Comparison of ELISA-based tyrosine kinase assays for screening EGFR inhibitors

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu,Tyr)l:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431 cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.     

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.     

Methacholine-induced bronchoconstriction in rats: effects of intravenous vs. aerosol delivery

Peták, Ferenc, Zoltán Hantos, ÁgnesAdamicza, Tibor Asztalos, and Peter D. Sly. Methacholine-inducedbronchoconstriction in rats: effects of intravenous vs. aerosoldelivery. J. Appl. Physiol. 82(5):1479-1487, 1997.To determine the predominant site of action ofmethacholine (MCh) on lung mechanics, two groups of open-chestSprague-Dawley rats were studied. Five rats were measured duringintravenous infusion of MCh (iv group), with doubling of concentrationsfrom 1 to 16 µg · kg¹ · min¹.Seven rats were measured after aerosol administration of MCh with dosesdoubled from 1 to 16 mg/ml (ae group). Pulmonary input impedance(ZL) between 0.5 and 21 Hz wasdetermined by using a wave-tube technique. A model containing airwayresistance (Raw) and inertance (Iaw) and parenchymal damping (G) andelastance (H) was fitted to theZL spectra. In the iv group, MChinduced dose-dependent increases in Raw [peak response 270 ± 9 (SE) % of the control level; P < 0.05] and in G (340 ± 150%;P < 0.05), with no increase inIaw (30 ± 59%) orH (111 ± 9%). In the ae group, thedose-dependent increases in Raw (191 ± 14%;P < 0.05) andG (385 ± 35%; P < 0.05) were associated with a significant increase in H (202 ± 8%; P < 0.05).Measurements with different resident gases [air vs. neon-oxygenmixture, as suggested (K. R. Lutchen, Z. Hantos, F. Peták,Á. Adamicza, and B. Suki. J. Appl.Physiol. 80: 1841-1849, 1996)] in thecontrol and constricted states in another group of rats suggested thatthe entire increase seen in G during the ivchallenge was due to ventilation inhomogeneity, whereas the aechallenge might also have involved real tissue contractions viaselective stimulation of the muscarinic receptors.

     

Mechanical impedance of the lung periphery

Hantos, Z., F. Peták, Á. Adamicza, T. Asztalos, J. Tolnai, and J. J. Fredberg. Mechanical impedance ofthe lung periphery. J. Appl. Physiol.83(5): 1595-1601, 1997.The mechanics of the regional airways andtissues was studied in isolated dog lobes by means of a modifiedwave-tube technique. Small-amplitude pseudorandom forced oscillationsbetween 0.1 and 48 Hz were applied through catheters wedged in2-mm-diameter bronchi in three regions of each lobe at translobarpressures (PL) of 10, 7, 5, 3, 2, and 1 cmH₂O. The measuredregional input impedances were fitted by a model containing theresistance (R₁) and inertance(I) of the regular (segmental) airways, the resistance of thecollateral channels (R₂), andthe damping (G) and elastance (H) of the local tissues. This model gavefar better fits to the data on impedance of the lung periphery thanwhen G and H were replaced by a single tissue compliance, whichexplains why interruption of segmental flow did not lead tomonoexponential pressure decay in previous studies. The interlobar andintralobar variances of the parameters were equally significant, andpoor correlations were found between the airway parametersR₁ andR₂ and between any airway andtissue parameter (e.g., R₁ and H).R₂ was on average ~10 timeshigher than R₁, although theR₂-to-R₁ratios and their dependencies on PL were regionally highlyvariable. However, for the total of 33 regions studied, thePL dependence was the same forR₁ and R₂, which may reflect similarmorphological structures for the regular and collateral airways. Thedependencies of G and H on PLshowed high interregional variations; generally, however, they assumedtheir minima at medium PL values(~5 cmH₂O).

     

Regulation of FasL by NF-kappaB and AP-1 in Fas-dependent thymineless death of human colon carcinoma cells

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS(-) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.     

Communication Patterns Within a Group of Shelter Dogs and Implications for Their Welfare

Keeping shelter dogs in groups provides them with a more socially and physically enriched environment, but eventually it may cause them stress. Understanding dogs' communication could help shelter staff recognize and prevent undesirable communicative patterns and encourage desirable ones. Therefore, the objective of this study was to determine communication patterns in a group of dogs in a shelter. The observed dogs were engaged in different classes of dyadic and group interactions. Certain dogs were frequently initiators of dyadic interactions, and different dogs were the recipients. The predominant form of dyadic interactions was a neutral one, and aggressive behavior was rarely observed. The tendency of certain dogs to interact continuously may represent a nuisance for less social individuals. All of the dogs participated in 3 defined classes of group interactions. At the group level, the dogs frequently interact vocally or olfactorily. A major welfare problem may be very vocal dogs because their vocalizations are noisy and broadcast far-reaching signals. The frequency of some group interactions was reduced by the amount of time the dogs had in the shelter.     

Synthesis and characterization of novel quinazoline type inhibitors for mutant and wild-type EGFR and RICK kinases

The development of selective protein kinase inhibitors has become an important area of drug discovery for the treatment of different diseases. We report the synthesis and characterization of a series of novel quinazoline derivatives against three therapeutically important and pharmacologically related kinases: 1) epidermal growth factor receptor (EGFR; wild type and mutant) in the field of cancer, 2) receptor-interacting caspase-like apoptosis-regulatory kinase (RICK) in the field of inflammation, and 3) pUL97 of human cytomegalovirus (HCMV). For reference purpose we have synthesized the four clinically relevant quinazolines, including the lead compounds, which we previously identified for RICK and pUL97. A total of 52 quinazoline derivatives were synthesized and tested on the basis of these leads to specifically target the hydrophobic pocket of the ATP-binding site. Selected compounds were tested on wild-type and mutant forms of EGFR, RICK, and pUL97 kinases; their logP and logS values for assessing suitability as drugs were calculated and hit or lead compounds identified.     

Prevention of bronchial hyperreactivity in a rat model of precapillary pulmonary hypertension

Background

The development of bronchial hyperreactivity (BHR) subsequent to precapillary pulmonary hypertension (PHT) was prevented by acting on the major signalling pathways (endothelin, nitric oxide, vasoactive intestine peptide (VIP) and prostacyclin) involved in the control of the pulmonary vascular and bronchial tones.

Methods

Five groups of rats underwent surgery to prepare an aorta-caval shunt (ACS) to induce sustained precapillary PHT for 4 weeks. During this period, no treatment was applied in one group (ACS controls), while the other groups were pretreated with VIP, iloprost, tezosentan via an intraperitoneally implemented osmotic pump, or by orally administered sildenafil. An additional group underwent sham surgery. Four weeks later, the lung responsiveness to increasing doses of an intravenous infusion of methacholine (2, 4, 8 12 and 24 μg/kg/min) was determined by using the forced oscillation technique to assess the airway resistance (Raw).

Results

BHR developed in the untreated rats, as reflected by a significant decrease in ED₅₀, the equivalent dose of methacholine required to cause a 50% increase in Raw. All drugs tested prevented the development of BHR, iloprost being the most effective in reducing both the systolic pulmonary arterial pressure (Ppa; 28%, p = 0.035) and BHR (ED₅₀ = 9.9 ± 1.7 vs. 43 ± 11 μg/kg in ACS control and iloprost-treated rats, respectively, p = 0.008). Significant correlations were found between the levels of Ppa and ED₅₀ (R = -0.59, p = 0.016), indicating that mechanical interdependence is primarily responsible for the development of BHR.

Conclusions

The efficiency of such treatment demonstrates that re-establishment of the balance of constrictor/dilator mediators via various signalling pathways involved in PHT is of potential benefit for the avoidance of the development of BHR.