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Kreimann M.,University of Greifswald | Brandt S.,University of Greifswald | Krauel K.,Institute For Immunologie Und Transfusionsmedizin | Block S.,University of Greifswald | And 4 more authors.
Blood | Year: 2014

The chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 anti-parallel β-sheets exceeding ∼30%(achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic. © 2014 by The American Society of Hematology.

Michael S.,University of Leipzig | Warstat C.,University of Leipzig | Michel F.,University of Leipzig | Yan L.,University of Bonn | And 3 more authors.
Purinergic Signalling | Year: 2010

Adenosine can show anti-inflammatory as well as pro-inflammatory activities. The contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. In this study, we examined the effect of the adenosine A2A receptor agonist CGS 21680 and the A2BR antagonist PSB-1115 on acute inflammation induced experimentally by 2,4,6-trinitrobenzenesulfonic acid (TNBS) on rat ileum/jejunum preparations. Pre-incubation of the ileum/jejunum segments with TNBS for 30 min resulted in a concentration-dependent inhibition of acetylcholine (ACh)-induced contractions. Pharmacological activation of the A2AR with CGS 21680 (0.1-10 μM) pre-incubated simultaneously with TNBS (10 mM) prevented concentration-dependently the TNBS-induced inhibition of the ACh contractions. Stimulation of A2BR with the selective agonist BAY 60-6583 (10 μM) did neither result in an increase nor in a further decrease of ACh-induced contractions compared to the TNBS-induced inhibition. The simultaneous pre-incubation of the ileum/jejunum segments with TNBS (10 mM) and the selective A2BR antagonist PSB-1115 (100 μM) inhibited the contraction-decreasing effect of TNBS. The effects of the A2AR agonist and the A2BR antagonist were in the same range as the effect induced by 1 μM methotrexate. The combination of the A2AR agonist CGS 21680 and the A2BR antagonist PSB-1115 at subthreshold concentrations of both agents found a significant amelioration of the TNBS-diminished contractility. Our results demonstrate that the activation of A2A receptors or the blockade of the A2B receptors can prevent the inflammation-induced disturbance of the ACh-induced contraction in TNBS pre-treated small intestinal preparations. The combination of both may be useful for the treatment of inflammatory bowel diseases. © 2009 Springer Science+Business Media B.V.

Muller D.,Martin Luther University of Halle Wittenberg | Adelsberger K.,Luft und Thermotechnik Bayreuth GmbH and Co. | Imming P.,Martin Luther University of Halle Wittenberg | Imming P.,Institute For Pharmazie
Synthetic Communications | Year: 2013

Thiols are indispensable for the preparation of many organic sulfur compounds. Their strong smell and use for gas leakage perception render it almost impossible to work with them without arousing public attention. Molar amounts of the very odoriferous thiol 2-methyl-2-propanethiol (t-butylthiol) are needed, for example, for the large-scale synthesis of two useful synthetic building blocks, 1,2,4,5-tetrakis(t-butylthio)benzene and tetramethylbenzo-2,2, 6,6-[1,2-d;4,5-d']bis[1,3]dithiol. We investigated an array of alternatives to circumvent the problem: (1) alternative thiols (primary, long-chain, tertiary thiols of larger molar mass); (2) exhaust cleaning methods (adsorption, oxidation, conversion to a salt); and (3) thermal exhaust treatment. Only combustion of the fumehood exhaust with domestic gas at 900 °C in a regenerative thermal oxidation unit was able to completely prevent the thiol smell from escaping. Supplemental materials are available for this article. Go to the publisher's online edition of Synthetic Communications® to view the free supplemental file. © 2013 Copyright Taylor and Francis Group, LLC.

Darras F.H.,Institute For Pharmazie | Pockes S.,Institute For Pharmazie | Huang G.,University of Wurzburg | Wehle S.,University of Wurzburg | And 5 more authors.
ACS Chemical Neuroscience | Year: 2014

Combination of AChE inhibiting and histamine H3 receptor antagonizing properties in a single molecule might show synergistic effects to improve cognitive deficits in Alzheimer's disease, since both pharmacological actions are able to enhance cholinergic neurotransmission in the cortex. However, whereas AChE inhibitors prevent hydrolysis of acetylcholine also peripherally, histamine H3 antagonists will raise acetylcholine levels mostly in the brain due to predominant occurrence of the receptor in the central nervous system. In this work, we designed and synthesized two novel classes of tri- and tetracyclic nitrogen-bridgehead compounds acting as dual AChE inhibitors and histamine H3 antagonists by combining the nitrogen-bridgehead moiety of novel AChE inhibitors with a second N-basic fragment based on the piperidinylpropoxy pharmacophore with different spacer lengths. Intensive structure-activity relationships (SARs) with regard to both biological targets led to compound 41 which showed balanced affinities as hAChE inhibitor with IC50 = 33.9 nM, and hH3R antagonism with Ki = 76.2 nM with greater than 200-fold selectivity over the other histamine receptor subtypes. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds and molecular dynamics studies to explain high affinity at the hH3R. © 2014 American Chemical Society.

Chen X.,Institute For Pharmazie | Chen X.,University of Wurzburg | Wehle S.,University of Wurzburg | Kuzmanovic N.,University of Regensburg | And 6 more authors.
ACS Chemical Neuroscience | Year: 2014

Photochromic cholinesterase inhibitors were obtained from cis-1,2-α-dithienylethene-based compounds by incorporating one or two aminopolymethylene tacrine groups. All target compounds are potent acetyl- (AChE) and butyrylcholinesterase (BChE) inhibitors in the nanomolar concentration range. Compound 11b bearing an octylene linker exhibited interactions with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Yet upon irradiation with light, the mechanism of interaction varied from one photochromic form to another, which was investigated by kinetic studies and proved "photoswitchable". The AChE-induced β-amyloid (Aβ) aggregation assay gave further experimental support to this finding: Aβ1-40 aggregation catalyzed by the PAS of AChE might be inhibited by compound 11b in a concentration-dependent manner and seems to occur only with one photochromic form. Computational docking studies provided potential binding modes of the compound. Docking studies and molecular dynamics (MD) simulations for the ring-open and -closed form indicate a difference in binding. Although both forms can interact with the PAS, more stable interactions are observed for the ring-open form based upon stabilization of a water molecule network within the enzyme, whereas the ring-closed form lacks the required conformational flexibility for an analogous binding mode. The photoswitchable inhibitor identified might serve as valuable molecular tool to investigate the different biological properties of AChE as well as its role in pathogenesis of AD in in vitro assays. © 2014 American Chemical Society.

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