Phenocopy – A Strategy to Qualify Chemical Compounds during Hit-to-Lead and/or Lead Optimization

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype-determined phenotype of another individual. The phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of biological systems with new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor β Receptor I (TGF-βR1) were qualified by high-throughput RNA expression profiling. This chemical genomics approach resulted in a precise time-dependent insight to the TGF-β biology and allowed furthermore a comprehensive analysis of each NCE's off-target effects. The evaluation of off-target effects by the phenocopy approach allows a more accurate and integrated view on optimized compounds, supplementing classical biological evaluation parameters such as potency and selectivity. It has therefore the potential to become a novel method for ranking compounds during various drug discovery phases

›Wort‹ und ›Stein‹. Differenz und Kohärenz kultureller Ausdrucksformen

›Wort‹ und ›Stein‹ bezeichnen grundsätzlich verschiedene Medien des kulturellen Gedächtnisses und der Ausdrucksformen der Künste. Sie stehen in spannungsreichen Beziehungen zueinander; gleichzeitig sind sie vielfach ineinandergearbeitet und aufeinander bezogen. Auch wenn die Kunst- und Zeichentheorie Hegels die Anregung zur Fragestellung des Bandes bot, so gilt das nicht für seine postulierte Hierarchie der Künste, die den paragone von Skulptur, Malerei, Dichtung und Musik seit der frühen Neuzeit fortsetzte. Vielmehr untersuchen die Beiträge die Bezugnahme und die Wechselwirkung der unterschiedlichen Ausdrucksformen. Dabei wird das mediale Spannungsverhältnis von ›Wort‹ und ›Stein‹ unter verschiedenen disziplinären und systematischen Perspektiven in den Blick genommen, die von der Ägyptologie und der Archäologie bis zu Literatur-, Sprach- und Zeichentheorie reichen

What Goes Around, Comes Around: Experimental Evidence on Exposed Lies

We experimentally investigate the optimal way to handle the uncovering of a noble lie, that is, a lie that supposedly is in the best interest of a given community. For this purpose, we analyze a public good game with feedback to group members on the average contributions of the other group members. The computer program inflates the feedback and shows higher than real average contributions to the high contributors. As shown by earlier studies, the partial feedback inflation increases the total payoff of the public good as it avoids the feeling of being a sucker for above average contributors. The lie is then uncovered and we continue with different feedback modes on contributions, some inflated, some true. We find that players respond similarly to both feedback modes. However, with true feedback, initial contributions in the second stage are significantly higher than with inflated feedback

Preclinical evaluation of long-acting muscarinic antagonists: comparison of tiotropium and investigational drugs. J Pharmacol Exp Ther 330: 660–668

ABSTRACT Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation caused by persistent inflammatory processes in the airways. An increased cholinergic tone mediates different pathophysiological features of COPD, such as bronchoconstriction and mucus hypersecretion, mostly through activation of the human muscarinic M 3 receptor (hM 3 ) subtype. Tiotropium bromide (Spiriva) is a well established muscarinic antagonist in the pharmacological management of COPD with a once-daily posology. The rationale behind the sustained bronchodilation obtained with tiotropium consists in its slow dissociation from hM 3 receptors. In this study, we performed a comprehensive preclinical comparison of tiotropium with other long-acting muscarinic antagonists (LAMAs) currently in clinical development, namely aclidinium bromide and glycopyrrolate. The different muscarinic antagonists were characterized for their 1) affinity toward the different human muscarinic receptor subtypes expressed in Chinese hamster ovary cells and kinetics of receptor dissociation, 2) potency in inhibiting the agonist-induced activation of muscarinic receptors through measurement of second messengers, and 3) efficacy and duration of bronchoprotection, as tested in a model of acetylcholine-induced bronchoconstriction in anesthetized dogs over a period of 24 h. All of the tested LAMAs showed high affinity and potency toward the hM 3 receptor (tiotropium, pA 2 ϭ 10.4; aclidinium, pA 2 ϭ 9.6; and glycopyrrolate, pA 2 ϭ 9.7). However, dissociation half-lives of the LAMAs from the hM 3 receptor differed significantly (tiotropium, t 1 ⁄2 ϭ 27 h; aclidinium, t 1 ⁄2 ϭ 10.7 h; and glycopyrrolate, t 1 ⁄2 ϭ 6.1 h). In line with their kinetic properties at the hM 3 , the tested LAMAs provided different levels of bronchoprotection in the in vivo setting 24 h after administration (tiotropium ϭ 35%, aclidinium ϭ 21%, and glycopyrrolate ϭ 0% at 24 h) when applied at equieffective doses

New Insights into the Wavelength Dependence of MALDI Mass Spectrometry

The interplay between the wavelength of the laser and the absorption profile of the matrix constitutes a crucial factor in matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Numerous studies have shown that typically best analytical results are obtained if the laser wavelength matches the UV absorption band of the matrix in the solid state well. However, many powerful matrices exhibit peak absorptions which differ notably from the standard MALDI laser wavelengths of 337, 349, and 355 nm, respectively. Here we used two wavelength-tunable lasers to investigate the MALDI wavelength dependence with a selected set of such matrices. We studied 3-hydroxypicolinic acid (3-HPA), 2,4,6-trihydroxyacetophenon (THAP), dithranol (1,8-dihydroxy-10<i>H</i>-anthracen-9-on), 2-(4′-hydroxybenzeneazo)­benzoic acid (HABA), and 6-aza-2-thiothymine (ATT). For analyte systems we investigated DNA oligomers (3-HPA), phospholipids (dithranol, THAP, HABA), and non-covalent peptide–peptide and protein–peptide complexes (ATT). We recorded analyte ion and total ion counts as a function of wavelength and laser fluence between 213 and 600 nm. Although the so-generated comprehensive heat maps generally corroborated the previously made findings, several fine features became notable. For example, despite a still high optical absorption exhibited by some of the matrices in the visible wavelength range, ion yields generally dropped strongly, indicating a change in ionization mechanism. Moreover, the non-covalent complexes were optimally detected at wavelengths corresponding to a relatively low optical absorptivity of the ATT matrix, presumably because of ejection of a particular cold MALDI plume. Our comprehensive data shed useful light into the MALDI mechanisms and could assist in further methodological advancement of the technique

Analysis of Free Fatty Acids by Ultraviolet Laser Desorption Ionization Mass Spectrometry Using Insect Wings as Hydrophobic Sample Substrates

Physiologically relevant free fatty acids (FFAs) were analyzed by UV-laser desorption/ionization orthogonal extracting time-of-flight mass spectrometry (LDI-oTOF-MS). Dissected wings from Drosophila melanogaster fruit flies were used as the hydrophobic, laser energy strongly absorbing sample substrates. Using untreated substrates produces predominantly molecular [M + K]⁺ ions of the FFAs, whereas other alkali metal adducts can be generated by treating the wings with the corresponding alkali hydroxide before spotting of analyte. Limits of detection for the positive ion mode were determined for mixtures of isolated FFAs to values in the low 10 pmol range. Specific values depend on chain length and degree of unsaturation. <i>R</i>² coefficients for the analysis of saturated FFAs were found to be generally close to 0.98 over about 3 orders of magnitude if an internal standard (15:0 FFA) was added. Semiquantitative analyses of mixtures containing unsaturated FFAs are also possible but require more effort on the calibration strategy. Notably, both saturated and (poly-)­unsaturated FFAs are detected sensitively in the presence of relatively high concentrations of other physiologically abundant lipids (phospholipids and triacyclglycerols). This simplifies screening of the FFA composition in crude tissue extracts. This feature is demonstrated by the analysis of a crude liver extract and that of fingermarks

Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines