Systematic interaction network filtering identifies CRMP1 as a novel suppressor of huntingtin misfolding and neurotoxicity

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins.DFG [SFB740, 740/2-11, SFB618, 618/3-09, SFB/TRR43 A7]; BMBF(NGFN-Plus) [01GS08169-73, 01GS08150, 01GS08108]; HDSA Coalition for the Cure; EU (EuroSpin) [Health-F2-2009-241498, HEALTH-F2-2009-242167]; Helmholtz Association (MSBN, HelMA) [HA-215]; FCT [IF/00881/2013]info:eu-repo/semantics/publishedVersio

Synthesis and Pharmacological Evaluation of Estrogen Receptor Ligands and Cyclooxygenase Inhibitors

Titelblatt Inhaltsverzeichnis 1\. Einleitung 1 2\. Problemstellung 27 3\. Synthese von N,N-Bis(benzyliden)arylmethandiaminen 30 4\. Synthese von meso-2,4,5-Triaryl-2-imidazolinen 32 5\. Synthese von 1,2,4-tri-, 1,2,5-tri- und 1,2,4,5-tetrasubstituierten 1H- Imidazolen 40 6\. Synthese von 1,2-Bis(4-hydroxyphenyl)-2-imidazolin und 1,2-Bis(4-hydroxyphenyl)-1H-imidazol 76 7\. Kernresonanz-Spektroskopische Untersuchungen 79 8\. UV-Spektren und Extinktionskoeffizienten von 1,2,4-tri- und 1,2,4,5-tetraarylierten 1H-Imidazolen und 1,2-Bis(4-hydroxyphenyl)-4H- indeno[2,3-d]imidazol 91 9\. Fluoreszenzspektroskopie 95 10\. Pharmakologische Untersuchungen 100 11\. Ergebnisse 105 12\. Diskussion 127 13\. Zusammenfassung 157 14\. Experimenteller Teil 161 15\. Abkürzungsverzeichnis 230 16\. Literaturverzeichnis 232Ausgehend vom meso-4,5-Bis(4-hydroxyphenyl)-2-imidazolin (15a), für dessen ortho-halogenierte Derivate 15b, 15c, 156 und 157 eine agonistische Wirkung am Estrogenrezeptor beschrieben ist, wurden meso-2,4,5-Triaryl-2-imidazoline 47a \- 47d dargestellt und ihre genaktivierende Wirkung untersucht. Dabei konnte für keine der Verbindungen eine agonistische oder antagonistische Wirkung festgestellt werden. Die Verbindungen wurden auf ihre antiproliferative Wirkung an der Mammakarzinomzellinie MCF-7 untersucht. Es zeigte sich, dass sowohl meso-2,4,5-Tris(4-hydroxyphenyl)-2-imidazolin (47a) als auch die halogenierten Derivate 47b \- 47d nur maginale Effekte auf das Zellwachstum in Konzentrationen von 1 &microM;, 5 &microM; und 10 &microM; in einem Zeitraum bis 260 h ausüben. Für eine Reihe von alkylierten Triarylheteroaromaten wurden in der Vergangenheit hohe Bindungsaffinitäten mit z.T. ausgeprägter Subtypspezifität zu einer Isoform des Estrogenrezeptors nachgewiesen. Ausgehend vom 1,2-Bis(4-hydroxyphenyl)-1H-imidazol (155) wurde der Einfluss von Alkyl- und Arylgruppen in den Positionen C(4) und C(5) sowie der para-Hydroxylierung und ortho-Halogenierung in den Arylen untersucht. Dabei wurden die stärksten agonistischen Genaktivierungen für das 1,2,4-Tris(4-hydroxyphenyl)-5-methyl-1H-imidazol (119), das 5-Ethyl-1,2,4-tris(4-hydroxyphenyl)-1H-imidazol (123) und das 1,2,4-Tris(4-hydroxyphenyl)-5-phenyl-1H-imidazol (127) nachgewiesen. Die Stärke der Aktivierung nahm vom Phenyl- über den Methyl- zum Ethylsubstituenten in der Position C(5) zu. Es zeigte sich, dass die in den Arylen an C(2) und C(4) ortho-halogenierten Derivate eine deutlich geringere Genaktivierung auslösten. Die Einführung einer para-ständigen Hydroxylgruppe am C(5)-Phenyl der Verbindung 127 hatte den Verlust der agonistischen Wirkung zur Folge. Für die am stärksten wirksame Verbindung 123 konnte gezeigt werden, dass sich die genaktivierende Wirkung mit dem Verlust einer der drei Hydroxylgruppen erniedrigt. Dabei wurde der stärkste Wirkungsverlust bei einer Dehydroxylierung am C(4)-Aryl, der schwächste am C(2)-Aryl beobachtet. Für das 1,2-Bis(4-hydroxyphenyl)-4H-indeno[2,3-d]imidazol (144) konnte eine antagonistische Wirkung nachgewiesen werden. Die Genaktivierung bei einer 17 -beta-Estradiol-Konzentration von 10-9 M wurde durch Substanzkonzentrationen von 10-7 \- 10-5 M um rund 50% reduziert. Für alle untersuchten 1H-Imidazole wurde eine minimale Bindungsaffinität zum Estrogenrezeptor beim Test mit Kalbsuterus-Cytosol als Estrogenrezeptorquelle gemessen. Alle 1H-Imidazole wurden einem Cytotoxizitätstest an MCF-7 Zellen unterworfen. Dabei zeigten 4-(2-Chlor-4-hydroxyphenyl)-1,2-bis(4-hydroxyphenyl)-1H-imidazol (117) und 2,4-Bis(2-chlor-4-hydroxyphenyl)-1-(4-hydroxyphenyl)-1H-imidazol (118) in Konzentrationen von 5 &microM; und 10 &microM; ab einer Inkubationszeit von drei Tagen einen antiproliferativen Effekt. Beide Verbindungen hemmten das Wachstum der hormonunabhängigen Mammakarzinomzelllinie MDA-MB 231 und der Leukämiezelllinien LAMA84 und SUP-B15. In den Kulturen von MDA-MB 231, LAMA84 und SUP-B15 Zellen, die mit diesen Verbindungen behandelt worden waren, wurde nach 5-tägiger Inkubation ssDNA nachgewiesen. Ein Apoptose-Test mit Annexin/Propidiumiodid im FACS nach 3-stündiger Inkubation verlief negativ. Für beide Verbindungen wurde die inhibitorische Wirkung an der Cyclooxygenase-1 und -2 bestimmt. In Konzentrationen von 1 &microM;, 10 &microM; und 200 &microM; wurde eine Hemmung der Aktivität der Cyclooxygenase-1 von 0%, 76% bzw. 99% festgestellt. An der Cyclooxygenase-2 wurde eine Hemmung der Enzymaktivität von 6%, 51% und 96% für die Verbindung 118 und von 8%, 31% und 84% für die Verbindung 117 in den gleichen Konzentrationen bestimmt. Das unchlorierte Derivat 115 zeigte dagegen in 10 &microM; nur eine Hemmung von 19% an der Cyclooxygenase-1 und von 17% an der Cyclooxygenase-2.Ortho-halogenated derivatives (15b, 15c, 156, 157) of meso-4,5-bis(4-hydroxyphenyl)-2-imidazoline (15a) are well known ligands for the estrogen receptor. One intention of this thesis was to investigate the influence of C(2)-substituents on the ER-activation. Therefore the gene activation were determined on the mammary carcinoma MCF-7 2a cells of meso-2,4,5-triaryl-2-imidazolines 47a \- 47d. The 2-imidazolines 47a \- 47d showed neither estrogenic or anti-estrogenic nor cytotoxic activity against ER-positiv MCF-7 cells. An other aim of this thesis was the study of the influence of alkyl and aryl substituents in 1,2-bis(4-hydroxyphenyl)-1H- imidazole (155) at C(4) and C(5) on the gene activation, according to recent publications which described triaryl hetero-aromatics as potent ligands for the estrogen receptor with partly a high selectivity for one subtype. Effects of para-hydroxylation and ortho-halogenation in the aryls were identified. The strongest gene activation was observed for the 1,2,4-tris(4-hydroxyphenyl)-5 -methyl-1H-imidazole (119), the 5-ethyl-1,2,4-tris(4-hydroxyphenyl)-1H- imidazole (123) and the 1,2,4-tris(4-hydroxyphenyl)-5-phenyl-1H-imidazole (127). The estrogenic activity increased from the phenyl over the methyl up to the ethyl substituent at C(4). The activity at the estrogen receptor was decreased by compounds bearing chlorine substituents in the aromatic rings at C(2) and C(4). It could be demonstrated, that the C(5)-phenol derivative of imidazole 127 had no agonistic activity. Contrary to this observation the partial de-hydroxylation of 123 reduced the agonistic potency. The analogous imidazole with a phenyl ring at N(1) was less active then the compound with a phenyl substituent at C(2). No activity was achieved by de-hydroxylation at C(4)-aryl. In this SAR study 1,2-bis(4-hydroxyphenyl)-4H- indeno[2,3-d]imidazole (144) was identified as an anti-estrogenic imidazole derivative. It reduced the gene activation of 17-beta-estradiol in 10-9 M of about 50% in concentrations from 10-7 to 10-5 M. For all 1H-imidazoles a minimal receptor binding affinity was observed by using calf uterus-cytosol as estrogen receptor source. The cytotoxic activities of imidazoles were evaluated on ER-positiv MCF-7 cells. Only the treatment with the 4-(2-chloro-4-hydroxyphenyl)-1,2-bis(4-hydroxyphenyl)-1H-imidazole (117) or the 2,4-bis(2-chloro-4-hydroxyphenyl)-1-(4-hydroxyphenyl)-1H-imidazole (118) in concentrations of 5 &microM; and 10 &microM; resulted in growth inhibition after three days of application. Compounds 117 and 118 inhibited the growth of ER-negative MDA-MB 231 and leukemia LAMA84 und SUP-B15 cells as well. Increase of single-stranded DNA in the cells as an indicator of apoptosis was detected after an incubation time of 5 days. As expected no apoptotic cells were shown after three hours treatment in an assay with annexine/propidiumiodide. Both 117 and 118 shown inhibitory activity on cyclooxygenase-1 und -2 enzymes. In concentrations of 1 &microM;, 10 &microM; and 200 &microM; a Cox-1-inhibition of 0%, 76% and 99% was determined, respectively. Cyclooxygenase-2 was inhibited of about 6%, 51% and 96% by imidazole 118 and of 8%, 31% and 84% by 117 in the same concentrations. The non-chlorinated analog 115 mediated at 10 &microM; only a minimal inhibition of cyclooxygenase-1 (19%) and cyclooxygenase-2 (17%). A distinct structure-activity relationship was made evident by this experiments. This thesis describes facile and practical syntheses of a series of imidazoles. A number of targeted compounds shown estrogenic and anti-estrogenic activity. Chlorinated non-estrogenic imidazoles of this series were also potent growth inhibitors of various cancer cell lines. In addition these imidazoles inhibited both isoforms of cycloxygenase

Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination

The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer’s disease (AD). In particular, small neurotoxic Aβ oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized D-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ oligomers. One of the hereby identified D-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD

Structure−Activity Relationship Study To Understand the Estrogen Receptor-Dependent Gene Activation of Aryl- and Alkyl-Substituted 1<i>H</i>-Imidazoles

A series of C5-substituted 1,2,4-triaryl-1H-imidazoles was synthesized. Their gene-activating properties were determined on estrogen receptor alpha positive MCF-7 breast cancer cells, stably transfected with the plasmid EREwtcluc (MCF-7-2a cells). The influence of 4-OH and 2-Cl substituents on the phenyl rings as well as the significance of a methyl, ethyl, or phenyl group at C5 on the estrogen receptor binding and the resulting gene activation in MCF-7-2a cells was studied. The alkyl and aryl groups at C5 of 1,2,4-tris(4-hydroxyphenyl)-1H-imidazole 1 increased the transactivation, while chlorine atoms on the phenyl rings diminished this effect. 5-Ethyl-1,2,4-tris(4-hydroxyphenyl)-1H-imidazole 9 was identified as the most active compound. Its excellent transcriptional activity did not only depend on the C5 ethyl group, but also on the three hydroxyl groups of the phenyl rings. Compounds (11−14) with a reduced number of hydroxyl groups displayed distinctly lower gene activation

Synthesis of optically active 1-(1-phenylethyl)imidazoles eerived from 1-phenylethylamine

The three-component reaction of (R)- or (S)-1-phenylethylamine (6), formaldehyde, and an -(hydroxyimino) ketone 5, i.e., 3-(hydroxyimino)butan-2-one (5a) or 2-(hydroxyimino)-1,2-diphenylethanone (5b), yields the corresponding enantiomerically pure 1-(1-phenylethyl)-1H-imidazole 3-oxide 7 in high yield (Schemes 2 and 3). The reactions are carried out either in MeOH or in AcOH. Smooth transformations of the N-oxides into optically active 1-(1-phenylethyl)-1H-imidazoles 10 and 2,3-dihydro-1-(1-phenylethyl)-1H-imidazole-2-thiones 11 are achieved by treatment of 7 with Raney-Ni and 2,2,4,4-tetramethyl-3-thioxocyclobutanone (12), respectively (Scheme 4)

Antitumor-Active Cobalt−Alkyne Complexes Derived from Acetylsalicylic Acid: Studies on the Mode of Drug Action

Cobalt−alkyne complexes are drugs with remarkable cytotoxicity. From the complexes tested up to now we selected the aspirin derivative [2-acetoxy-(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS) as the lead compound. To get more insight into the mode of action, we systematically modified the alkyne ligand and determined the cytotoxic properties of the resulting cobalt complexes. Further investigations were performed on the drug lipophilicity, the cellular uptake into MCF-7 and MDA-MB 231 breast cancer cells, the DNA-binding efficacy, and the nuclear drug content. The ability to inhibit glutathione reductase and cyclooxygenase (COX) enzymes, the binding to the estrogen receptor, and the induction of apoptotic processes were examined for selected compounds. Interestingly, the most antitumor active compounds were potent COX inhibitors (COX-1 and COX-2). The presented results indicate that cobalt−alkyne complexes of the Co-ASS type, represent a new class of organometallic cytostatics with a mode of drug action in which COX inhibition probably plays a major role

Synthesis and Pharmacological Evaluation of 1<i>H</i>-Imidazoles as Ligands for the Estrogen Receptor and Cytotoxic Inhibitors of the Cyclooxygenase

The 1H-imidazoles 7a−e were synthesized and tested for biological activity in vitro. The results pointed to a clear structure−activity relationship. The introduction of an ethyl chain at C5 of the 1,2,4-tris(4-hydroxyphenyl)-1H-imidazole 7a caused hormonal activity in estrogen receptor positive MCF-7-2a cells. An o-chlorine substituent in the phenolic rings at C2 and C4 as realized in 7b and 7c increased the antiproliferative effects against human breast cancer cell lines MCF-7 and MDA-MB 231. Additionally, both compounds showed strong inhibitory effects on cyclooxygenase enzymes. Therefore, a mode of action including the interference in the arachidonic acid cascade might be possible

Mixing A beta(1-40) and A beta(1-42) peptides generates unique amyloid fibrils

Recent structural studies show distinct morphologies for the fibrils of Aβ(1-42) and Aβ(1-40), which are believed not to co-fibrillize. We describe here a novel, structurally-uniform 1 : 1 mixed fibrillar species, which differs from both pure fibrils. It forms preferentially even when Aβ(1-42) : Aβ(1-40) peptides are mixed in a non-stoichiometric ratio.status: publishe