and profiled them using EJ-DR assays. These compounds were found to be potent alt-NHEJ inhibitors, with reduced CCB activity, as well as attenuated hERG and CYP450 inhibition. We then tested the pharmacokinetic parameters of the synthesized analogues and validated their ability to cross the blood-brain barrier (BBB) and accumulate in mouse brain tissue, at levels similar to that observed with mibefradil. Finally, through the knockdown of DNA damage response (DDR) proteins in the high-throughput imaging-based assay, we identified potential targets or regulators of mibefradil, which phenocopied the selective inhibition of alt-NHEJ over HR. Overall, this study provides a framework for the development of superior mibefradil analogues, with reduced off-target effects and improved potency. These analogues could further be tested as radiosensitizers in To support the rapid screening of mibefradil analogues, we adapted the previously published EJ-DR assay from our group to a 384-well imaging-based assay . The EJ-An overview of the workflow of this assay is presented in Various doses of mibefradil were tested in this imaging-based EJ-DR assay to validate assay performance. Consistent with previous studies, mibefradil selectively inhibited alt-NHEJ with an IC50 of approximately 4 μM, confirming that the assay accurately detects and quantifies the effects of the drug. In addition to quantifying HR and alt-NHEJ activities, cytotoxicity in the absence of DSB ligands (CT) was also evaluated by quantifying the number of Hoeschst 33342-stained cells, allowing us to better understand the margin between on-target and presumable off-target, DNA repair-independent effects. IC50 values of HR inhibition, alt-NHEJ inhibition, and CT were used to calculate the “selectivity index” (SI)  and alt-NHEJ selectivity defined as HR IC50/alt-NHEJ IC50. We demonstrated that mibefradil showed expected patterns of selective alt-NHEJ inhibition over HR as previously described, i.e., alt-NHEJ IC50 ~ 4 μM, HR IC50 > 60 μM, SI = > 15, validating the robustness of this system to screen mibefradil analogues  Figure .v3.1, Cav3.2, Cav3.3) but inhibits L-type calcium channels (Cav1.2) with much lower potency [Structure-activity relationship (SAR) studies were performed to identify structural elements of mibefradil essential for its alt-NHEJ inhibition and CCB function. The SAR studies focused on modifications to the four major moieties in mibefradil, the substituted tetrahydronaphthalene core (pink), the tertiary amine linker (orange), benzimidazole ring (purple), and the methoxyacetate group (blue) . Mibefra potency . The met potency , 20. We To determine whether the other three moieties were responsible for alt-NHEJ inhibition, we synthesized three broad categories of analogues: (1) Replacement of the tetrahydronaphthalene group with a tetrahydroisoquinoline moiety, (2) Replacement of the benzimidazole moiety with other heterocyclic aromatic groups, (3) Replacement of the basic amine linkage with an amide linkage . RepresehERG is a potassium ion channel responsible for repolarization of the cardiac action potential. Inhibition of hERG blocks the repolarization of cardiac cells, which leads to QT prolongation and can predispose individuals to arrhythmias . In our hERG), or the ratio of hERG IC50 to the alt-NHEJ IC50. Notably, all three analogues contained a five-membered heterocyclic pyrrolidine, which suggests that reducing the basicity of the tertiary amine is a viable approach to reducing hERG inhibition. Following the synthesis of mibefradil analogues with reduced hERG activity, we undertook a similar rational drug design approach to synthesizing mibefradil analogues with reduced CYP inhibition.As such, we aimed to reduce the basicity of the tertiary amine and increase lipophilicity of the side groups as summarized in in vitro inhibition of both CYP enzymes by mibefradil. Our data confirmed that mibefradil is a potent inhibitor of both CYP3A4 (IC50 = 0.8 μM) and CYP2D6 (IC50 = 0.6 μM). Our aim was to reduce activity against CYPs using rational drug design and previous SAR information based on common CYP inhibitors, while retaining the alt-NHEJ activity of the analogues [Mibefradil was previously FDA approved for hypertension and then withdrawn owing to potent drug-drug interactions with a wide range of drugs including β-blockers, simvastatin, digoxin, methylprednisolone, diltiazem, and cyclosporin leading to severe toxicities including cardiogenic shock, myopathy, renal failure, and death , 24–26. nalogues , 32. Prenalogues , 34.in vitro using human liver microsomes along with their alt-NHEJ inhibition (CYP). All three analogues showed a >10-fold decrease in CYP3A4 inhibition while YU252376 showed a ~3-fold decrease in CYP2D6 inhibition. Our data demonstrates that the methoxyacetate group may not be as critical to CYP inhibition as previously thought, compared to the benzimidazole ring, as YU252376 showed meaningful reduction in CYP inhibition with modifications to only the amine group and aromatic side chains [in vitro and in vivo pharmacokinetics of the optimized compounds.To modulate the CYP3A4 and CYP2D6 activity, we synthesized 29 analogues using the following two strategies: (1) Retaining the tetrahydronaphthalene core while modifying the basicity of the central amine or reducing the lipophilicity of aromatic side groups, and (2) Modifying the tetrahydronaphthalene core to explore alternate scaffolds. For the first approach, we synthesized analogues using a similar scaffold as that in hibition , 4C. Fige chains . After sin vivo studies. We first tested the metabolic stability of the selected analogues in mouse liver microsomes. Phase I metabolic enzymes in liver microsomes account for ~80% of total drug metabolism. Testing the half-lives of the analogues incubated with liver microsomes allows for the estimation of the anticipated plasma exposure of the drug and its intrinsic clearance. Those with a lower half-life would be expected to attain a lower steady-state plasma concentration compared with an equipotent drug with a higher half-life. We profiled the mouse liver microsome half-life of mibefradil, twelve analogues, and previously FDA-approved drugs which are known to be metabolized in these microsomes i.e., Propranolol, Verapamil, Terfenadine, and Imipramine , maximum concentration achieved (Cmax), and half-life (t1/2) (max << 100 ng/mL) and did not achieve detectable brain exposure. Notably, YU252386 showed improved brain exposure compared to mibefradil (~8-fold) and this concentration was greater than the alt-NHEJ IC50 (~3-fold), suggesting that this dose could inhibit alt-NHEJ in the brain. Correspondingly, YU252386 showed a markedly reduced concentration in the plasma compared with mibefradil (~0.3-fold), which could imply specificity to brain tissue. YU252386 showed a half-life of ~2.5 hours while mibefradil showed a half-life of ~8 hours in the brain tissue. This suggests that YU252386 could require more frequent dosing to achieve a meaningful and sustained inhibition of alt-NHEJ. However, given that the percent coverage of the in vitro alt-NHEJ IC50 of YU252398 at the Cmax in the brain is 300%, and this analogue is highly brain penetrant, this may not prove to be a shortcoming for future in vivo studies. The results so far demonstrate the successful identification of a novel analogue of mibefradil, YU252386, which shows improved potency, reduced off-target effects, and increased specificity towards the brain in vivo.Female athymic nude mice were orally dosed with 30 mg/kg of YU252222 (a methyl ester prodrug was used), YU252377 and YU252386, and plasma and brain concentration were recorded at 1, 2, 4, 8, 12, 24 hours post dosing. These measurements were used to calculate various pharmacokinetic parameters critical to bioavailability including time to reach maximum concentration  plot, the schematic for which is shown in The criteria for choosing hits from the DNA repair screen included: (1) Inhibition of either alt-NHEJ and HR with a relative value of alt-NHEJ and HR inhibition < 1, (2) Greater inhibition of alt-NHEJ compared to HR, i.e., a ratio of inhibition of alt-NHEJ to HR inhibition < 0.8, (3) Cell viability greater than 80%, i.e., cytotoxicity > 0.8. Top hits included RPA2, POLA, DDB1, TYMS, CHEK1, mTOR, TTRAP, FEN1, UBE2A, PARP3, and ATRX.We followed up the DDR screen with a focused deconvolution screen where four siRNAs comprising SMARTpool in the primary screen were tested individually for the top eleven hits . mTOR i. Each ofWe report the synthesis and validation of novel analogues of the potent T-type and L-type CCB, mibefradil, for potential use as radiosensitizers in GBM. Radiation induces both single-strand breaks (SSBs), double-strand breaks (DSBs), as well as a number of complex lesions which require HR for their repair. Alt-NHEJ functions as a back-up pathway for HR for the repair of these complex lesions and DSBs, which, if unrepaired, can be lethal to the cell. We previously reported that mibefradil showed potent and selective inhibition of alt-NHEJ over HR. However, while mibefradil was previously FDA approved for use in hypertension, it was subsequently withdrawn from the market owing to multiple off-target effects including hERG channel inhibition leading to QT prolongation and arrhythmias as well as inhibition of CYP enzymes leading to severe drug-drug interactions. To minimize these off-target effects, while retaining DNA repair inhibition activity, we developed novel SAR approaches and reported the synthesis of over 140 mibefradil analogues. We developed a 384-well imaging-based EJ-DR assay to rapidly screen the DNA repair activity of the synthesized analogues, and validated the results of this assay using mibefradil and NU 7441 as controls. This assay also closely recapitulated the results of the original flow cytometry-based EJ-DR assay and as such, represents an improvement on the previous assay to screen a larger library of compounds more rapidly.Following the development of this assay, we performed preliminary SAR studies to determine which moieties were critical for the alt-NHEJ inhibition by mibefradil. We determined that the tetrahydronaphthalene core and tertiary amine in mibefradil contributed to its alt-NHEJ function, while the benzimidazole ring and the methoxyacetate group (responsible for the CCB ability), were non-essential. This was an important finding, which could also aid the development of more selective CCB analogues. Mibefradil has both T-type and L-type CCB activity, however it is still unclear which calcium channel is crucial for its unique cardiovascular effects. The analogue YU252377 showed only a slight reduction in its L-type blocking over mibefradil while a > 10-fold decrease in T-type channel activity . Testingmax and hERG inhibition IC50 of compounds in pre-clinical development would allow for a reduction in the hERG-associated cardiotoxicity [in vivo hERG inhibition studies may be required to validate the reduction in this off-target effect. Further modifications to the aromatic side chains and the central amine of the synthesized analogues may be required to widen the therapeutic margin. We undertook a similar approach to minimize the CYP3A4 and CYP2D6 inhibition of mibefradil and synthesized 29 analogues with >10-fold reduction in CYP3A4 activity and ~3-fold reduction in CYP2D6 activity. The syntheses strategies we employed for reducing CYP inhibition included modifications to the tetrahydronaphthalene core, which have been previously unexplored in SAR studies with mibefradil. We utilized alternate scaffolds adapted from the structures of loperamide and proadifen, which we had previously identified as selective alt-NHEJ inhibitors [After determining preliminary SAR, we began to further understand the structure-liability relationship (SLR) with the mibefradil pharmacophore. Using the structures of common hERG inhibitors and other previously established SAR approaches, we synthesized 48 analogues with increased lipophilicity of the aromatic side chains and reduced basicity of the central amine. We were able to reduce hERG inhibition by ~5-fold, while retaining alt-NHEJ activity. While this was a significant improvement over the highly potent hERG inhibition by mibefradil, a report in 2003 suggested that a 30-fold margin between the Ctoxicity . Prior tin vitro and in vivo pharmacokinetic studies. YU252386 showed the greatest improvement in pharmacokinetic profile over mibefradil including an ~8-fold increase in brain exposure and a ~0.3-fold decrease in plasma exposure. These pharmacokinetic attributes make it the most suitable for future in vivo radiosensitization studies. Prior to performing such studies, it will be critical to determine optimal timing and dosing regimens to achieve sufficient brain tissue penetration of the compound before administering radiotherapy.After successfully reducing the known off-target liabilities of mibefradil, we selected the top 12 analogues for further To determine the critical proteins involved in this unique regulation of alt-NHEJ, we performed a high-throughput siRNA screen to determine which DDR proteins phenocopied the DNA repair activity of mibefradil. Interestingly, siRNAs targeting POLQ, the critical alt-NHEJ gene, showed a specificity for alt-NHEJ over HR. All four siRNAs targeting mTOR also showed a selective inhibition of alt-NHEJ over HR. After validating the knockdown of mTOR, we also tested three mTOR inhibitors, AZD 3147 (mTORC1/2i), AZD 8055 (mTORi), and Everolimus (mTORC1i), in the EJ-DR assay, which showed similar results. We then interrogated kinases downstream of mibefradil including S6K, CDK4/6, and CHK1, however, knockdown or small molecule inhibition of these targets did not explain the alt-NHEJ activity of mTOR. After testing the knockdown of a number of proteins in the DDR pathway, we discovered that knockdown and inhibition of Akt closely phenocopied mibefradil and mTOR. Previous reports have presented a contradictory view on the regulation of the Akt pathway by mibefradil with one report describing the activation of PI3K/Akt/mTOR by mibefradil while another report demonstrated mibefradil reduced the phosphorylation of Akt , 37. Thiin vivo study in clinically relevant GBM models.The use of DNA repair inhibitors as radiosensitizers in GBM could represent a viable approach to achieve better response owing to the range of DDR pathways activated in response to radiation-induced DNA damage. Additionally, the identification of selective inhibitors of alt-NHEJ could also be tested in other settings where alt-NHEJ activity is critical, such as in HR-deficient tumors , 45. The2.The EJ-DR cell line was created in the U2OS cells (a human osteosarcoma cell line), and contained a stably integrated EJ-RFP plasmid to measure alt-NHEJ repair and a DR-GFP plasmid to measure HR repair, and has been previously described . The EJ-Calcium channel blocking studies were performed by Charles River Laboratories in Chinese Hamster Ovary (CHO) and HEK293 cell lines stably expressing L-Type and T-type calcium channels, respectively, under tetracycline induction in accordance with a previously published study . Data acThe EJ-DR cells were plated in 384-well imaging plates at 2500 cells/well. 24 hours later, the DSB ligands, Triamcinolone Acetonide  and Shield-1 (Takara Bio Inc.), responsible for inducing the DSBs, were added for a final concentration of 100 nM TA and 1 μM Shield-1. Test compounds were also added at the same time using Echo 550 Acoustic Dispenser (Labcyte). A separate plate was seeded to test for cytotoxicity of the test compounds in the absence of the DSB ligands. All EJ-DR experiments included the internal controls mibefradil (Tocris Bioscience) and DNA protein kinase catalytic subunit inhibitor . The DSB ligands were washed out 24 hours after addition. 96 hours after addition of the ligands and test compounds, the media was washed off and cells were incubated with Hoechst nuclear stain in Live Cell Imaging Solution (Thermo Fisher Scientific). Plates were rinsed and prepared for immediate live cell imaging using GE IN Cell 2200 (Cytiva Life Sciences). Images were analyzed using InCell Analyzer software (Cytiva Life Sciences). Nuclei were segmented based on the Hoechst staining, and the intensity of the GFP and RFP signals in control populations were measured and binned into histograms to determine signal intensity threshold. The GFP and RFP intensity of all wells was then analyzed, and the percent of positive cells with GFP and RFP intensity above the threshold was calculated to determine the induction of signal. The DAPI cell counts were used to determine the relative cytotoxicity of test compounds in the absence of induced DNA damage.Mibefradil analogues were generated as described in Supplementary Materials and Methods.Automated whole cell voltage clamp hERG inhibition assays were performed in Human hERG (Kv11.1) expressed in CHO-K1 cells optimized for automated patch clamp studies by Sophion Bioscience A/S .CYP inhibition assays were performed by Eurofins Scientific. Test compounds were incubated with human liver microsomes, followed by compound detection using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) .Microsomal half-life studies were performed at Eurofins Scientific using mouse liver microsomes and test compounds were incubated for 0, 15, 30, 45, and 60 minutes. Compound concentrations were measured at these time-points using Liquid Chromatography with tandem mass spectrometry (LC-MS-MS). Pharmacokinetic studies were performed by orally dosing test compounds at 30 mg/kg dissolved in 50% PEG400 and 50% water in female athymic nude mice. Plasma and brain tissue concentration were measured using mass spectrometry (MS).On Target-Plus Smart-pool siRNAs (Horizon Discovery) targeting key DNA repair and control genes were reconstituted as 1 μM stock solutions in 1X siRNA buffer made from 5X siRNA buffer (Thermo Fisher Scientific) (Supplementary Table 3). Deconvoluted siRNAs were reconstituted as 10 μM stock solutions in 1X siRNA buffer. The EJ-DR cells were seeded at 1000 cells/well in a 384-well plate and transfected with 10 pmol of Smart-pool siRNAs along with a non-targeting control. 72 hours after transfection, the DSB ligands were added along with mibefradil as a control. 24 hours after DSB ligand addition, the plates were rinsed and 72 hours later the plates were imaged and analyzed as described above.RNA samples were lysed in TRIzol (Invitrogen) and then reverse transcribed using the TaqMan RT-qPCR kit according to manufacturer’s instructions by ARQ Genetics. TaqMan fluorescent probes for mTOR and GAPDH were used and mRNA expression was quantified using ΔΔCt comparison normalized to the GAPDH control.t-test and described significant as *p < 0.05, **p < 0.01, ***p < 0.001.Data are presented as the mean ± SEM and the analyses were performed using Microsoft Excel and GraphPad Prism Software. Statistical comparisons were conducted using Student’s two-tailed