RESUMEN
Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Approximately 10% of AS individuals harbor UBE3A genes with point mutations, frequently resulting in the expression of full-length E6AP variants with defective E3 activity. Since E6AP exists in two states, an inactive and an active one, we hypothesized that distinct small molecules can stabilize the active state and that such molecules may rescue the E3 activity of AS-derived E6AP variants. Therefore, we established an assay that allows identifying modulators of E6AP in a high-throughput format. We identified several compounds that not only stimulate wild-type E6AP but also rescue the E3 activity of certain E6AP variants. Moreover, by chemical cross-linking coupled to mass spectrometry we provide evidence that the compounds stabilize an active conformation of E6AP. Thus, these compounds represent potential lead structures for the design of drugs for AS treatment.
Asunto(s)
Síndrome de Angelman/genética , Mutación Puntual , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Conformación Proteica , Ubiquitina-Proteína Ligasas/químicaRESUMEN
In order to investigate the possibilities of Fischer glycosylation towards the synthesis of bromoalkylglycosides we performed a variety of different reactions resulting in a small library of 16 different glycosides. Using standardized reaction conditions we could gain a broad range of results from small to higher yields. Finally we randomly selected three reactions and performed them with higher amounts of bromoalcohol resulting in significantly better yields, showing the optimization potential of these basic research work.
Asunto(s)
Bromo/química , Glicósidos/química , Glicósidos/síntesis química , Técnicas de Química Sintética , GlicosilaciónRESUMEN
Diadenosine polyphosphates (ApnAs) such as diadenosine tri- and tetraphosphates are formed in prokaryotic as well as eukaryotic cells. Since upon stress intracellular ApnA concentrations increase, it was postulated that ApnAs are alarmones triggering stress-adaptive processes. The major synthesis pathway of ApnAs is assumed to be a side reaction of amino acid activation. How this process is linked to stress adaptation remains enigmatic. The first step of one of the most prominent eukaryotic post-translational modification systems-the conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubl) to target proteins-involves the formation of an adenylate as intermediate. Like ApnA formation, Ub and Ubl conjugation is significantly enhanced during stress conditions. Here, we demonstrate that diadenosine tri- and tetraphosphates are indeed synthesized during activation of Ub and Ubls. This links one of the most prevalent eukaryotic protein-modification systems to ApnA formation for the first time.
Asunto(s)
Fosfatos de Dinucleósidos/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Biocatálisis , Cromatografía Líquida de Alta Presión , Fosfatos de Dinucleósidos/análisis , Humanos , Espectrometría de Masas , Mutagénesis , Proteínas Recombinantes/biosíntesis , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/genéticaRESUMEN
Poly(ADP-ribose) polymerase-1 (PARP-1) is an important target in cancer therapy. We present the synthesis of novel disaccharide nucleoside analogues that resemble the central motif of poly(ADP-ribose) and test their inhibitory effects on human PARP-1. Some compounds show inhibition of enzymatic activity in vitro and thus might be interesting for further investigations.
Asunto(s)
Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli Adenosina Difosfato Ribosa/análogos & derivados , Poli Adenosina Difosfato Ribosa/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Técnicas de Química Sintética , Disacáridos/síntesis química , Disacáridos/química , Disacáridos/farmacología , Descubrimiento de Drogas , Humanos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/síntesis química , Poli Adenosina Difosfato Ribosa/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/síntesis químicaRESUMEN
The intracellular concentration of diadenosine tetraphospate (Ap4A) increases upon exposure to stress conditions. Despite being discovered over 50 years ago, the cellular functions of Ap4A are still enigmatic. If and how the varied Ap4A is a signal and involved in the signaling pathways leading to an appropriate cellular response remain to be discovered. Because the turnover of Ap4A by Ap4A cleaving enzymes is rapid, small molecule inhibitors for these enzymes would provide tools for the more detailed study of the role of Ap4A. Here, we describe the development of a high-throughput screening assay based on a fluorogenic Ap4A substrate for the identification and optimization of small molecule inhibitors for Ap4A cleaving enzymes. As proof-of-concept we screened a library of over 42â¯000 compounds toward their inhibitory activity against the Ap4A phosphorylase (Rv2613c) of Mycobacterium tuberculosis (Mtb). A sulfanylacrylonitril derivative with an IC50 of 260 ± 50 nM in vitro was identified. Multiple derivatives were synthesized to further optimize their properties with respect to their in vitro IC50 values and their cytotoxicity against human cells (HeLa). In addition, we selected two hits to study their antimycobacterial activity against virulent Mtb to show that they might be candidates for further development of antimycobacterial agents against multidrug-resistant Mtb.
Asunto(s)
Fosfatos de Dinucleósidos/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Nucleotidiltransferasas/metabolismo , Supervivencia Celular , Fosfatos de Dinucleósidos/química , Inhibidores Enzimáticos/química , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Nucleotidiltransferasas/genética , Unión ProteicaRESUMEN
Three different building blocks have been synthesised and used for the synthesis of linear triazole linked pseudo oligosaccharides with copper(I)-catalysed cycloaddition (CuAAC). Ethynylferrocene has been used as analytical probe to improve the UV/Vis properties and HPLC methods have been used and optimised for the analysis of the pseudo oligosaccharides. The smallest ones have been isolated and characterised by analytical HPLC, NMR, ESI-MS and elemental analysis.
Asunto(s)
Compuestos Ferrosos/química , Sondas Moleculares/química , Oligosacáridos/análisis , Oligosacáridos/síntesis química , Triazoles/química , Conformación Molecular , Oligosacáridos/químicaRESUMEN
A series of novel sulfonamide substituted heteroleptic salan titanium(IV)-bis-chelates complexed to 2,6-pyridinedicarboxylic acid were synthesized, structurally characterized and evaluated for their anticancer activity against two human carcinoma cell lines. All cytotoxic complexes showed complete inhibition of cell growth at active concentration, two complexes based on pyrrolidine and azepane substituted sulfonamides displayed IC50 values below 1.7 µM and are more cytotoxic than cisplatin in both tested cell lines. The azepane substituted complex [L3Ti(dipic)] exhibited excellent activity with an IC50 value of 0.5 ± 0.1 µM in Hela S3 and 1.0 ± 0.1 µM in Hep G2.