Calorimetric vs. van't Hoff binding enthalpies from isothermal titration calorimetry: Ba2+-crown ether complexation

The 1:1 complexation reaction between Ba(2+) and 18-crown-6 ether is re-examined using isothermal titration calorimetry (ITC), with the goal of clarifying previously reported discrepancies between reaction enthalpies estimated directly (calorimetric) and indirectly, from the temperature dependence of the reaction equilibrium constant K (van't Hoff). The ITC thermograms are analyzed using three different non-linear fit models based on different assumptions about the data error: constant, proportional to the heat and proportional but correlated. The statistics of the fitting indicate a preference for the proportional error model, in agreement with expectations for the conditions of the experiment, where uncertainties in the delivered titrant volume should dominate. With attention to proper procedures for propagating statistical error in the van't Hoff analysis, the differences between Delta H(cal) and Delta H(vH) are deemed statistically significant. In addition, statistically significant differences are observed for the Delta H(cal) estimates obtained for two different sources of Ba(2+), BaCl(2) and Ba(NO(3))(2). The effects are tentatively attributed to deficiencies in the standard procedure in ITC of subtracting a blank obtained for pure titrant from the thermogram obtained for the sample.     

N-[18F]fluoroethylpiperidin-4-ylmethyl butyrate: a novel radiotracer for quantifying brain butyrylcholinesterase activity by positron emission tomography

In Alzheimer's disease, cerebral cortical butyrylcholinesterase (BChE) activity is reported to be elevated. Our aim was to develop a novel (18)F-labeled tracer for quantifying cerebral BChE activity by positron emission tomography. With in vitro screening of N-[(14)C]ethylpiperidin-3- and 4-ylmethyl esters, N-[(14)C]ethylpiperidin-4-ylmethyl butyrate was selected as a lead for (18)F-labeling, affording N-[(18)F]fluoroethylpiperidin-4-ylmethyl butyrate. The (18)F-labeled butyrate showed the required properties for in vivo BChE measurement, that is, the lipophilic nature of the authentic ester, high specificity to BChE, a moderate hydrolysis rate, and the hydrophilic nature of the metabolite.     

Design and synthesis of Rho kinase inhibitors (I)

Several structurally unrelated scaffolds of the Rho kinase inhibitor were designed using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. A docking simulation using the ligand-binding pocket of the Rho kinase model helped to comprehensively understand and to predict the structure-activity relationship of the inhibitors. This understanding was useful for developing new Rho kinase inhibitors of higher potency and selectivity. We identified several potent platforms for developing the Rho kinase inhibitors, namely, pyridine, 1H-indazole, isoquinoline, and phthalimide.     

A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer

Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.     

Identification of a Gene Involved in the Synthesis of a Dipeptidyl Peptidase IV Inhibitor in Aspergillus oryzae

WYK-1 is a dipeptidyl peptidase IV inhibitor produced by Aspergillus oryzae strain AO-1. Because WYK-1 is an isoquinoline derivative consisting of three l-amino acids, we hypothesized that a nonribosomal peptide synthetase was involved in its biosynthesis. We identified 28 nonribosomal peptide synthetase genes in the sequenced genome of A. oryzae RIB40. These genes were also identified in AO-1. Among them, AO090001000009 (wykN) was specifically expressed under WYK-1-producing conditions in AO-1. Therefore, we constructed wykN gene disruptants of AO-1 after nonhomologous recombination was suppressed by RNA interference to promote homologous recombination. Our results demonstrated that the disruptants did not produce WYK-1. Furthermore, the expression patterns of 10 genes downstream of wykN were similar to the expression pattern of wykN under several conditions. Additionally, homology searches revealed that some of these genes were predicted to be involved in WYK-1 biosynthesis. Therefore, we propose that wykN and the 10 genes identified in this study constitute the WYK-1 biosynthetic gene cluster.     

Characterization of the expression and cell-surface localization of transmembrane protein 132A

The aim of this work was to study the in vitro effects of δ-lactone 1, δ-lactam 3 and their enaminone derivatives 2 and 4, synthesized in our laboratory, on the proliferative responses of human lymphocytes, Th1 and Th2 cytokine secretion and intracellular redox status. Peripheral blood lymphocytes were isolated using differential centrifugation on a density gradient of Histopaque. They were cultured with mitogen concanavalin A (Con A) and with different concentrations of the compounds 1, 2, 3 and 4 (0.1-10 μM). Proliferation (MTT assay), IL-2, INFγ and IL-4 (Elisa kits), oxidative markers (intracellular glutathione, hydroperoxide and carbonyl protein contents) and cytotoxic effect (micronucleus test) were determined. The compounds 1 and 2 are immunosuppressive and decrease IL-2, INFγ and IL-4 secretion with a shift away from Th2 response to Th1 phenotype. The compounds 3 and 4 were immunostimulant and increased cytokine secretion with a shift away from Th1 response to Th2. The introduction of an enamine group to 1 and 3 to provide 2 and 4 seemed to attenuate their immunological properties. These immunomodulatory properties were, however, accompanied by an increase in lymphocyte intracellular oxidative stress, especially with 1 and 2 at high concentrations. In conclusion, the compounds 1, 2, 3 and 4 could be used to provide cell-mediated immune responses for novel therapies in T-cell mediated immune disorders.