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Inhibition Pattern (inhibition + pattern)
Selected AbstractsBiochemical characterization and inhibitor discovery of shikimate dehydrogenase from Helicobacter pyloriFEBS JOURNAL, Issue 20 2006Cong Han Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shikimate pathway. It catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate, and has been developed as a promising target for the discovery of antimicrobial agent. In this report, we identified a new aroE gene encoding SDH from Helicobacter pylori strain SS1. The recombinant H. pylori shikimate dehydrogenase (HpSDH) was cloned, expressed, and purified in Escherichia coli system. The enzymatic characterization of HpSDH demonstrates its activity with kcat of 7.7 s,1 and Km of 0.148 mm toward shikimate, kcat of 7.1 s,1 and Km of 0.182 mm toward NADP, kcat of 5.2 s,1 and Km of 2.9 mm toward NAD. The optimum pH of the enzyme activity is between 8.0 and 9.0, and the optimum temperature is around 60 °C. Using high throughput screening against our laboratory chemical library, five compounds, curcumin (1), 3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H -chromen-7-yl 3-chlorobenzoate (2), butyl 2-{[3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H -chromen-7-yl]oxy}propanoate (3), 2-({2-[(2-{[2-(2,3-dimethylanilino)-2-oxoethyl]sulfanyl}-1,3-benzothiazol-6-yl)amino]-2-oxoethyl}sulfanyl)- N -(2-naphthyl)acetamide (4), and maesaquinone diacetate (5) were discovered as HpSDH inhibitors with IC50 values of 15.4, 3.9, 13.4, 2.9, and 3.5 µm, respectively. Further investigation indicates that compounds 1, 2, 3, and 5 demonstrate noncompetitive inhibition pattern, and compound 4 displays competitive inhibition pattern with respect to shikimate. Compounds 1, 4, and 5 display noncompetitive inhibition mode, and compounds 2 and 3 show competitive inhibition mode with respect to NADP. Antibacterial assays demonstrate that compounds 1, 2, and 5 can inhibit the growth of H. pylori with MIC of 16, 16, and 32 µg·mL,1, respectively. This current work is expected to favor better understanding the features of SDH and provide useful information for the development of novel antibiotics to treat H. pylori -associated infection. [source] Kinetic mechanism for p38 MAP kinase ,FEBS JOURNAL, Issue 18 2005A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate p38 Mitogen-activated protein kinase alpha (p38 MAPK,) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPK,, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1,10) that at pH 7.5, 10 mm Mg2+ and 27 °C p38 MAPK, phosphorylates ATF2,115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (,,,-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPK, with dissociation constants of KA = 360 µm, KI = 240 µm, and KI > 2000 µm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPK,,ATF2,115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd , 70 µm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2,115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a ,-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPK, inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations >,100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor. [source] Synthesis and pp60c-Src Tyrosine Kinase Inhibitory Activities of Novel Indole-3-Imine and Amine Derivatives Substituted at N1 and C5ARCHIV DER PHARMAZIE, Issue 6 2009Zuhal Kiliç Abstract A series of novel 1,3,5-trisubstituted indole derivatives, namely, N -benzyl 5-phenyl indole-3-imine, N -benzyl-5-(p -fluorophenyl)indole-3-imine and their corresponding amine congeners, were designed and synthesized as pp60c-Src tyrosine kinase inhibitors, and their inhibitory activities toward pp60c-Src tyrosine kinase were evaluated by in-vitro kinase assay. Pre-screening at two doses of compounds against kinase target revealed that, except for the N -benzyl-5-phenyl indole imine derivatives 7a,7d, all indole derivatives show the target inhibition at varying levels. Consequently, the compounds, 8c, 8f, 8g, and 8h, were selected for prescreening tests. The dose-response curves for up to six concentrations (250 to 7.8 ,M) of the active compounds were obtained by tyrosine kinase assay and the four-parameter logistic analysis of these data resulted in the IC50s of 4.69, 74.79, 75.06, and 84.23 ,M for compounds 8c, 8f, 8g, and 8h, respectively. Therefore, compound 8c, 1-(1-benzyl-5-phenyl-1H -indole-3-yl)- N -(4-fluorobenzyl)methanamine·HCl, was the promising inhibitor for pp60c-Src, followed by compounds 8g and 8h. Under the same conditions, compound 8f did not provide any reasonable inhibition pattern to be considered as active compound. Therefore, among all four active compounds, compound 8f was not found suitable for further analysis. [source] The in vitro Inhibitory Potential of Trade Herbal Products on Human CYP2D6-Mediated Metabolism and the Influence of EthanolBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 5 2007Bent H. Hellum Herbal components were extracted from commercially available products in a way that ensured the same composition of constituents in the extract as in the original trade products. c-DNA baculovirus expressed CYP2D6 was used with dextromethorphan as substrate. Quinidine was included as a positive control inhibitor. A validated high performance liquid chromatography methodology was used to quantify the formation of dextrorphan (product of dextromethorphan O-demethylation). Ethanol showed a biphasic effect on CYP2D6 metabolism, increasing initially the CYP2D6 activity with 175% of control up to a concentration of 1.1%, where after ethanol linearly inhibited the CYP2D6 activity. All the investigated herbs inhibited CYP2D6 activity to some extent, but only St. John's wort, common sage and common valerian were considered possible candidates for in vivo clinically significant effects. They showed IC50 values of 0.07 ± 7 × 10,3 mg/ml, 0.8 ± 0.05 mg/ml and 1.6 ± 0.2 mg/ml, respectively. St. John's wort inhibited CYP2D6-mediated metabolism in an uncompetitive manner, while common valerian and common sage in a non-competitive manner demonstrated interherb differences in inhibition patterns and differences when compared to the more homogenous competitive inhibitor quinidine. Common valerian was the only herb that showed a mechanistic inhibition of CYP2D6 activity and attention should be paid to a possible toxicity of this herb. [source] Antitumor activity of sequence-specific alkylating agents: Pyrolle-imidazole CBI conjugates with indole linkerCANCER SCIENCE, Issue 3 2006Ken-ichi Shinohara DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2,6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of , log GI50 (50% growth inhibition concentration) for compounds 1,6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the , log GI50 of compound 1 and its conjugates 2,6 showed extremely low values (R < 0). These results suggest that differences in the sequence specificity of DNA alkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis. (Cancer Sci 2006; 97: 219,225) [source] Sensitization and allergy to turnip rape: a comparison between the Finnish and French children with atopic dermatitisACTA PAEDIATRICA, Issue 2 2009S Poikonen Abstract Aim: Finnish children with atopic dermatitis (AD) are frequently sensitized and show positive food challenge to turnip rape. We examined whether French children are also allergic to this oilseed plant and whether mustard could be the cross-reacting allergen. Methods: Turnip rape and mustard challenge was performed to 14 Finnish and 14 French children with atopic dermatitis and positive skin prick test to turnip rape. Specific IgE antibodies were measured by ImmunoCAP and enzyme-linked immunosorbent assay (ELISA). Results: Open labial or oral challenge to turnip rape was positive in 14 (100%) Finnish and five (36%) French children and mustard challenge in five Finnish and five French children. IgE antibodies to oilseed rape and mustard were slightly more frequent in the Finnish (100% and 93%) than in the French (93% and 71%) children but rare (4%) in the 28 matched controls. The same findings were true for IgE antibodies to purified 2S albumin allergens, which showed similar cross-wise IgE inhibition patterns. Conclusion: French children with atopic dermatitis show IgE antibodies to turnip rape, oilseed rape and mustard similarly to the Finnish children. 2S albumin allergens in the seeds of these plants are highly cross-reactive and therefore, they all could be important sensitizers in children with atopic dermatitis. [source] | |||||||||||||