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

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)

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

Aggregation state of monomeric Aβ(1–42) after 1 h incubation.

<p>For optimization of D3 with peptide microarrays, the peptide microarrays were incubated with 5 μM initially monomeric Aβ(1–42) for 1 h at room temperature. The aggregation state of this Aβ preparation was analyzed by density gradient centrifugation followed by 16% Tricine-SDS-PAGE. FITC-Aβ(1–42) was detected via FITC fluorescence and was only detectable in the first four lanes, which represent mainly monomeric and oligomeric FITC-Aβ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153035#pone.0153035.ref014" target="_blank">14</a>].</p

TEM of Aβ-DB3 and -DB3DB3 co-complexes.

<p>10 μM initial monomeric Aβ(1–42) without (A) and with 10 μM DB3 (B) or 5 μM DB3DB3 (C) were coincubated for 24 h. Subsequently, the samples were absorbed onto formval/carbon coated copper grids and negative stained with 1% uranyl acetate. The images were obtained using a transmission electron microscope (TEM). Scale bar: 0.25 μm.</p

Amino acid sequences of D3 and DB1 to DB5.

<p>Amino acid sequences of D3 and DB1 to DB5.</p

K_D determination of DB3 and DB3DB3 to monomeric Aβ using biolayer interferometry (BLI).

<p>N-terminally biotinylated Aβ(1–42) monomers were immobilized on streptavidin biosensors and the binding of DB3 and DB3DB3 was detected. Representative double referenced sensorgrams of a dilution series of DB3 (A) and DB3DB3 (B) are shown, including the equilibrium dissociation constants (K_D) as means ± SD of data recorded in triplicate. For steady state analysis Langmuir´s 1:1 binding model was applied. Representative fits of DB3 (C) and DB3DB3 (D) are depicted with the corresponding corrected <i>R</i>².</p

Inhibition of Aβ fibril formation and Aβ aggregation disassemble by DB3 and DB3DB3.

<p>A) Monomeric Aβ(1–42) (400 nM) was mixed with different concentrations of DB3 (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 μM) and the aggregation state of Aβ was analyzed using an Aβ aggregate specific ELISA. For DB3DB3 half of the molar concentrations compared to DB3 were used. Aβ without DB3 and DB3DB3 addition was taken as control. For DB3 an EC₅₀ of 6 μM was calculated using a logistic fit model. DB3DB3 inhibited the formation of Aβ fibrils more efficiently with an EC₅₀ of 7 nM. B) The disassembly properties of DB3 and DB3DB3 were measured using an Aβ aggregation specific ELISA. Monomeric Aβ(1–42) (400 nM) was preincubated in order to form fibrils and mixed with nine different concentrations of DB3 (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 μM). For DB3DB3, the molar concentrations were half those used for DB3. For DB3 an EC₅₀ of 2.5 μM was determined. DB3DB3 disassembled Aβ aggregates at the lowest concentration (10 nM). Thus, the EC₅₀ could not be determined, but is < 10 nM. All data were determined in triplicate. The Mann-Whitney-U-test was performed for statistical analysis. * <i>p</i>< 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001</p