Src Tyrosine Kinase Inhibitor Research Articles

A novel boron-conjugated SRC inhibitor for Proton Boron Capture Therapy in glioblastoma treatment

The ability of protons to deliver the maximum dose at the tumor region can work synergistically with boron atoms to emit alpha particles, enhancing therapy effects with less damage to healthy tissue. Protons and boron nuclear fusion reaction is the principle for the so-called Proton Boron Capture Therapy, that can contribute to high therapy efficiency by using smaller flux than conventional proton therapy, especially for radioresistant brain tumors such as glioblastoma. Glioblastoma is the most infiltrating and aggressive tumor of the brain with a very low life expectancy, ranging from 6 to 24 months. In this study we evaluated the protons combined effectwithanovelboron-conjugated compound, which is a pyrazolo[3,4-d]pyrimidine derivative, acting as SRC tyrosine kinase inhibitor. Indeed, this drug includes a boron cluster, that can be directed in tumor cells using the ATP-competitive mechanism of the pyrazolo[3,4 d]pyrimidine ring in activated SRC form of glioblastoma cells, achieving tumor uptake of boron for Proton Boron Capture Therapy reaction. This preliminary study showed interesting results that could offer important contributions for further experiments.

Exploring the benefits of in-diet versus repeated oral dosing of saracatinib (AZD0530) in chronic studies: insights into pharmacokinetics and animal welfare.

Saracatinib/AZD0530 (SAR), a Src tyrosine kinase inhibitor, mitigates seizure-induced brain pathology in epilepsy models upon repeated oral dosing. However, repeated dosing is stressful and can be challenging in some seizing animals. To overcome this issue, we have incorporated SAR-in-Diet and compared serum pharmacokinetics (PK) and brain concentrations with conventional repeated oral dosing. Saracatinib in solution or in-diet was stable at room temperature for >4 weeks (97 ± 1.56%). Adult Sprague Dawley rats on SAR-in-Diet consumed ~1.7 g/day less compared to regular diet (16.82 ± 0.6 vs. 18.50 ± 0.5 g/day), but the weight gain/day was unaffected (2.63 ± 0.5 g/day vs. 2.83 ± 0.2 g/day). Importantly, we achieved the anticipated SAR dose range from 2.5-18.7 mg/kg of rat in response to varying concentrations of SAR-in-Diet from 54 to 260 ppm of feed, respectively. There was a strong and significant correlation between SAR-in-Diet dose (mg/kg) and serum saracatinib concentrations (ng/ml). Serum concentrations also did not vary significantly between SAR-in-Diet and repeated oral dosing. The hippocampal saracatinib concentrations derived from SAR-in-Diet treatment were higher than those derived after repeated oral dosing (day 3, 546.8 ± 219.7 ng/g vs. 238.6 ± 143 ng/g; day 7, 300.7 ± 43.4 ng/g vs. 271.1 ± 62.33 ng/g). Saracatinib stability at room temperature and high serum and hippocampal concentrations in animals fed on SAR-in-Diet are useful to titer the saracatinib dose for future animal disease models. Overall, test drugs in the diet is an experimental approach that addresses issues related to handling stress-induced variables in animal experiments.

The effects of Src tyrosine kinase inhibitor, saracatinib, on the markers of epileptogenesis in a mixed-sex cohort of adult rats in the kainic acid model of epilepsy.

Neurodegeneration and neuroinflammation are key processes of epileptogenesis in temporal lobe epilepsy (TLE). A considerable number (∼30%) of patients with epilepsy are resistant to currently available antiseizure drugs and thus there is a need to develop adjunct therapies to modify disease progression. A vast majority of interventional strategies to treat TLE have utilized males which limits the translational nature of the studies. In this study, we investigated the effects of repeated low-dose kainic acid (KA) injection on the initial status epilepticus (SE) and the effects of Src kinase inhibitor, saracatinib (SAR/AZD0530; 20 mg/kg, oral, daily for 7 days), in a mixed-sex cohort of adult Sprague Dawley rats during early epileptogenesis. There were no sex differences in response to KA-induced SE, and neither did the stage of estrus influence SE severity. KA-induced SE caused significant astrogliosis and microgliosis across the hippocampus, piriform cortex, and amygdala. SAR treatment resulted in a significant reduction of microgliosis across brain regions. Microglial morphometrics such as branch length and the endpoints strongly correlated with CD68 expression in the vehicle-treated group but not in the SAR-treated group, indicating mitigation by SAR. KA-induced SE caused significant neuronal loss, including parvalbumin-positive inhibitory neurons, in both vehicle (VEH) and SAR-treated groups. SAR treatment significantly mitigated FJB-positive neuronal counts as compared to the VEH group. There was an increase in C3-positive reactive astrocytes in the VEH-treated group, and SAR treatment significantly reduced the increase in the piriform cortex. C3-positive astrogliosis significantly correlated with CD68 expression in the amygdala (AMY) of VEH-treated rats, and SAR treatment mitigated this relationship. There was a significant increase of pSrc(Y419)-positive microglia in both KA-treated groups with a statistically insignificant reduction by SAR. KA-induced SE caused the development of classical glial scars in the piriform cortex (PIR) in both KA-treated groups, while SAR treatment led to a 42.17% reduction in the size of glial scars. We did not observe sex differences in any of the parameters in this study. SAR, at the dose tested in the rat kainate model for a week in this study mitigated some of the markers of epileptogenesis in both sexes.

Nonfunctional TGF-β/ALK1/ENG signaling pathway supports neutrophil proangiogenic activity in hereditary hemorrhagic telangiectasia.

The transforming growth factor β (TGF-β)/ALK1/ENG signaling pathway maintains quiescent state of endothelial cells, but at the same time, it regulates neutrophil functions. Importantly, mutations of this pathway lead to a rare autosomal disorder called hereditary hemorrhagic telangiectasia (HHT), characterized with abnormal blood vessel formation (angiogenesis). As neutrophils are potent regulators of angiogenesis, we investigated how disturbed TGF-β/ALK1/ENG signaling influences angiogenic properties of these cells in HHT. We could show for the first time that not only endothelial cells, but also neutrophils isolated from such patients are ENG/ALK1 deficient. This deficiency obviously stimulates proangiogenic switch of such neutrophils. Elevated proangiogenic activity of HHT neutrophils is mediated by the increased spontaneous degranulation of gelatinase granules, resulting in high release of matrix-degrading matrix metalloproteinase 9 (MMP9). In agreement, therapeutic disturbance of this process using Src tyrosine kinase inhibitors impaired proangiogenic capacity of such neutrophils. Similarly, inhibition of MMP9 activity resulted in significant impairment of neutrophil-mediated angiogenesis. All in all, deficiency in TGF-β/ALK1/ENG signaling in HHT neutrophils results in their proangiogenic activation and disease progression. Therapeutic strategies targeting neutrophil degranulation and MMP9 release and activity may serve as a potential therapeutic option for HHT.

Receptor-specific contributions of caveolae, PKC, and Src tyrosine kinase to serotonergic and adrenergic regulation of Kv channels and vasoconstriction

AimsCaveolae are invaginated, Ω-shaped membrane structures. They are now recognized as portals for signal transduction of multiple chemical and mechanical stimuli. Notably, the contribution of caveolae has been reported to be receptor-specific. However, details of how they differentially contribute to receptor signaling remain unclear. Main methodsUsing isometric tension measurements, patch-clamping, and western blotting, we examined the contribution of caveolae and their related signaling pathways to serotonergic (5-HT2A receptor-mediated) and adrenergic (α1-adrenoceptor-mediated) signaling in rat mesenteric arteries. Key findingsDisruption of caveolae by methyl-β-cyclodextrin effectively blocked vasoconstriction mediated by the 5-HT2A receptor (5-HT2AR), but not by the α1-adrenoceptor. Caveolar disruption selectively impaired 5-HT2AR-mediated voltage-dependent K+ channel (Kv) inhibition, but not α1-adrenoceptor-mediated Kv inhibition. In contrast, both serotonergic and α1-adrenergic effects on vasoconstriction, as well as Kv currents, were similarly blocked by the Src tyrosine kinase inhibitor PP2. However, inhibition of protein kinase C (PKC) by either GO6976 or chelerythrine selectively attenuated the effects mediated by the α1-adrenoceptor, but not by 5-HT2AR. Disruption of caveolae decreased 5-HT2AR-mediated Src phosphorylation, but not α1-adrenoceptor-mediated Src phosphorylation. Finally, the PKC inhibitor GO6976 blocked Src phosphorylation by the α1-adrenoceptor, but not by 5-HT2AR. Significance5-HT2AR-mediated Kv inhibition and vasoconstriction are dependent on caveolar integrity and Src tyrosine kinase, but not on PKC. In contrast, α1-adrenoceptor-mediated Kv inhibition and vasoconstriction are not dependent on caveolar integrity, but rather on PKC and Src tyrosine kinase. Caveolae-independent PKC is upstream of Src activation for α1-adrenoceptor-mediated Kv inhibition and vasoconstriction.

FUNDC1: a key mediator of adenosine A2BR activation-induced inhibition of cardiac mitophagy under ischemia/reperfusion conditions.

Mitophagy is an essential factor in mitochondrial quality control and myocardial ischaemia/reperfusion (I/R) injury protection. Because adenosine A2B receptor (A2BR) activation exerts a major role in reducing myocardial I/R injury, the effects of adenosine A2BR activation on cardiac mitophagy under reperfusion conditions were investigated. 110 adult Wistar rats (7-10 w), weighing 250-350 grams, were cultured in specific-pathogen-free (SPF) conditions before experiments. All hearts were removed and reperfused by Langendorff device. Six hearts with coronary flow (CF) values >28 or <10 mL/min were excluded. Others were arbitrarily divided into the following groups: sham operation group, I/R group, BAY60-6583 (BAY) (1-1,000 nM) + I/R group, PP2 + BAY + I/R group. After ischemia in rats, reperfusion was performed. H9c2 cells were placed in an imitated ischemic environment followed by Tyrode's solution to stimulate hypoxia/reoxygenation (H/R) injury. The mitochondrial fluorescence indicator MitoTracker Green and lysosomal fluorescence indicator LysoTracker Red were used to examine mitochondria and lysosomes, respectively. Colocalization of mitochondrial and autophagy marker proteins was determined by immunofluorescence. Autophagic flow currents were tested by Ad-mCherry-GFP-LC3B. Protein-protein interactions were predicted using a database and analyzed by co-immunoprecipitation. Autophagy marker protein, mitophagy marker protein, and mitophagy protein FUNDC1 were detected by immunoblotting. Compared with those in the I/R group, myocardial autophagy and mitophagy were suppressed by the selective adenosine A2BR agonist BAY, and this effect was inhibited by the selective Src tyrosine kinase inhibitor PP2, indicating that adenosine A2BR activation could inhibit myocardial autophagy and mitophagy by activating Src tyrosine kinase. In support, in H9c2 cells, the selective Src tyrosine kinase inhibitor PP2 inhibited the effect of BAY on TOM20 with LC3 or mitochondria with lysosomes colocalization and autophagy flow. Here, we showed that mitochondrial FUNDC1 co-precipitated with Src tyrosine kinase after BAY was added. Consistently, the immunofluorescence and western blotting results demonstrated that compared to that in the H/R group, the expression of mitochondrial FUNDC1 was reduced by BAY, but this effect was reversed by PP2. Adenosine A2BR activation may inhibit myocardial mitophagy by downregulating expression of the mitochondrial FUNDC1 by activating Src tyrosine kinase under I/R conditions and could increase the interaction between Src tyrosine kinase and FUNDC1.

A6 PROTEASE-ACTIVATED RECEPTOR (PAR)-2 ACTIVATION ENHANCES EPITHELIAL WOUND HEALING MIGRATION THROUGH SRC AND RAC1 PATHWAYS

Abstract Background Protease-activated receptors (PARs) and their activating enzymes have been postulated to play a role in inflammatory bowel disease (IBD) pathogenesis, but the specific roles of PAR2 in disease initiation and progression remain unclear. PAR2 activation has both pro-proliferative and pro-migratory effects and could be involved with the restoration of the epithelial barrier following injury. We previously showed that PAR2 activation increases epithelial wound healing through ERK, PI3K, and JNK pathways. However, the role of Src kinase and RhoGTPases, which PAR2 also activates, is not known. Purpose We hypothesized that PAR-2 activation induces wound healing in intestinal epithelial cells through Src and Rac1 activity. Method Circular wounds were made in T84 colonic epithelial cell monolayers. Wounded monolayers were treated with the PAR2 activating peptide, 2-furoyl-LIGRLO (2fLI, 5 μM), or the inactive control reverse-sequence peptide, 2-furoyl-OLRGIL (2fO, 5 μM), and live-cell imaging was used to record wound healing over a 24-hr period. Proliferation and apoptosis were measured using EdU and TUNEL assays, respectively. The mechanism of action was evaluated using inhibitors of Src (PP2), EGFR (PD153035), MLCK (ML-7), ROCK (Y-27632), Rac1 (NSC 23766), and Cdc42 (ML141), and western blot (WB) was used to confirm the protein levels of p-Src (Y416), p-ERK1/2, p-JNK, and p-PI3K. For immunofluorescence, images of E-cadherin and F-actin were taken to capture the entire wound border and surrounding cells. Result(s) PAR2 activation by 2fLI promoted wound healing compared to 2fO or vehicle control at the 24-hr time point (p&amp;lt;0.05). PAR2 activation had no effect on proliferation at the wound edge and did not affect apoptosis but did enhance lamellipodia/filopodia formation (p&amp;lt;0.001). These findings indicate that PAR2 reprograms the cells toward a migratory rather than a proliferative phenotype. When we investigated the mechanisms of action, the Src tyrosine kinase inhibitor, PP2, blocked PAR2-induced wound healing (p&amp;lt;0.0001). PAR2 activation increased Src phosphorylation (Y416, p&amp;lt;0.05) and the immunofluorescence showed a rise in actin cable formation at the wound edge and a reduction in lamellipodia/filopodia formation in the group treated with PP2 when compared to 2fLI (p&amp;lt;0.001). Although PAR2 activation increases the phosphorylation of ERK1/2 and JNK, this did not happen through Src. Furthermore, PAR2 did not increase the phosphorylation of PI3K as confirmed by WB analysis. Inhibition of EGFR (PD153035), MLCK (ML-7), ROCK (Y-27632), and Cdc42 (ML-141) did not alter PAR2-induced wound healing (p&amp;gt;0.05). In contrast, Rac1 inhibition by NSC23766 completely abrogated the PAR2-induced wound healing (p&amp;lt;0.05). Conclusion(s) PAR2 activation drives wound healing via Src tyrosine kinase and Rac1 activities. These findings provide a further mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases. Disclosure of Interest None Declared

ImmunoPET Imaging Identifies the Optimal Timepoint for Combination Therapy in Xenograft Models of Triple-Negative Breast Cancer.

(1) Purpose: The glycoprotein non-metastatic melanoma B (gpNMB) is a type 1 transmembrane protein that is overexpressed in numerous cancers, including triple-negative breast cancer (TNBC). Its overexpression is associated with lower overall survival of patients with TNBC. Tyrosine kinase inhibitors such as dasatinib can upregulate gpNMB expression, which has the potential to enhance therapeutic targeting with anti-gpNMB antibody drug conjugates such as glembatumumab vedotin (CDX-011). Our primary aim is to quantify the degree and identify the timeframe of gpNMB upregulation in xenograft models of TNBC after treatment with the Src tyrosine kinase inhibitor, dasatinib, by longitudinal positron emission tomography (PET) imaging with the 89Zr-labeled anti-gpNMB antibody ([89Zr]Zr-DFO-CR011). The goal is to identify the timepoint at which to administer CDX-011 after treatment with dasatinib to enhance therapeutic efficacy using noninvasive imaging. (2) Methods: First, TNBC cell lines that either express gpNMB (MDA-MB-468) or do not express gpNMB (MDA-MB-231) were treated with 2 μM of dasatinib in vitro for 48 h, followed by Western blot analysis of cell lysates to determine differences in gpNMB expression. MDA-MB-468 xenografted mice were also treated with 10 mg/kg of dasatinib every other day for 21 days. Subgroups of mice were euthanized at 0-, 7-, 14-, and 21-days post treatment, and tumors were harvested for Western blot analysis of tumor cell lysates for gpNMB expression. In a different cohort of MDA-MB-468 xenograft models, longitudinal PET imaging with [89Zr]Zr-DFO-CR011 was performed before treatment at 0 (baseline) and at 14 and 28 days after treatment with (1) dasatinib alone (2) CDX-011 (10 mg/kg) alone, or (3) sequential treatment of dasatinib for 14 days then CDX-011 to determine changes in gpNMB expression in vivo relative to baseline. As a gpNMB-negative control, MDA-MB-231 xenograft models were imaged 21 days after treatment with dasatinib, combination of CDX-011 and dasatinib, and vehicle control. (3) Results: Western blot analysis of MDA-MB-468 cell and tumor lysates showed that dasatinib increased expression of gpNMB in vitro and in vivo at 14 days post treatment initiation. In PET imaging studies of different cohorts of MDA-MB-468 xenografted mice, [89Zr]Zr-DFO-CR011 uptake in tumors (SUVmean = 3.2 ± 0.3) was greatest at 14 days after treatment initiation with dasatinib (SUVmean = 4.9 ± 0.6) or combination of dasatinib and CDX-011 (SUVmean= 4.6 ± 0.2) compared with that at baseline (SUVmean = 3.2 ± 0.3). The highest tumor regression after treatment was observed in the combination-treated group with a percent change in tumor volume relative to baseline (%CTV) of -54 ± 13 compared with the vehicle control-treated group (%CTV = +102 ± 27), CDX-011 group (%CTV = -25 ± 9.8), and dasatinib group (%CTV = -23 ± 11). In contrast, the PET imaging of MDA-MB-231 xenografted mice indicated no significant difference in the tumor uptake of [89Zr]Zr-DFO-CR011 between treated (dasatinib alone or in combination with CDX-011) and vehicle-control groups. (4) Conclusions: Dasatinib upregulated gpNMB expression in gpNMB-positive MDA-MB-468 xenografted tumors at 14 days post treatment initiation, which can be quantified by PET imaging with [89Zr]Zr-DFO-CR011. Furthermore, combination therapy with dasatinib and CDX-011 appears to be a promising therapeutic strategy for TNBC and warrants further investigation.

Autophagy Inhibition Enhances Anti-Glioblastoma Effects of Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors.

Drug resistance presents a major obstacle to the successful treatment of glioblastoma. Autophagy plays a key role in drug resistance, particularly in relation to targeted therapy, which has prompted the use of autophagy inhibitors to increase the effectiveness of targeted therapeutics. The ability of two Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306, to induce autophagy was evaluated in the human glioblastoma cell line U87 and its multidrug-resistant counterpart U87-TxR. Autophagy markers were assessed by flow cytometry, microscopy, and Western blot, and induction of autophagy by these compounds was demonstrated after 3 h as well as 48 h. The effects of Si306 and pro-Si306 on cell proliferation and cell death were examined in the presence or absence of autophagy inhibition by bafilomycin A1. Combined treatments of Si306 and pro-Si306 with bafilomycin A1 were synergistic in nature, and the inhibition of autophagy sensitized glioblastoma cells to Src tyrosine kinase inhibitors. Si306 and pro-Si306 more strongly inhibited cell proliferation and triggered necrosis in combination with bafilomycin A1. Our findings suggest that modulation of Si306- and pro-Si306-induced autophagy can be used to enhance the anticancer effects of these Src tyrosine kinase inhibitors and overcome the drug-resistant phenotype in glioblastoma cells.

A62 PROTEASE-ACTIVATED RECEPTOR-2 ACTIVATION ENHANCES EPITHELIAL WOUND HEALING THROUGH SRC PATHWAY-DEPENDENT CELL MIGRATION

BackgroundProtease-activated receptors (PARs) and their activating enzymes play a role in inflammatory bowel disease (IBD) pathogenesis, but the specific roles of PAR2 in disease initiation and progression remain unclear. Interestingly, PAR2 activation has both pro-proliferative and pro-migratory effects and could be involved with restoration of the epithelial barrier following injury. We previously showed that the PAR2 activation increase the epithelial wound healing through ERK, PI3K and JNK pathways. However, the role of Src kinase, which is also activated by PAR2, is not known.AimsWe hypothesized that PAR-2 activation induces a wound healing response in intestinal epithelial cells through Src activity.MethodsCircular wounds were made in T84 colonic epithelial cells monolayers. Wounded monolayers were treated with the PAR2 activating peptide, 2-furoyl-LIGRLO (2fLI, 5 μM), or the inactive control reverse-sequence peptide, 2-furoyl-OLRGIL (2fO, 5 μM), and live cell imaging was used to record wound closure over a 12 or 24-hr period. The specificity of PAR2 was assessed with a PAR2 inhibitor (GB88). Proliferation and cytotoxicity were measured using EdU and TUNEL assays. The mechanism of action was evaluated using inhibitors of FAK (PF57328 and FAK14) and Src (PP2) and western blot (WB) was used to confirm the protein levels [p-FAK (Y397; Y576/577) and p-Src (Y416)]. For immunofluorescence, images of E-cadherin and F-actin or p-FAK were taken to capture the entire wound border and surrounding cells.ResultsPAR2 activation by 2fLI promoted wound closure compared to 2fO or vehicle control at the 12 and 24 hr timepoints ( p<0.05). PAR2’s ability to enhance wound closure was blocked with the PAR2 inhibitor, GB88 ( p<0.01). PAR2 activation had no effect on proliferation at the wound-edge and did not cause apoptosis, but enhanced lamellipodia/filopodia formation ( p<0.001), indicating that PAR2 might program the cells toward a migratory phenotype rather than a proliferative phenotype. When we investigated the mechanisms of action, PAR2 activation did not induce focal adhesion kinase (FAK) expression in T84 cells and inhibition of FAK by selective inhibitors (PF573288 and FAK14) did not alter PAR2-induced wound healing. The immunofluorescence for p-FAK (Y397) and WB (Y937 and Y576/577) confirmed these results. However, the Src tyrosine kinase inhibitor (PP2, p<0.0001) inhibited PAR2-induced wound healing. PAR2 activation increased Src phosphorylation (Y416, p<0.05).ConclusionsPAR2 activation drives wound healing in part via Src tyrosine kinase activity, but independently of FAK activity. These findings provide a further mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases.Funding AgenciesCAG, CIHR

Sphingosine-1-phosphate induces Ca&lt;sup&gt;2+&lt;/sup&gt; mobilization via TRPC6 channels in SH-SY5Y cells and hippocampal neurons

Sphingosine-1-phosphate (S1P) is a widely expressed biologically active sphingolipid that plays an important role in cell differentiation, migration, proliferation, metabolism and apoptosis. S1P activates various signaling pathways, some of which evoke Ca&lt;sup&gt;2+&lt;/sup&gt; signals in the cytosol. Few studies have focused on the mechanism by which S1P evokes Ca&lt;sup&gt;2+&lt;/sup&gt; signals in neurons. Here, we show that S1P evokes global Ca&lt;sup&gt;2+&lt;/sup&gt; signals in SH-SY5Y cells and hippocampal neurons. Removal of extracellular calcium largely abolished the S1P-induced increase in intracellular Ca&lt;sup&gt;2+&lt;/sup&gt;, suggesting that the influx of extracellular Ca&lt;sup&gt;2+&lt;/sup&gt; is the major contributor to this process. Moreover, we found that S1P-induced Ca&lt;sup&gt;2+&lt;/sup&gt; mobilization is independent of G protein-coupled S1P receptors. The TRPC6 inhibitor SAR7334 suppressed S1P-induced calcium signals, indicating that the TRPC6 channel acts as the downstream effector of S1P. Using patch-clamp recording, we showed that S1P activates TRPC6 currents. Two Src tyrosine kinase inhibitors, Src-I1 and PP2, dramatically inhibited the activation of TRPC6 by S1P. Taken together, our data suggest that S1P activates TRPC6 channels in a Src-dependent way to induce Ca&lt;sup&gt;2+&lt;/sup&gt; mobilization in SH-SY5Y cells and hippocampal neurons.

Saracatinib, a Src Tyrosine Kinase Inhibitor, as a Disease Modifier in the Rat DFP Model: Sex Differences, Neurobehavior, Gliosis, Neurodegeneration, and Nitro-Oxidative Stress.

Diisopropylfluorophosphate (DFP), an organophosphate nerve agent (OPNA), exposure causes status epilepticus (SE) and epileptogenesis. In this study, we tested the protective effects of saracatinib (AZD0530), a Src kinase inhibitor, in mixed-sex or male-only Sprague Dawley rats exposed to 4–5 mg/kg DFP followed by 2 mg/kg atropine and 25 mg/kg 2-pralidoxime. Midazolam (3 mg/kg) was given to the mixed-sex cohort (1 h post-DFP) and male-only cohort (~30 min post-DFP). Saracatinib (20 mg/kg, oral, daily for 7 days) or vehicle was given two hours later and euthanized eight days or ten weeks post-DFP. Brain immunohistochemistry (IHC) showed increased microgliosis, astrogliosis, and neurodegeneration in DFP-treated animals. In the 10-week post-DFP male-only group, there were no significant differences between groups in the novel object recognition, Morris water maze, rotarod, or forced swim test. Brain IHC revealed significant mitigation by saracatinib in contrast to vehicle-treated DFP animals in microgliosis, astrogliosis, neurodegeneration, and nitro-oxidative stressors, such as inducible nitric oxide synthase, GP91phox, and 3-Nitrotyrosine. These findings suggest the protective effects of saracatinib on brain pathology seem to depend on the initial SE severity. Further studies on dose optimization, including extended treatment regimen depending on the SE severity, are required to determine its disease-modifying potential in OPNA models.

Phosphorylation of Na+,K+-ATPase at Tyr10 of the \u03b11-Subunit is Suppressed by AMPK and Enhanced by Ouabain in Cultured Kidney Cells

Na+,K+-ATPase (NKA) is essential for maintenance of cellular and whole-body water and ion homeostasis. In the kidney, a major site of ion transport, NKA consumes ~ 50% of ATP, indicating a tight coordination of NKA and energy metabolism. AMP-activated protein kinase (AMPK), a cellular energy sensor, regulates NKA by modulating serine phosphorylation of the α1-subunit, but whether it modulates other important regulatory phosphosites, such as Tyr10, is unknown. Using human kidney (HK-2) cells, we determined that the phosphorylation of Tyr10 was stimulated by the epidermal growth factor (EGF), which was opposed by inhibitors of Src kinases (PP2), tyrosine kinases (genistein), and EGF receptor (EGFR, gefitinib). AMPK activators AICAR and A-769662 suppressed the EGF-stimulated phosphorylation of EGFR (Tyr1173) and NKAα1 at Tyr10. The phosphorylation of Src (Tyr416) was unaltered by AICAR and increased by A-769662. Conversely, ouabain (100 nM), a pharmacological NKA inhibitor and a putative adrenocortical hormone, enhanced the EGF-stimulated Tyr10 phosphorylation without altering the phosphorylation of EGFR (Tyr1173) or Src (Tyr416). Ouabain (100–1000 nM) increased the ADP:ATP ratio, while it suppressed the lactate production and the oxygen consumption rate in a dose-dependent manner. Treatment with ouabain or gene silencing of NKAα1 or NKAα3 subunit did not activate AMPK. In summary, AMPK activators and ouabain had antagonistic effects on the phosphorylation of NKAα1 at Tyr10 in cultured HK-2 cells, which implicates a role for Tyr10 in coordinated regulation of NKA-mediated ion transport and energy metabolism.Graphical

Protease-activated receptor-2 activation enhances epithelial wound healing via epidermal growth factor receptor

ABSTRACT The intestinal barrier function relies on the presence of a single layer of epithelial cells. Barrier dysfunction is associated with the inflammatory bowel diseases (IBD). Understanding the mechanisms involved in intestinal wound healing in order to sustain the barrier function has a great therapeutic potential. Activation of protease-activated receptor-2 (PAR2) induces COX-2 expression in intestinal epithelial cells via EGFR transactivation. COX-2 is well known for its protective effects in the gastrointestinal tract. Therefore, we hypothesized that PAR-2 activation induces a wound healing response in intestinal epithelial cells through COX-2-derived lipid mediators and EGFR transactivation. Immunofluorescence and calcium assay were used to characterize CMT-93 mouse colonic epithelial cell line for PAR2 expression and its activity, respectively. Treatment with PAR2 activating peptide 2-furoyl-LIGRLO-NH2 (2fLI), but not by its inactive reverse-sequence peptide (2fO) enhanced wound closure in scratch wounded monolayers. The EGFR tyrosine kinase inhibitor (PD153035), broad-spectrum matrix metalloproteinase inhibitor (GM6001) and Src tyrosine kinase inhibitor (PP2) inhibited PAR2-induced wound healing. However, PAR2 activation did not induce COX-2 expression in CMT-93 cells and inhibition of COX-2 by COX-2 selective inhibitor (NS-398) did not alter PAR2-induced wound healing. In conclusion, PAR2 activation drives wound healing in CMT-93 cells via EGFR transactivation. Matrix metalloproteinases and Src tyrosine kinase activity may involve in EGFR transactivation and PAR2-induced wound healing is independent of COX-2 activity. These findings provide a mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases.

Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells.

Background: Glioblastoma (GBM) highly expresses Src tyrosine kinase involved in survival, proliferation, angiogenesis and invasiveness of tumor cells. Src activation also reduces reactive oxygen species (ROS) generation, whereas Src inhibitors are able to increase cellular ROS levels. Methods: Pro-oxidative effects of two pyrazolo[3,4-d]pyrimidine derivatives—Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306—were investigated in human GBM cells U87 and patient-derived GBM-6. ROS production and changes in mitochondrial membrane potential were assessed by flow cytometry. The expression levels of superoxide dismutase 1 (SOD1) and 2 (SOD2) were studied by Western blot. DNA damage, cell death induction and senescence were also examined in GBM-6 cells. Results: Si306 and pro-Si306 more prominently triggered ROS production and expression of antioxidant enzymes in primary GBM cells. These effects were followed by mitochondrial membrane potential disruption, double-strand DNA breaks and senescence that eventually led to necrosis. Conclusion: Src kinase inhibitors, Si306 and pro-Si306, showed significant pro-oxidative potential in patient-derived GBM cells. This feature contributes to the already demonstrated anti-glioblastoma properties of these compounds in vitro and in vivo and encourages clinical investigations.

Implementation of DoE and Risk-Based Enhanced Analytical Quality by Design Approach to Stability-Indicating RP-HPLC Method for Stability Study of Bosutinib.

Bosutinib is a small-molecule BCR-ABL and Src tyrosine kinase inhibitor used for the treatment of chronic myelogenous leukemia. According to published literature, no stability-indicating reversed-phase (RP)-HPLC method has been reported for the estimation of bosutinib. A stability-indicating RP-HPLC method has been developed for the stability study of bosutinib, using a risk- and design of experiments (DoE)-based enhanced analytical quality by design (AQbD) approach. The risk-based AQbD approach was applied by risk parameter identification and risk assessment by risk priority number (RPN) ranking and filtering, as per International Conference on Harmonization (ICH) Q9 guidelines. The DoE-based AQbD approach was implemented by response surface analysis using a central composite design. The risks of critical method risk parameters were mitigated by navigation of design space and framing of control strategy. Chromatographic separation was performed using a C18 column and acetonitrile-1.0% triethylamine (v/v) in water (pH 7.0, adjusted by ortho-phosphoric acid). The developed method was validated according to ICH Q2 (R1). The developed and validated method was applied for the assay of bosutinib in pharmaceutical dosage forms. The developed method was extended for an oxidative degradation kinetic study of bosutinib at different pH conditions. The developed RP-HPLC method can be used as an analytical tool for quality control and stability studies of pharmaceutical dosage forms of bosutinib in the pharmaceutical industry. The development and validation of a stability-indicating RP-HPLC method for the estimation of bosutinib by implementation of a DoE- and risk-based enhanced AQbD approach. The method was applied to assay pharmaceutical dosage forms and in an oxidative degradation kinetic study.

Multi-omics data integration and network-based analysis drives a multiplex drug repurposing approach to a shortlist of candidate drugs against COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is undeniably the most severe global health emergency since the 1918 Influenza outbreak. Depending on its evolutionary trajectory, the virus is expected to establish itself as an endemic infectious respiratory disease exhibiting seasonal flare-ups. Therefore, despite the unprecedented rally to reach a vaccine that can offer widespread immunization, it is equally important to reach effective prevention and treatment regimens for coronavirus disease 2019 (COVID-19). Contributing to this effort, we have curated and analyzed multi-source and multi-omics publicly available data from patients, cell lines and databases in order to fuel a multiplex computational drug repurposing approach. We devised a network-based integration of multi-omic data to prioritize the most important genes related to COVID-19 and subsequently re-rank the identified candidate drugs. Our approach resulted in a highly informed integrated drug shortlist by combining structural diversity filtering along with experts’ curation and drug–target mapping on the depicted molecular pathways. In addition to the recently proposed drugs that are already generating promising results such as dexamethasone and remdesivir, our list includes inhibitors of Src tyrosine kinase (bosutinib, dasatinib, cytarabine and saracatinib), which appear to be involved in multiple COVID-19 pathophysiological mechanisms. In addition, we highlight specific immunomodulators and anti-inflammatory drugs like dactolisib and methotrexate and inhibitors of histone deacetylase like hydroquinone and vorinostat with potential beneficial effects in their mechanisms of action. Overall, this multiplex drug repurposing approach, developed and utilized herein specifically for SARS-CoV-2, can offer a rapid mapping and drug prioritization against any pathogen-related disease.

Regulation of Fibroblast Cell Polarity by Src Tyrosine Kinase.

Src protein tyrosine kinases (SFKs) are a family of nonreceptor tyrosine kinases that are localized beneath the plasma membrane and are activated during cell adhesion, migration, and elongation. Due to their involvement in the activation of signal transduction cascades, SFKs have been suggested to play important roles in the determination of cell polarity during cell extension and elongation. However, the mechanism underlying Src-mediated polarity formation remains unclear. The present study was performed to investigate the mechanisms underlying Src-induced cell polarity formation and cell elongation using Src knockout fibroblasts (SYFs) together with an inhibitor of Src. Normal and Src knockout fibroblasts were also transfected with a wild-type c-Src, dominant negative c-Src, or constitutively active c-Src gene to analyze the changes in cell morphology. SYF cells cultured on a glass substrate elongated symmetrically into spindle-shaped cells, with the formation of focal adhesions at both ends of the cells. When normal fibroblasts were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, they elongated into symmetrical spindle-shaped cells, similar to SYF cells. These results suggest that cell polarity during extension and elongation may be regulated by SFKs and that the expression and regulation of Src are important for the formation of polarity during cell elongation.

Inhibition of Akt/mTOR pathway overcomes intrinsic resistance to dasatinib in triple-negative breast cancer

Currently, the only therapeutic choice for the treatment of triple-negative breast cancer (TNBC) is chemotherapy. In TNBC, despite strong preclinical data, clinical trials of molecular targeted drugs, such as the Src tyrosine kinase inhibitor dasatinib, have failed because of the heterogeneity of TNBC cells. Here, we examined the mechanism of intrinsic resistance to dasatinib in five TNBC cell lines. First, we divided the TNBC cell lines into those sensitive or resistant to dasatinib and found that activation of Src was inhibited in all of the cell lines. In contrast, we found that dasatinib inhibited Akt phosphorylation in only the dasatinib-sensitive cell lines. Consequently, we found that combination treatment with dasatinib and an inhibitor of Akt or mTOR suppressed cell proliferation more than did either monotherapy in the dasatinib-resistant cell lines. Finally, to mimic intrinsic resistance, we established a dasatinib-tolerant TNBC cell line. In this cell line, the combinational effect of Akt/mTOR inhibition with dasatinib was observed, as it was in the cell lines with intrinsic resistance. Together, the present results show that the effect of dasatinib in TNBC is independent of Src inhibition, and that Akt/mTOR inhibition might be an effective strategy to overcome TNBC cells with intrinsic dasatinib resistance.

SRC Tyrosine Kinase Inhibitor and X-rays Combined Effect on Glioblastoma Cell Lines.

Glioblastoma (GBM) is one of the most lethal types of tumor due to its high recurrence level in spite of aggressive treatment regimens involving surgery, radiotherapy and chemotherapy. Hypoxia is a feature of GBM, involved in radioresistance, and is known to be at the origin of treatment failure. The aim of this work was to assess the therapeutic potential of a new targeted c-SRC inhibitor molecule, named Si306, in combination with X-rays on the human glioblastoma cell lines, comparing normoxia and hypoxia conditions. For this purpose, the dose modifying factor and oxygen enhancement ratio were calculated to evaluate the Si306 radiosensitizing effect. DNA damage and the repair capability were also studied from the kinetic of γ-H2AX immunodetection. Furthermore, motility processes being supposed to be triggered by hypoxia and irradiation, the role of c-SRC inhibition was also analyzed to evaluate the migration blockage by wound healing assay. Our results showed that inhibition of the c-SRC protein enhances the radiotherapy efficacy both in normoxic and hypoxic conditions. These data open new opportunities for GBM treatment combining radiotherapy with molecularly targeted drugs to overcome radioresistance.