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.

Profiling of the drug resistance of thousands of Src tyrosine kinase mutants uncovers a regulatory network that couples autoinhibition to the dynamics of the catalytic domain

SUMMARYProtein kinase inhibitors are effective cancer therapies, but acquired resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations with model studies and in the clinic, we still lack a comprehensive understanding of kinase inhibitor resistance. Here, we measured the resistance of thousands of Src tyrosine kinase mutants to a panel of ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src’s catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src’s phosphotransferase activity were prone to the development of resistance. Unexpectedly, a resistance-prone cluster of residues that are on the top face of the N-terminal lobe of the catalytic domain contributes to Src autoinhibition by reducing the dynamics of the catalytic domain, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how comprehensive profiling of drug resistance can be used to understand potential resistance pathways and uncover new mechanisms of kinase regulation.

Src activates retrograde membrane traffic through phosphorylation of GBF1

The Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP Exchange factor (GEF) GBF1 and small GTPase Arf1. Here we show that Src induces the formation of tubular transport carriers containing GALNTs. The kinase phosphorylates GBF1 on 10 tyrosine residues; two of them, Y876 and Y898 are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes GBF1 binding to the GTPase; molecular modeling suggests partial melting of the Sec7 domain and intramolecular rearrangement. GBF1 mutants defective for these rearrangements prevent binding, carrier formation and GALNTs relocation, while phosphomimetic GBF1 mutants induce tubules. In sum, Src promotes GALNTs relocation by promoting GBF1 binding to Arf1. Based on residue conservation, similar regulation of GEF-Arf complexes by tyrosine phosphorylation could be a conserved and wide-spread mechanism.

Toward the Synthesis of (–)-TAN-2483B Lactam Analogues

<p>Natural products continue to be an abundant source of lead compounds for drug discovery and development. (–)-TAN-2483A and (–)-TAN-2483B, isolated from the culture of a filamentous fungus, incorporate an unusual furo[3,4-b]pyran-5-one scaffold. TAN-2483A was initially reported to inhibit the c-Src tyrosine kinase enzyme, a potential anticancer target, and parathyroid hormone-induced bone resorption. TAN-2483B, on the other hand, was not isolated in sufficient quantities for biological testing. The synthesis of TAN-2483B is therefore desirable from a drug discovery perspective. Several analogues of TAN-2483B that are functionalised at the propenyl sidechain have previously been synthesised in the Harvey group and have shown promising biological activity. For example, the (Z)-ethyl ester analogue showed micromolar inhibition of HL-60 cells and Bruton’s tyrosine kinase, a protein involved in B-cell maturation that is implicated in certain cancers. The lactone moiety of TAN-2483B and its sidechain analogues, however, appears to be unstable to nucleophilic attack. The aim of this thesis was to investigate the viability of a synthetic route toward lactam analogues of TAN-2483B. It was proposed that substituting the lactone for a lactam would increase the stability of the compound in nucleophilic media. Moreover, the lactam nitrogen may provide a site for further functionalisation of the compound for future structure-activity relationship studies. Because installation of the (Z)-ethyl ester sidechain via Wittig conditions has previously been found to be more facile than installation of the (E)-propenyl sidechain found in the natural product, investigations into forming the lactam ring system were carried out on the ethyl ester advanced intermediates. Reductive amination of a ketone intermediate was envisaged to install the amine prior to a palladium-catalysed carbonylation/lactam formation step. The promising bioactivity of the (Z)-ethyl ester analogue was anticipated to be retained in the target lactam analogues. It was found that the substrates of the proposed reductive amination, the advanced ketone intermediates, were incompatible with the tested conditions, presumably due to base sensitivity. Three by-products from the reductive amination experiments were isolated and tentatively characterised by NMR spectroscopy and HRMS. An alternative route toward lactam analogues of TAN-2483B, via intermediate amines accessed by the substitution of an activated alcohol, was briefly investigated with encouraging results. Further optimisation of the synthetic route toward analogues of TAN-2483B was also achieved. Removal of a purification step enabled the more expedient two-step synthesis of a diol intermediate. The two-step transformation to (Z)- and (E)-ethyl ester intermediates, via sodium periodate-mediated diol cleavage and Wittig olefination, proceeded in the highest yield obtained to date. Investigations into the desilylation of a trimethylsilyl-protected acetylene were also conducted. Although lactam analogues of TAN-2483B were not obtained in this study, progress was made toward their synthesis. The alternative route toward amines that was briefly explored here appears promising, and work is ongoing in the Harvey group to access lactam (and other) analogues of TAN-2483B, in addition to the natural product itself.</p>

Toward the Synthesis of (–)-TAN-2483B Lactam Analogues

<p>Natural products continue to be an abundant source of lead compounds for drug discovery and development. (–)-TAN-2483A and (–)-TAN-2483B, isolated from the culture of a filamentous fungus, incorporate an unusual furo[3,4-b]pyran-5-one scaffold. TAN-2483A was initially reported to inhibit the c-Src tyrosine kinase enzyme, a potential anticancer target, and parathyroid hormone-induced bone resorption. TAN-2483B, on the other hand, was not isolated in sufficient quantities for biological testing. The synthesis of TAN-2483B is therefore desirable from a drug discovery perspective. Several analogues of TAN-2483B that are functionalised at the propenyl sidechain have previously been synthesised in the Harvey group and have shown promising biological activity. For example, the (Z)-ethyl ester analogue showed micromolar inhibition of HL-60 cells and Bruton’s tyrosine kinase, a protein involved in B-cell maturation that is implicated in certain cancers. The lactone moiety of TAN-2483B and its sidechain analogues, however, appears to be unstable to nucleophilic attack. The aim of this thesis was to investigate the viability of a synthetic route toward lactam analogues of TAN-2483B. It was proposed that substituting the lactone for a lactam would increase the stability of the compound in nucleophilic media. Moreover, the lactam nitrogen may provide a site for further functionalisation of the compound for future structure-activity relationship studies. Because installation of the (Z)-ethyl ester sidechain via Wittig conditions has previously been found to be more facile than installation of the (E)-propenyl sidechain found in the natural product, investigations into forming the lactam ring system were carried out on the ethyl ester advanced intermediates. Reductive amination of a ketone intermediate was envisaged to install the amine prior to a palladium-catalysed carbonylation/lactam formation step. The promising bioactivity of the (Z)-ethyl ester analogue was anticipated to be retained in the target lactam analogues. It was found that the substrates of the proposed reductive amination, the advanced ketone intermediates, were incompatible with the tested conditions, presumably due to base sensitivity. Three by-products from the reductive amination experiments were isolated and tentatively characterised by NMR spectroscopy and HRMS. An alternative route toward lactam analogues of TAN-2483B, via intermediate amines accessed by the substitution of an activated alcohol, was briefly investigated with encouraging results. Further optimisation of the synthetic route toward analogues of TAN-2483B was also achieved. Removal of a purification step enabled the more expedient two-step synthesis of a diol intermediate. The two-step transformation to (Z)- and (E)-ethyl ester intermediates, via sodium periodate-mediated diol cleavage and Wittig olefination, proceeded in the highest yield obtained to date. Investigations into the desilylation of a trimethylsilyl-protected acetylene were also conducted. Although lactam analogues of TAN-2483B were not obtained in this study, progress was made toward their synthesis. The alternative route toward amines that was briefly explored here appears promising, and work is ongoing in the Harvey group to access lactam (and other) analogues of TAN-2483B, in addition to the natural product itself.</p>

Crosstalk between KRAS, SRC and YAP Signaling in Pancreatic Cancer: Interactions Leading to Aggressive Disease and Drug Resistance

Pancreatic ductal adenocarcinoma (PDAC), the predominant form of pancreatic cancer, remains a devastating disease. The purpose of this review is to highlight recent literature on mechanistic and translational developments that advance our understanding of a complex crosstalk between KRAS, YAP and Src tyrosine kinase family (SFK) in PDAC development and maintenance. We discuss recent studies indicating the importance of RAS dimerization in signal transduction and new findings showing that the potent pro-oncogenic members of the SFK phosphorylate and inhibit RAS function. These surprising findings imply that RAS may not play a crucial role in maintaining certain subtypes of PDAC. In support of this interpretation, current evidence indicates that the survival of the basal-like subtype of PDAC is less dependent on RAS but relies, at least in part, on the activity of YAP/TAZ. Based on current evidence, we propose that SFK propels PDAC cells to a state of high metastasis, epithelial-mesenchymal transition (EMT) and reduced dependence on KRAS signaling, salient features of the aggressive basal-like/squamous subtype of PDAC. Strategies for PDAC treatment should consider the opposite effects of tyrosine phosphorylation on KRAS and SFK/YAP in the design of drug combinations that target these novel crosstalk mechanisms and overcome drug resistance.

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.

Time Series Analysis on the Conformational Change of c-Src Tyrosine Kinase

c-Src tyrosine kinase plays an important role in signal transduction pathways, where its activity is regulated by phosphorylation of the two tyrosine residues. We performed targeted molecular dynamics simulation to obtain trajectory of conformational change from inactive to active form. To investigate the conformational change of c-Src tyrosine kinase, we applied network analysis to time series of correlation among residues. The time series of correlation between residues during the conformational change generated by targeted molecular dynamic simulation. With centrality measures such as betweenness centrality, degree centrality, and closeness centrality, we observed a few important residues that significantly contribute to the conformational change of c-Src tyrosine kinase for the different time steps.

Regulation of Src tumor activity by its N-terminal intrinsically disordered region

The membrane anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.