Die Deregulation des Transkriptionsfaktors Myc ist ein charakteristisches Merkmal für eine Vielzahl von humanen Tumoren. Durch die transkriptionelle Aktivierung von Genen, die im Zusammenhang mit Metabolismus, Translation und Proliferation stehen, wird dadurch das Tumorwachstum begünstigt. Myc bildet zudem mit dem Zinkfinger-Protein Miz1 einen Komplex, der hemmend auf die Transkription von Zielgenen wirkt. Bisher sind nur wenige Myc/Miz1-reprimierte Zielgene bekannt. In der vorliegenden Arbeit konnten genomweit die DNA-Bindestellen von Myc und Miz1 durch Chromatin-Immunpräzipitationen gefolgt von Hochdurchsatzsequenzierung in einer Zervixkarzinomzelllinie bestimmt werden. Es konnte gezeigt werden, dass Myc an Promotoren aller drei RNA-Polymerasen sowie in enhancer-Regionen bindet, während Miz1 Kernpromotoren von RNA-Polymerase II- und III-transkribierten Genen besetzt. reChIP-Experimente zeigten, dass Myc und Miz1 als Komplex an Promotoren von Zielgenen binden. Zudem wurde ein Miz1-DNA-Bindemotiv identifiziert und der transaktivierende Einfluss von Miz1 auf Gene mit diesem Motiv nachgewiesen. Das überwiegende Vorhandensein von Myc/Max-Komplexen führt zu einer Transaktivierung von E-Box-haltigen Promotoren. Andererseits erfolgt die transkriptionelle Repression von Myc/Miz1-Zielgenen an Promotoren, an denen der Myc/Miz1-Komplex vorherrscht. In aktuellen Publikationen konnte gezeigt werden, dass nach mitogener Stimulation von Lymphozyten es zu einer Erhöhung der Myc-Expression kommt, wodurch Myc als ein genereller Transkriptionsaktivator fungiert, der alle Gene gleichermaßen induziert. Trotz hoher Myc-Mengen in Tumorzellen konnte die generelle Myc-vermittelte Transaktivierung nicht nachgewiesen werden. Zusätzlich zur Myc-abhängigen Transaktivierung von E-Box-haltigen Genen, z. B. beteiligt an Translation und RNA-Prozessierung, und der Miz1-vermittelten transkriptionellen Aktivierung von Genen mit Miz1-Motiv (z. B. involviert in Autophagie), konnte entgegen dem Modell der generellen Genamplifikation durch Myc eine Myc/Miz1-abhängige Repression von Zielgenen belegt werden. Die neu gewonnenen Erkenntnisse des Bindeverhaltens des Myc/Miz1-Komplexes und der daraus resultierenden transkriptionellen Regulation von Myc/Miz1-Zielgenen ermöglichen ein besseres Verständnis der Myc-Funktion in Tumorzellen und könnte zur Verbesserung von Tumortherapien führen. / Deregulation of the transcription factor Myc is a characteristic feature of a variety of human tumors. The Myc-dependent transcriptional activation of genes involved in metabolism, translation and proliferation therefor promotes tumor growth. Additionally, Myc forms a complex with the zinc finger protein Miz1, which represses transcription of target genes. So far, only a limited number of Myc/Miz1-repressed genes is known. Within the present thesis DNA binding sites of Myc and Miz1 were mapped genome-wide using chromatin immunoprecipitations followed by high-throughput sequencing in a cervical cancer cell line. It could be shown that Myc binds to promoters of all three RNA polymerases as well as to enhancer regions, whereas Miz1 binding sites could be found only in core promoters of RNA polymerase II and III transcribed genes. reChIP experiments illustrated binding of Myc and Miz1 as a complex on DNA. Additionally, a DNA binding motif of Miz1 was identified and furthermore it was possible to verify the transctivating influence of Miz1 on genes carrying that motif in the promoter. On E-box containing promoters the predominantly existence of Myc/Max complexes resulted in transactivation of the respective genes. Otherwise, transcriptional repression of Myc/Miz1 target genes occured at promoters where the Myc/Miz1 complex dominates. Recent publications have illustrated that after mitogenic stimulation of primary lymphocytes, Myc expression is enhanced, whereby Myc serves as a general transcriptional activator that induces the expression of virtually all genes. Although Myc levels are high in tumor cells that general mechanism of Myc-mediated transactivation could not be verified. Additionally to the Myc-dependent transactivation of E-box-containing genes, e. g. involved in translation and RNA processing, and Miz1-mediated transcriptional activation of genes containing a Miz1 binding motif (e. g. autophagy-related genes), and in opposition to the general amplifier model a Myc/Miz1-dependent repression of target genes could be proven. The obtained evidences concerning DNA binding properties of the Myc/Miz1 complex as well as the resulting transcriptional regulation of Myc/Miz1 target genes facilitates a better understanding of Myc function in tumor cells and could leed to better anti-tumor therapies.

Preclinical methods for the identification and characterization of molecules for development into new cancer drugs were investigated. Based on repurposing, i.e. the exploration of currently prescribed drugs for new indications, and as a result of a new high throughput screening (HTS) approach, the benzimidazoles benomyl and carbendazim, the alkaloid cryptolepine and the acridine acriflavine were found interesting to characterize using these methods. In mice the benzimidazoles inhibited 3H-thymidine incorporation in tissues with high cell renewal, with benomyl being more active than carbendazim.  They were rapidly absorbed with highest amounts seen in the liver, kidneys and gastro-intestinal lumen as evidenced from distribution of 14C-labeled drugs. In human tumour cell lines, the benzimidazoles showed a similar activity pattern but benomyl was more potent. This was true also in tumour cells from patients but carbendazim was relatively more active against solid tumours. Analyses of drug activity cross-resistance patterns and of drug activity – gene expression correlations in a cell line panel suggested multiple mechanisms of action for the benzimidazoles. Cryptolepine was widely distributed to tissues in vivo in the mice. It was more potent than the benzimidazoles in tumour cells, with highest activity in haematological malignancies but some patient samples of breast, colon and non small-cell lung cancer were sensitive. Cross-resistance analysis indicated cryptolepine to be a topoisomerase II inhibitor whereas drug activity – gene expression correlations suggested additional mechanisms of action. HTS on 2 000 molecules in colon cancer cell lines and normal cells identified acriflavine as a hit molecule, subsequently shown to have unprecedented activity against colorectal cancer tumour cells in patient tumour samples. Connectivity map analysis, based on drug induced gene expression perturbation patterns in a tumour cell line, indicated acriflavine to be a topoisomerase inhibitor, subsequently confirmed in a plasmid relaxation assay. In conclusion, repurposing of drugs and HTS using stringent activity criteria followed by preclinical characterization might contribute to more efficient development of new cancer drugs.

The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

The recently-emerged field of chemical genomics is centered on the use of small molecules to perturb biological systems as a means of investigating their function. In order to employ this approach for the study of plant-pathogen interactions, I established an assay in which Arabidopsis thaliana seedlings are grown in liquid media in 96-well plates. Inoculation of these seedlings with a virulent strain of the bacterial phytopathogen Pseudomonas syringae resulted in macroscopic bleaching of the cotyledons of these seedlings. This symptom was used as the basis for high-throughput chemical genomic screens aimed at identifying small molecules that protect Arabidopsis seedlings from infection. One of the first chemicals identified through this screen was the sulfanilamide compound sulfamethoxazole (Smex). This compound was later shown to also reduce the susceptibility of both Arabidopsis and wheat to infection by the fungal pathogen Fusarium graminearum, suggesting a broad spectrum of activity. More detailed investigations of Smex indicated that the protective activity of this compound did not derive from antimicrobial effects, and that this activity was not executed through common defence-related signalling pathways. The folate biosynthetic pathway enzyme dihydropteroate synthase is a known target of sulfanilamides, and it does appear to contribute to Smex-induced disease resistance, albeit in a folate-independent manner. In order to identify downstream mediators of Smex activity, I initiated two forward genetic screens intended to recover mutants with altered sensitivity to Smex in a seedling growth assay. Interestingly, while these screens yielded mutants with striking Smex sensitivity phenotypes, disease resistance phenotypes were not altered. Gene expression profiling of Arabidopsis tissues treated with Smex prior to bacterial inoculation suggested that this compound generally affects lipid signalling. Altogether, it is evident that Smex elicits a complex set of responses in Arabidopsis with apparently non-overlapping phenotypic outputs.

Biocatalysts offer advantages over their chemical counterparts in terms of their high enantioselectivity and the opportunity to develop more environmentally friendly processes. However, the widespread adoption of biocatalytic processes is hampered by the long development times for enzymes with novel and sufficient activity and adequate stability under operating conditions. Protein engineering, while extremely useful for modifying the properties of protein catalysts in select cases, still cannot be performed rapidly enough for many applications. In order for biocatalysts to become a competitive alternative to chemical catalysts, new tools to make the tailoring of biocatalysts by protein engineering methods speedier and more efficient are necessary. The aim of this work was to develop methods to aid in the faster production of novel biocatalysts. Protein engineering involves two steps: the generation of diversity and the screening or selection of variants with the desired properties. Both of these must be targeted to create a faster protein engineering process. In the case of the former, this work sought to clone and overexpress some template enzymes which would create smaller, more manageable libraries of mutants with a higher likelihood of function by the manipulation of a few focused amino acid residues. For the latter, this work developed and validated a Monte-Carlo simulation model of pooling to increase screening throughput and created a set of vectors to aid in high-throughput screening by eliminating unwanted mutants from the assay procedure entirely.

Thesis (M.S.)--Ball State University, 2009. / Title from PDF t.p. (viewed on Nov. 30, 2009). Includes bibliographical references (p. 74-80).

Ricin and Shiga toxin type 1 are potent cytotoxins known as ribosome inhibition proteins, abbreviated RIPs. Proteins of this family shut down protein synthesis by removing a critical adenine in the conserved stem-loop structure of 28S rRNA. Due to its exquisite cytotoxicity, the plant toxin ricin has been used as a biological warfare agent. Although great achievement has been made on ricin research, including catalytic mechanism and structure analysis, there is still no specific treatment available for ricin exposure. In addition, ricin A chain inhibitors may also be useful against the homologous bacterial proteins shiga toxins, which are responsible for dysentery, and diseases related to food poisoning, including hemolytic uremic syndrome. Previous study on RTA inhibitor search has provided a number of substrate analog inhibitors, all of which, however, are weaker inhibitors. Therefore, the goal of this work is to improve the binding affinity of known inhibitors and to discovery new scaffolds for inhibitor discovery and development. In this work, multiple approaches were employed for this purpose, including optimizing known inhibitors and searching new inhibitors by Virtual Drug Screening (VDS) and High Throughput Screening (HTS). A number of new RTA inhibitors were discovered by these strategies, which provide a variety of pharmacophores for RTA inhibitor design, and also added a new line of evidence for VDS as an advanced technology for drug discovery and development. / text

Asymmetric synthesis has become an important tool to prepare enantiomerically pure compounds because it avoids the wasteful discarding of the undesired enantiomer. Combinatorial libraries allow for much faster screening for new and better asymmetric catalysts/auxiliaries, but they generate a large number of samples whose enantiomeric excess (ee) cannot be determined rapidly. This bottleneck currently limits the applicability of such approaches. We propose here the use of faster optical techniques for the determination of ee using common instrumentation, such as UV-vis spectrophotometers, and circular dichroism (CD) spectrophotometers. Our methods are easily transitioned to the microwell format commonly used in parallel/combinatorial chemistry endeavors, just by using common microplate readers: this allows for an even more rapid analysis of samples and a seamless integration in a high-throughput workflow. We have shown that enantioselective indicator displacement assays can be developed to determine ee in a high-throughput fashion utilizing either a UV-vis spectrophotometer or a 96-well plate reader. Two chiral receptors and a commercial pH indicator were used to enantioselectively discriminate α-amino acids by monitoring the degree of indicator displacement. The two receptors were able to enantioselectively discriminate 13 of the 17 analyzed α-amino acids and accurately determine ee values of independent test samples with the use of ee calibration curves. Moreover, a sample of valine was synthesized through an asymmetric reaction, whose ee was then determined with our assay and compared to chiral HPLC and 1H NMR chiral shift reagent analysis, with excellent correlation. An artificial neural network was also successfully employed in the analyses, as an alternative to ee calibration curves. Both techniques consistently produced results accurate enough for preliminary determination of ee in a rapid manner, allowing for high throughput screening (HTS) of asymmetric reactions. The use of circular dichroism spectroscopy with chiral BINAP was also explored to enantioselectively discriminate α-chiral ketones. The ketones were derivatized with pyridyl hydrazines to produce hydrazones, which were then bound to enantiomerically pure [Cu(I)(BINAP)]+, forming diastereomeric complexes with differential steric interactions leading to different degrees of twist in the BINAP moiety and characteristic signatures in the CD spectrum, as a function of sample ee. / text

EZH2 is a SET domain-containing methyltransferase and the catalytic component of the multimeric Polycomb- group (PcG) protein complex, PRC2. When in complex with other PRC2 members (EED, SUZ12, AEBP2, and RBBP4), EZH2 catalyzes methylation of H3K27, a histone modification associated with transcriptional repression and developmental regulation. As several PRC2 components are upregulated or mutated in a variety of human cancers, efforts to discover small-molecule modulators of PRC2 and understand its regulation may yield therapeutic insights. Identification of small-molecule probes with distinct chemotypes, MOAs, and selectivity profiles are not only of great value, but necessary in establishing comprehensive probe sets capable of illuminating the various roles of EZH2 in oncogenesis. Here we describe efforts to identify and characterize small-molecule modulators of PRC2 and further understand its regulation. Chapter II outlines the expression and purification of 5-component PRC2 (EZH2-EED-SUZ12-AEBP2-RBBP4) and the establishment of biochemical and cellular HTS assays. These assays were used to screen a diverse set of small molecules (>120,000), identifying biochemical PRC2 inhibitors and activators (described in Chapter III). One biochemical PRC2 inhibitor, BRD1835, appeared to inhibit PRC2 activity through a novel artifactual mechanism involving interaction with peptide substrate, leading to apparent peptide-competitive behavior and putative cellular activity (described in Chapter IV). The characterization of novel biochemical PRC2 activators, BRD3934 and BRD8284, is discussed in Chapter V. Chapter VI describes the use of an HCS assay to identify known bioactive compounds that alter intracellular levels of H3K27me3 through modulating H3K27me3-connected regulatory nodes or by targeting PRC2 directly. These efforts led to the discovery that an antifungal agent, miconazole, is capable of activating PRC2 activity in vitro, while a mucolytic agent, bromhexine, selectively ablates cellular H3K27me3 levels through targeting an activity distinct from PRC2. Finally, Chapter VII discusses novel PRC2-connected crosstalk mechanisms identified through screening libraries of uniquely modified histone peptides for their ability to bind or support methylation by PRC2. These studies enhance our understanding of PRC2 regulation by revealing the effects of H3R26 and H3K23me1 modifications on enzymatic activity, implicating their respective methyltransferases in PRC2 regulation.

Plant hormones act in a complex network where their pathways regulate and interact to control different mechanisms, such as development and stress responses. This crosstalk between hormones can be exploited by pathogens to suppress plant defense responses and thereby increase pathogen growth. Pseudomonas syringae pathogenicity is reliant on a Type III secretion system (TTSS) that acts as a specialized injection apparatus to deliver virulence proteins, known as type III effectors (TTEs), into the plant cell cytosol. In my work, I have screened hormone inducible promoter::GUS transgenic Arabidopsis thaliana lines against a P. syringae TTE library in order to identify TTEs involved in the perturbation of hormone signaling in planta. Through this screen I identified two P. syringae TTEs, HopAK1 and HopAL1, both belonging to the same bacterial strain P. syringae pv. maculicola ES4326. I found that HopAK1 can sensitize A. thaliana plants to auxin. On the other hand, HopAL1 activates auxin signaling. Monitoring of auxin signaling was done using transgenic DR5::GUS plants. Both TTEs render the plant susceptible to bacterial infection, highlighting a potential relationship between increased auxin signaling and virulence.