The non-receptor tyrosine kinase activity of fusion gene BCR-ABL derived oncoproteins is the key factor responsible for development and progress of Philadelphia positive (Ph+) chronic myeloid leukemia (CML). In the search for a superior and novel peptide-based inhibitor of Bcr-Abl, here I investigated a naturally occurring molecule, called apoptin. Apoptin is a 13.6 kDa protein derived from chicken anemia virus (CAV) and known to induce apoptosis in a wide range of transformed but not in primary cells. Apoptin is a protein without any reported structural and/or functional homolog and is an interesting candidate to initiate protein-protein interactions and subsequent downstream effects. Initially by an array-based analysis I found that apoptin interacts with the SH3 domain of Abl. By high stringency pull-down and co-immunoprecipitation assays the apoptin and Bcr-Abl interaction was further confirmed. Subsequently, a set of apoptin and Bcr-Abl deletion mutants were used to map this interaction precisely that mainly occurred between a proline rich domain of apoptin and the SH3 domain of Bcr-Abl. I further investigated the role of apoptin on Bcr-Abl. Apoptin was able to modify the phosphorylation of a series of targets (e.g. CrkL, STAT5, c-Myc) downstream of Bcr-Abl kinase. In addition, I used computational algorhythms for protein modeling to study the 3D structure of apoptin and it’s docking with Bcr-Abl at the molecular level. In controlled studies using the 2-pheny-laminopyrimidine derived specific tyrosine kinase inhibitor Imatinib® I found that apoptin has comparable effects on CML cells, suggesting that the interacting segment of the apoptin molecule acts as an adaptor and negatively regulates the Bcr-Abl kinase by deactivating many cell proliferation and anti-apoptotic pathways in CML cells. Briefly, this work provides important insights towards the development of peptide based tyrosine kinase inhibitors as new anti-cancer agents.

The non-receptor tyrosine kinase activity of fusion gene BCR-ABL derived oncoproteins is the key factor responsible for development and progress of Philadelphia positive (Ph+) chronic myeloid leukemia (CML). In the search for a superior and novel peptide-based inhibitor of Bcr-Abl, here I investigated a naturally occurring molecule, called apoptin. Apoptin is a 13.6 kDa protein derived from chicken anemia virus (CAV) and known to induce apoptosis in a wide range of transformed but not in primary cells. Apoptin is a protein without any reported structural and/or functional homolog and is an interesting candidate to initiate protein-protein interactions and subsequent downstream effects. Initially by an array-based analysis I found that apoptin interacts with the SH3 domain of Abl. By high stringency pull-down and co-immunoprecipitation assays the apoptin and Bcr-Abl interaction was further confirmed. Subsequently, a set of apoptin and Bcr-Abl deletion mutants were used to map this interaction precisely that mainly occurred between a proline rich domain of apoptin and the SH3 domain of Bcr-Abl. I further investigated the role of apoptin on Bcr-Abl. Apoptin was able to modify the phosphorylation of a series of targets (e.g. CrkL, STAT5, c-Myc) downstream of Bcr-Abl kinase. In addition, I used computational algorhythms for protein modeling to study the 3D structure of apoptin and it’s docking with Bcr-Abl at the molecular level. In controlled studies using the 2-pheny-laminopyrimidine derived specific tyrosine kinase inhibitor Imatinib® I found that apoptin has comparable effects on CML cells, suggesting that the interacting segment of the apoptin molecule acts as an adaptor and negatively regulates the Bcr-Abl kinase by deactivating many cell proliferation and anti-apoptotic pathways in CML cells. Briefly, this work provides important insights towards the development of peptide based tyrosine kinase inhibitors as new anti-cancer agents.

Yes / Vandetanib is an orally available tyrosine kinase inhibitor used in the treatment of cancer. The current synthesis proceeds via an unstable 4-chloroquinazoline, using harsh reagents, in addition to requiring sequential protection and deprotection steps. In the present work, use of the Dimroth rearrangement in the key quinazoline forming step enabled the synthesis of Vandetanib in nine steps (compared to the previously reported 12–14). / This work was supported by the Cancer Research UK-Cancer Imaging Centre (grant: C1060/ A16464), the Institute of Cancer Research and the University of Hull.

In dieser Arbeit wurden zwei Klassen von analog-sensitiven (AS) Kinaseinhibitoren, welche wichtige pharmakologische Instrumente für die Untersuchung von Signalwegen darstellen, durch den Einbau von lichtempfindlichen Komponenten in ihre Struktur photoschaltbar gemacht. Die Einführung eines Cyclohexa-1,3-diens anstelle des zentralen Benzolrings im Staralog-Grundgerüst ermöglicht eine reversible 6π Elektrozyklisierung analog zu Diarylethenen (DAEs). Nach der Entwicklung eines Synthesewegs führte die iterative Verbesserung des strukturellen Designs schließlich zu einer Reihe von Photoschaltern, die in reinem Wasser bestrahlt werden können. Eine detaillierte Untersuchung des photochemischen Verhaltens ergab effizientes Schalten für beide Bestrahlungsprozesse, was auf hohe Quantenausbeuten und eine ausreichende Bandenseparation zwischen beiden Isomeren zurückzuführen ist. Die vorgestellte Studie demonstriert das Potenzial von DAE-basierten Kinaseinhibitoren und stellt eine vielversprechende Möglichkeit dar, um die momentanen Einschränkungen anderer Photoschalterklassen zu überwinden. Um die katalytische Aktivität von TgCDPK1 zu kontrollieren, wurden auf Grundlage des 5-Aminopyrazol-4-carboxamid Gerüsts photoschaltbare Kinaseinhibitoren entwickelt, die auf einer lichtinduzierten E/Z Isomerisierung eines Arylazopyrazols basieren. Aus einer Bibliothek von synthetisierten Inhibitoren wurden die effektivsten Derivate identifiziert, die ein optimales Verhältnis zwischen guten photochemischen Eigenschaften und Affinität gegenüber der Kinase aufweisen. Tatsächlich konnte so eine reversible Kontrolle der katalytischen Aktivität von TgCDPK1 durch die Anwendung von Licht verschiedener Wellenlängen erreicht werden. Es wurde gezeigt, dass die Wirksamkeit dieser Inhibitoren stark mit der Größe des Gatekeeper-Rests korreliert, was die Möglichkeit einer universellen Anwendung dieses Konzepts für die selektive lichtinduzierte Kontrolle von AS Kinasen in der Zukunft eröffnet. / In this work two general classes of analog-sensitive (AS) kinase inhibitors, which constitute pharmacological key elements for studying kinase-mediated signaling pathways, were rendered photoresponsive by implementing two photoswitchable moieties into the scaffold of their corresponding parent inhibitors. Photoswitchability was introduced to the staralog inhibitor scaffold by implementing a cyclohexa-1,3-diene capable of undergoing 6πelectrocyclization analog to diarylethenes (DAEs). A robust synthetic route was established, and iterative improvement of the structural design eventually led to a set of photoswitches which can be readily operated in pure water. In depth investigation of the photochemical behavior revealed high quantum yields for both processes resulting in efficient photoswitching and sufficient band separation between both isomers. This investigation illustrates the potential of DAE-based kinase inhibitors and presents an attractive strategy to address current limitations of other photoswitch classes and currently employed DAE motifs. Furthermore, photoswitchable kinase inhibitors based on the 5-aminopyrazolo-4-carboxamide (PCA) scaffold were used to control the catalytic activity of TgCDPK1 utilizing light-induced structural changes of an arylazopyrazole. Within a library of synthesized small molecule inhibitors, the best candidates exhibiting an optimal balance between good photochemical properties and target affinity were identified. A substantial difference in IC50 values was achieved between both photostationary states and consequently, the catalytic activity of TgCDPK1 was controlled effectively in a reversible fashion by applying light of different wavelengths. The potency of these inhibitors strongly correlates with the size of the gatekeeper residue, highlighting the potential of these photoswitchable inhibitors for the selective light-induced control over AS kinases in the future.

The Pim family of serine/threonine protein kinases (Pim 1, 2, and 3) contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy.

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

Tyrosine kinases regulate fundamental signal pathways in cells including cell proliferation, motility, and differentiation. The kinase activity is tightly controlled in normal cells but is usually excessive activated in cancers. Several tyrosine kinase inhibitors are used in cancer therapies nowadays. Our novel tyrosine kinase inhibitor, 1J-309, is a multiple kinase inhibitor that targets several receptors including vascular endothelial growth factor receptors (VEGFRs). We find 1J-309 dramatically reduces cell proliferation of VEGFR3+/VEGF-C+ A549 human lung cancer cells by decreasing the expression of CDK1 and cyclin B1 following growth arrest at G2/M phase. After long term drug treatment, 1J-309 causes cell death. Moreover, 1J-309 represses CDK1 expression at early stage but it does not change CDK1 RNA expression and protein stability. Additionally, 1J-309 significantly decreases the migration ability of A549 cells. 1J-309 also reduces gelatin-related invasion potency. The AKT and p38 MAPK activity are significantly repressed by 1J-309 and it dramatically drives the expression of tumor suppressor, p53, at low-dose treatment. Our results demonstrate that 1J-309 significantly attenuates cell proliferation by inducing G2/M growth arrest, reduces the invasion and migration potency, and promotes a dramatic increase of p53 in A549 cells.

No description available.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 140-170).