Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors.Peer reviewe

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.Peer reviewe

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors

γ-H2AX Kinetics as a Novel Approach to High Content Screening for Small Molecule Radiosensitizers

Persistence of γ-H2AX after ionizing radiation (IR) or drug therapy is a robust reporter of unrepaired DNA double strand breaks in treated cells.DU-145 prostate cancer cells were treated with a chemical library ±IR and assayed for persistence of γ-H2AX using an automated 96-well immunocytochemistry assay at 4 hours after treatment. Hits that resulted in persistence of γ-H2AX foci were tested for effects on cell survival. The molecular targets of hits were validated by molecular, genetic and biochemical assays and in vivo activity was tested in a validated Drosophila cancer model.We identified 2 compounds, MS0019266 and MS0017509, which markedly increased persistence of γ-H2AX, apoptosis and radiosensitization in DU-145 cells. Chemical evaluation demonstrated that both compounds exhibited structurally similar and biochemical assays confirmed that these compounds inhibit ribonucleotide reductase. DNA microarray analysis and immunoblotting demonstrates that MS0019266 significantly decreased polo-like kinase 1 gene and protein expression. MS0019266 demonstrated in vivo antitumor activity without significant whole organism toxicity.MS0019266 and MS0017509 are promising compounds that may be candidates for further development as radiosensitizing compounds as inhibitors of ribonucleotide reductase

Rhodium(I) catalyzed hydroboration

The stereochemical outcomes and trends of catalysis of hydroboration of alkenes by catecholborane using rhodium(I) catalysts is examined. For 1,2-chirality transfer in simple allylic alcohol derivatives with aliphatic substituents on the chiral center the syn diastereoisomer is formed preferentially, this result is stereocomplementary to the stereoselectivities observed in reaction with hindered alkyl borane reagents. Syn diastereoselectivity, in the catalyzed reaction, is found to increase with increasing electron withdrawing power of the substituent on oxygen and with its steric bulk. The observed stereochemical outcome of the catalyzed reactions is postulated to be the result of binding of the substrate to the low valent metal catalyst in such a way that it behaves as the best ligand for a transition metal, i.e. such that the $\pi$* of the alkene lowered by perturbation with the lowest energy $\sigma$* of a bond on the chiral center (electronic effect). This occurs when bond to the oxygen substituent is aligned with the lobes of the $\pi$* orbital. Steric interactions dictate that the metal bonds opposite the alcohol substituent, and the larger this group is, the larger its preference to occupy this position (steric effect). N-(benzyltosyl) allyl amines were subjected to catalyzed and uncatalyzed hydroboration conditions. The catalyzed reactions give syn selectivity as anticipated. However 9-BBN also gives syn products preferentially which was not expected from analogy of the behavior of this reagent with allylic alcohol derivatives. Borane-THF complex gives consistantly high anti selectivity with these substrates. Transfer of chirality from a monochiral rhodium catalyst to several simple prochiral alkenes is described. The efficiency of this enantioselective hydroboration process is strongly dependant on the type monochiral bisphosphine used to form the catalyst. A new class of monochiral bisphosphine which contains stereogenic phosphorus centers, and a chiral backbone linking them, were synthesized by alkylation of unsymmetrically substituted phosphide ions with 1,4-Ditosyl-2,3-O-isopropylidene-L-threitol. Separation of the resulting epimeric (at phosphorus) compounds was achieved by flash chromatography of molybdenum carbonyl complexes. Decomplexation to give pure diastereoisomers is carried by treatment with sodium naphthenalide

Cone Beam CT Imaging of the Paranasal Region with a Multipurpose X-ray System—Image Quality and Radiation Exposure

Besides X-ray and fluoroscopy, a previously introduced X-ray scanner offers a 3D cone beam option (Multitom Rax, Siemens Healthcare). The aim of this study was to evaluate various scan parameters and post-processing steps to optimize image quality and radiation exposure for imaging of the parasinus region. Four human cadaver heads were examined with different tube voltages (90–121 kV), dose levels (DLs) (278–2180 nGy) and pre-filtration methods (none, Cu 0.2 mm, Cu 0.3 mm and Sn 0.4 mm). All images were reconstructed in 2 mm slice thickness with and without a metal artifact reduction algorithm in three different kernels. In total, 80 different scan protocols and 480 datasets were evaluated. Image quality was rated on a 5-point Likert scale. Radiation exposure (mean computed tomography volume index (CTDIvol) and effective dose) was calculated for each scan. The most dose-effective combination for the diagnosis of sinusitis was 121 kV/DL of 278/0.3 mm copper (CTDIvol 1.70 mGy, effective dose 77 µSv). Scan protocols with 121 kV/DL1090/0.3 mm copper were rated sufficient for preoperative sinus surgery planning (CTDIvol 4.66 mGy, effective dose 212 µSv). Therefore, sinusitis and preoperative sinus surgery planning can be performed in diagnostic image quality at low radiation dose levels with a multipurpose X-ray system

Limits in reaching the anhydrous state of wood and cellulose

Open access funding provided by Norwegian Institute of Bioeconomy Research. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.Water-sorption studies and certain organic chemistry reactions require water removal from cellulosic samples. This is hindered by the strong interaction of cellulosic materials with water, and it remains uncertain if a completely anhydrous state can be reached under common drying conditions. Here, different drying conditions were applied to wood and cellulose, and the residual moisture contents were quantified either gravimetrically or by coulometric Karl-Fischer titration. Vacuum-drying at 103 °C and ≤ 1 mbar for at least 360 min decreased the moisture content to ≤ 0.04%. However, in automated sorption balances, drying at atmospheric pressure under dry air or nitrogen flow left some samples with more than 1% moisture content. The residual moisture content obtained under dry gas flow was temperature dependent. Increasing the temperature up to 55 °C decreased the residual moisture content and cooling resulted in a moisture re-uptake, presumably due to small quantities of water vapor in the surrounding atmosphere. These effects must be considered in fundamental studies on water interactions of cellulosic materials.Peer reviewe