Biochemical assay-based selectivity profiling of clinically relevant kinase inhibitors on mutant forms of EGF receptor

Gefitinib and erlotinib are potent EGFR tyrosine kinase inhibitors (potentially) useful for the treatment of non-small-cell lung cancer (NSCLC). Clinical responses, however, in NSCLC patients have been linked to the presence of certain activating mutations of EGFR. We used an ELISA-based biochemical assay to confirm the selective inhibitory efficacy of gefitinib and erlotinib on the activated mutant receptor. Our results are in line with the clinical observations providing evidence for the predictive power of the kinase assay. Four additional compounds were also investigated: CI-1033 and EKB-569 had dramatic inhibitory effects on all EGFR forms, whereas PD153035 and AG1478 were active on wild-type and activating mutant protein. In docking simulations with wild-type EGFR, our inhibitory data are in good agreement with the binding scores. These data confirm that anilinoquinazolines are good starting structures for the next generation of selective drugs against mutant EGFR, whereas CI-1033 and EKB-569 may represent advances for patients with both wild-type and anilinoquinazoline-resistant mutant tumors.     

Prp19/Pso4 Is an Autoinhibited Ubiquitin Ligase Activated by Stepwise Assembly of Three Splicing Factors

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Chemotactic selection of Tetrahymena pyriformis GL induced with histamine, di-iodotyrosine or insulin

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (EQ) is a synthetic antioxidant used for preventing rancidity in animal foodstuffs. Three groups of ten fish were given a diet containing respectively 75 (control group with the commercial food), 200 and 400 ppm EQ for 16 days. The control group had a plasma osmolality and chloride concentration within the normal range of marine teleosts, but sodium concentrations of only about 110 mM, indicating the presence in the plasma of substantial amounts of another cation. Fish given food with 400 ppm EQ displayed a 70 mM increase in the plasma concentration of sodium. This indicates that EQ has disturbed the iono-regulatory mechanisms, probably by reducing the ATP production or inhibiting directly the Na/K-ATPase in the gills. The large increase in plasma sodium concentration was not accompanied by any significant increase in plasma osmolality, indicating that at least a part of the sodium added to the plasma is made osmotically inactive. In spite of the elevated plasma sodium concentration, the sodium content of erythrocytes of the 400-ppm EQ fish was reduced to half, while the content of calcium was unaffected. The transmembrane energy gradient of sodium in the EQ exposed turbot obviously increased, allowing them to use a sodium coupled antiport system to keep the cellular calcium content low when the Ca-ATPases is blocked. A mechanism of this kind is also likely to be important to turbot that experience hypoxia under natural conditions. The 400-ppm group also displayed a substantial increase in liver weight, but the physiological significance of this effect is not clear. The leucocyte counts indicated the absence of obvious immunological effects.     

Functional Organization of the Inverted Repeats of IS30

The mobile element IS30 has 26-bp imperfect terminal inverted repeats (IRs) that are indispensable for transposition. We have analyzed the effects of IR mutations on both major transposition steps, the circle formation and integration of the abutted ends, characteristic for IS30. Several mutants show strikingly different phenotypes if the mutations are present at one or both ends and differentially influence the transposition steps. The two IRs are equivalent in the recombination reactions and contain several functional regions. We have determined that positions 20 to 26 are responsible for binding of the N-terminal domain of the transposase and the formation of a correct 2-bp spacer between the abutted ends. However, integration is efficient without this region, suggesting that a second binding site for the transposase may exist, possibly within the region from 4 to 11 bp. Several mutations at this part of the IRs, which are highly conserved in the IS30 family, considerably affected both major transposition steps. In addition, positions 16 and 17 seem to be responsible for distinguishing the IRs of related insertion sequences by providing specificity for the transposase to recognize its cognate ends. Finally, we show both in vivo and in vitro that position 3 has a determining role in the donor function of the ends, especially in DNA cleavage adjacent to the IRs. Taken together, the present work provides evidence for a more complex organization of the IS30 IRs than was previously suggested.Mobile DNA elements have been described in most organisms and represent a considerable proportion of their genetic material. These elements play an important role in the evolution of the host genome due to their capacities to generate DNA rearrangements and influence the expression of neighboring genes. Their ability to form compound transposons contributes to the sequestering and dispersion of accessory genes, such as those specifying resistance to antibiotics, virulence, and various catabolic activities. The simplest mobile elements are the bacterial insertion sequences (ISs), which typically harbor one or two open reading frames (ORF) coding for the transposase (Tpase). More than 2,400 ISs have been described and classified into families (IS Finder, http://www-is.biotoul.fr/) on the basis of similarities in their genetic organization and Tpases (30). The terminal inverted repeats (IRs) are essential for the transposition of most ISs. The IRs, together with the Tpase, form a complex where the cleavage and strand transfer reactions occur. The IRs generally contain two functional modules: the internal region serves as the binding site of Tpase, while the terminal part is required for DNA cleavage and the strand transfer process (2). Besides these principal cis-acting elements, some ISs carry additional regulatory DNA sequences in the IRs or in the subterminal regions (18).The IS30 family currently comprises more than 80 elements distributed throughout the Gram-positive and Gram-negative bacteria and the Archaea (IS Finder, http://www-is.biotoul.fr). IS30 (1, 5), the founding element of the family, is 1,221 bp long and has 26-bp imperfect IRs (the left end of the IR [IRL] and the right end of the IR [IRR]; Fig. ​Fig.1A)1A) and one ORF with a coding capacity for a 44.3-kDa Tpase. The element has a preference for two distinct types of target sequences: the natural hot spots (HSs), characterized by a 24-bp symmetric consensus (23), and the IRs of the element itself (21, 22). Potential helix-turn-helix motifs (HTH) responsible for HS and IR targeting are located in the N-terminal region of the Tpase (19). While the first motif, HTH1, is required only for transposition into the HS sequences, the conserved H-HTH2 motif is essential for both IR and HS targeting (15, 19).Open in a separate windowFIG. 1.Transposition assays for comparing the IS30-based transposons composed of simple IRs. (A) Comparison of the IS30 IR sequences. Dots indicate matching bases. (B) Schematic representation of the intermolecular transposition pathway. The graph shows the two major steps characteristic for IS30 transposition (steps 1 and 2). The transposon donor plasmid and its derivative, the circular transposon (thin line), carry the 26-bp IRs of IS30 (boxes with open and filled triangles representing IRL and IRR, respectively). The Cm^r gene flanking the transposon in the donor plasmid is shown as a gray box. The target plasmid (dotted line) carries the GOHS hot spot sequence (cross-hatched box). (C) Transposition frequencies of IS30-based transposons with different combinations of the IRs. The graph shows the overall frequency of transposition into the hot spot (steps 1 and 2) and the frequency of the major steps assayed separately. Data were obtained from at least three parallel experiments.IS30 transposition occurs through two major steps (14, 24) (Fig. ​(Fig.1B).1B). The first is the formation of an active intermediate by joining of the IRs. This process involves the Tpase-catalyzed cleavage of one strand at the 3′ IS end, which then attacks the same strand 2 bp outside the other IR. This strand transfer generates a single-strand bridge between the ends and leads to a figure-eight structure (33). This active transposition intermediate carrying the joined IRs probably proceeds via replicative resolution, as described for IS911 (11, 25) and IS2 (16). The resolution can lead to the circularization of a single IS or to the formation of a head-to-tail repeat of two IS30 copies. In the second step of transposition, the active forms interact with the target DNA, resulting in the known transposition products: simple insertion, deletion, inversion, or replicon fusion (14, 24).In this work, we describe the modularity of the IR ends of IS30 by analyzing several mutants. According to our results, the IS30 IRs can be divided into functional regions that are differently involved in the main transposition steps. We show that positions 2 and 3 play a pivotal role in cleavage of the ends and, consequently, in their donor function. While the terminal part (1 to 17 bp) of the IRs is indispensable for both major steps, the internal region, i.e., the binding site for the N-terminal part of Tpase (20 to 26 bp), appears to be required only for the junction formation. Although the exact role of the terminal part of IRs is less clear, several mutations in this region considerably affected both the junction formation and integration. The fact that the internal IR region is not involved in the integration suggests that the Tpase binds to other sequences during this reaction.     

Development and characterization of a FIA system for selective assay of l-ascorbic acid in food samples

An amperometric detector and an enzymatic reaction were combined for the measurement of l-ascorbic acid. The enzyme cell (containing immobilized ascorbate oxidase) was connected to a flow injection analyzer (FIA) system with a glassy carbon electrode as an amperometric detector. During optimization and measurements two sample injectors were used, one before and one after the enzyme cell, thus eliminating the background interferences. Subtraction of the signal area given in the presence of enzyme from the one given in the absence of enzyme was applied for measuring analyte concentrations and calibration at 400 mV. Analysis capacity of system is 25 samples/hour. The relative standard deviation (RSD) was below 5% (5 times repeated, 400 μmol/L conc.), linearity up to 400 μmol/L, limit of detection (LOD) 5 μmol/L, fitting of calibration curve in 25–400 μmol/L range was R ² = 0.99.     

Spiropiperidine CCR5 antagonists

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.     

Effect of thymol on calcium handling in mammalian ventricular myocardium

Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, this effect was significant from 50 microM, whereas the thymol-induced calcium release only from 100 microM. In single ryanodine receptors incorporated into artificial lipid bilayer thymol induced long lasting openings, having mean open times increased with 3 orders of magnitude, however, the specific conductance of the channel remained unaltered. This effect of thymol was not voltage-dependent and failed to prevent the binding of ryanodine. In conclusion, the negative inotropic action of thymol can be explained by reduction in calcium content of the sarcoplasmic reticulum due to the combination of the thymol-induced calcium release and inhibition of the calcium pump. The calcium-sensitizer effect, observed at lower thymol concentrations, indicates that thymol is likely to interact with the contractile machinery also.