Design,synthesis, and molecular modelling of pyridazinone and phthalazinone derivatives as protein kinases inhibitors

The design and synthesis of pyridazinone and phthalazinone derivatives are described. Newly synthesized compounds were tested on a panel of four kinases in order to evaluate their activity and potential selectivity. In addition, the promising compounds were tested on four cancer cell lines to examine cytotoxic effects. The compounds inhibited DYRK1A and GSK3 with different activity. SAR analysis and docking calculations were carried out to aid in the interpretation of the results. Taken together, our findings suggest that pyridazinone and phthalazinone scaffolds are interesting starting points for design of potent GSK3 and DYRK1A inhibitors.     

Cdk2-dependent phosphorylation of p21 regulates the role of Cdk2 in cisplatin cytotoxicity

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21(WAF1/Cip1) was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21(S78D). Mutant p21(S78D) was an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.     

Repellent and insecticidal activities of Melia azedarach L. against cotton leafworm, Spodoptera littoralis (Boisd.)

A crude acetone extract and oil of ripe fruits from Melia azedarach L. were evaluated against the 2nd and 4th instar larvae of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae). Both oil and extract exhibited highly significant growth inhibition at all concentrations tested, while the oil of M. azedarach recorded higher insecticidal activity against both instars than the crude extract. GC-MS analysis of the oil revealed the presence of linoleic acid methyl ester, oleic acid methyl ester, and free oleic acid as the main components in addition to hexadecanol, palmitic acid, methyl esters of stearic acid and myristic acid. Fatty acids and their esters were not only the main constituents of essential oil from the ripe fruits of M. azedarach, but also mainly responsible for the insecticidal and growth inhibition activity against S. littoralis.     

The only elements required by plants that are deficient in seawater are nitrogen,phosphorous and iron

It has been known for some time that mangroves grow in the intertidal areas of desert countries where fresh water flows into the sea, but this phenomenon has not been understood. In Eritrea we observed that mangroves grow where infrequent rains flow into the Red Sea. We theorized that the fresh water must bring elements needed for plant growth that are absent in seawater. We compared the composition of seawater to that of Zarrouk’s algae medium. All the elements in algae medium are in sufficient quantity in seawater except for nitrogen, phosphorous and iron. If we supply these elements we can grow any plant that can grow in seawater. We have also begun planting plants that can grow in sea water in the Sahara desert with sea water irrigation and fertilization with nitrogen, phosphorus and iron. We believe this will be major step in reducing hunger and poverty in the world.     

Roles of three transporters, CbcXWV, BetT1, and BetT3, in Pseudomonas aeruginosa choline uptake for catabolism

Pseudomonas aeruginosa uses the quaternary amine choline as a carbon source, osmoprotectant, and macromolecular precursor. The importance of choline in P. aeruginosa physiology is highlighted by the presence of multiple known and putative choline transporters encoded within its genome. This report describes the relative roles of three choline transporters, the ABC transporter CbcXWV and two symporters, BetT1 and BetT3, in P. aeruginosa growth on choline under osmotic conditions that are physiologically relevant to eukaryotic hosts. The increased lag phases exhibited by the ΔbetT1 and ΔbetT1 ΔbetT3 mutants relative to the wild type upon transfer to medium with choline as a sole carbon source suggested roles for BetT1 and BetT3 in cells newly exposed to choline. BetT3 and CbcXWV, but not BetT1, were sufficient to support growth on choline. betT1 and betT3 expression was regulated by the repressor BetI and choline, whereas cbcXWV expression was induced by the activator GbdR and glycine betaine. The data support a model in which, upon transfer to a choline-based medium, the glycine betaine derived from choline taken up by BetT1 and BetT3 promotes subsequent GbdR-mediated cbcXWV induction. Furthermore, growth data indicated that the relative contributions of each transporter varied under different conditions, as BetT1 and CbcXWV were the primary choline transporters under hypo-osmolar conditions whereas BetT3 was the major choline transporter under hyperosmolar conditions. This work represents the first systematic approach to unravel the mechanisms of choline uptake in P. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.     

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94-99%) and blastocyst (90-96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94-98%) and blastocysts (91-93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes.     

Functional and morphological study of cultured pancreatic islets treated with cyclosporine

Cyclosporine A (CsA), a potent immunosuppressive drug, has been found to induce glucose intolerance through its toxic effect on the endocrine pancreas. It is not exactly known whether CsA has a direct effect on the endocrine pancreas or induces its effect indirectly. The present study was therefore undertaken to examine the function and morphology of isolated pancreatic islets when they are directly exposed in vitro to CsA. Pancreatic islets were isolated from adult male Lewis rats using collagenase ductal perfusion technique. The islets were separated with the discontinuous Ficoll gradient technique and further purified by hand picking of the non-islet tissue. The islets were cultured in RPMI-1640, pH 7.4 and maintained at 37 degrees C in a humid atmosphere of 5% (v/v) carbon dioxide in air. Cyclosporine was added to the culture medium to give a final concentration of 1 microg/ml (therapeutic dose), 5 microg/ml (toxic dose), or vehicle (control). Islets were harvested at 1, 4 and 10 days of culture and processed for functional or histological study. The functional study of the islets cultured with 1 microg/ml CsA showed insulin and C-peptide contents similar to those of the control islets. The islets cultured with 5 microg/ml CsA showed a marked decrease in insulin and C-peptide contents. Glucose-dependent insulin release was variable. C-peptide release was lower than that of the control following both the therapeutic and toxic doses of CsA. Phase contrast microscopy showed that the islets cultured with 1 microg/ml CsA were mostly normal looking with a well-defined regular periphery; a few islets had ill-defined or irregular peripheries. The islets cultured with 5 microg/ml CsA had ill-defined irregular peripheries at 1 day, and were dense and forming clumps at 4 and 10 days following culture. There was a decrease in the islet number following the therapeutic dose; the decrease was more following the toxic dose of CsA. The islet diameters increased after the therapeutic dose, but slightly decreased following the toxic dose of CsA. Islets showed a weakly positive immunoperoxidase reaction for insulin that was weaker following the toxic dose of CsA. It is concluded that CsA has a direct effect on B-cells that was proved by the functional and morphological changes seen in the pancreatic islets cultured in vitro.     

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.