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1
Etter, Jonathan Parker. "Development of Inhibitors in the IL-6/GP130/JAK/STAT Pathway as Therapeutic Agents." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376525461.
2
Ghafoory, Shima. "Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer." Thesis, Mälardalen University, Mälardalen University, School of Sustainable Development of Society and Technology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-7797.
Abstract:
Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers.
Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells.
Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer.
Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors.
The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection.
At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project.
Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer
3
Berrabah, Sofia. "Etude de nouvelles cibles thérapeutiques dans les lymphomes compliquant la maladie cœliaque." Electronic Thesis or Diss., Université Paris Cité, 2021. http://www.theses.fr/2021UNIP5201.
Abstract:
La maladie cœliaque réfractaire de type II (MCRII), autrement appelé lymphome intraépithélial, est une complication rare mais sévère de la maladie cœliaque caractérisée par une expansion clonale d'une population particulière de lymphocytes intraépithéliaux (LIE) innés, présents dans l'intestin normal chez l'Homme comme chez la souris. Notre laboratoire a montré que cette population particulière de LIE innés partage des caractéristiques communes à celles des lymphocytes T et des cellules NK. Ces « LIE iCD3+ innés » sont caractérisées par une expression de CD3 au niveau intracellulaire mais pas à la surface, de récepteurs NK et présentent des réarrangements des gènes codant le récepteur T. En outre, le laboratoire a montré que ces cellules se développent dans l'épithélium intestinal à partir de précurseurs de la moëlle osseuse en réponse à une combinaison de signaux induits à travers la voie NOTCH et l'interleukine 15. Durant la lymphomagénèse, les LIE iCD3+ innés acquièrent des mutations somatiques gain-de-fonction dans JAK1et/ou STAT3. Ces mutations pourraient favoriser l'expansion clonale des LIE iCD3+ mutés aux dépens des lymphocytes T normaux résidents en leur conférant une sensibilité accrue à l'interleukine 15 (IL-15), une cytokine surexprimée dans l'intestin des patients. Ainsi, notre hypothèse est que ces mutations ont un rôle central dans l'initiation de la lymphomagénèse dans un contexte de production chronique d'IL-15 et, de ce fait, représentent une cible thérapeutique. Le premier objectif de ma thèse a été d'étudier l'intérêt des inhibiteurs de la voie JAK/STAT dans le traitement de la MCRII. Dans un premier temps, nous avons testé in vitro différents inhibiteurs de JAK/STAT sur des lignées cellulaires IL-15-dépendantes issues soit de LIE de MCRII soit de LIE T normaux. Nous avons démontré que ces drogues inhibent la prolifération et la phosphorylation de STAT3 et augmentent l'apoptose cellulaire aussi bien dans les LIE MCRII que dans les LIE T normaux. Dans un second temps, nous avons généré un modèle de xénogreffe en injectant des cellules issues de biopsies intestinales ou du sang d'un patient MCRII dans des souris immunodéficientes surexprimant l'IL-15 humaine dans l'épithélium intestinal (Rag-/-Gc-/-IL-15TgE ou IRGC) afin de tester l'efficacité des inhibiteurs de JAK/STAT in vivo. Le traitement des souris xénogreffées par le ruxolitinib, inhibiteur de JAK1/JAK2, a permis une diminution de la fréquence et du nombre ainsi que de l'activité cytotoxique des cellules tumorales humaines et une amélioration de l'état général des souris. Ces résultats encourageants restent à confirmer. Le second objectif de ma thèse a été de vérifier si la mutation pD661V de STAT3 était suffisante pour induire le développement de la MCRII dans un contexte de surproduction d'IL-15 dans des souris IRGC. Nous avons généré avec succès les LIE iCD3+ innés murins semblables aux LIE iCD3+ innés humaines à partir de précurseurs communs aux cellules lymphoïdes (CLP) en combinant un signal NOTCH et IL-15. Nous avons ensuite transduit les CLP avec un vecteur rétroviral contenant Stat3 sauvage ou muté (D661V). Les cellules transduites ont alors été injectées chez des souris IRGC suivies pendant 8 semaines. Les résultats préliminaires ont montré que les LIE iCD3+ innés se logent préférentiellement dans l'intestin mais aucun développement d'un lymphome intraépithélial n'a été observé au bout de 8 semaines suggérant que la mutation pD661V de STAT3 seule ne suffit pas en présence d'IL-15 à induire in vivo un lymphome intraépithélial. Ces résultats préliminaires sont toutefois à reproduire et à confirmer. Le modèle mise en place pour l'étude de STAT3 va désormais être utilisé afin d'évaluer la contribution respective de mutations canoniques de JAK1 et STAT3 et des autres mutations récurrentes retrouvées dans le lymphome intraépithélialRefractory coeliac disease type II (RCDII), also called intraepithelial lymphoma, is a rare but severe complication of coeliac disease characterized by the clonal expansion of a small subset of innate intraepithelial lymphocytes (IEL), present in the normal human and murine intestine. Our lab has shown that this population displays shared features between T and natural killer (NK) cells. These so-called iCD3+ innate IEL are mainly characterized by intracellular expression of CD3, which is not detected at the cell surface, expression of NK receptors as well as DNA rearrangement of T cell receptor genes. Our lab has also shown that iCD3+ innate IEL originate from bone marrow precursors through coordinated NOTCH1 and interleukin (IL)-15 signals. During lymphomagenesis, iCD3+ innate IEL of most RCDII patients were shown to have acquired somatic gain-of-function mutations in JAK1 and/or STAT3 that confer increased sensitivity to interleukin-15, a cytokine overexpressed in the intestine of coeliac patients, thereby promoting their clonal expansion. Thus, our hypothesis is that JAK1/STAT3 mutations play a key role in initiating lymphomagenesis associated to coeliac disease in an IL-15-rich environment and that they could represent an attractive therapeutic target.The first objective of my thesis was to study the interest of JAK/STAT inhibitors for RCDII treatment. First, we have tested in vitro different JAK/STAT inhibitors on IL-15-dependent RCDII or normal IEL-T cell lines. We have shown that these inhibitors decrease the proliferation and phosphorylation of STAT3 and increase cellular apoptosis in both RCDII and normal T cell lines. Secondly, we have established a xenograft model based on the injection of cells derived from biopsy or blood from one RCDII patient into immunodeficient mice overexpressing the human IL-15 transgene in their gut epithelium (Rag-/-Gc-/- IL-15TgE; IRGC) to test the efficacy of JAK/STAT inhibitors in vivo. Treatment of xenografted mice with ruxolitinib, a potent inhibitor of JAK1/JAK2 decreased the frequency, number and cytotoxic potential of human tumoral cells and allowed clinical restoration. These preliminary results are encouraging but need to be confirmed. The second objective of my thesis was to test whether the Stat3 pD661V mutation is sufficient to induce the intraepithelial lymphoma in an IL-15-rich context in IRGC mice. We have successfully generated murine iCD3+ innate IEL in vitro, resembling their human counterparts from common lymphoid precursors by combining NOTCH and IL-15 signals. We then transduced CLP with a retroviral vector containing wild-type or mutated Stat3 pD661V. The transduced cells were injected into IRGC mice that subsequently were followed-up during a period of 8 weeks. In vitro generated iCD3+ innate IEL preferentially homed to the intestine. However, no development of intraepithelial lymphoma was observed suggesting that the Stat3 pD661V variant alone is not sufficient to induce the intraepithelial lymphoma. These preliminary results need to be reproduced and confirmed. The murine model used to test the role of STAT3 will now be used to evaluate the respective contribution of canonical mutations in JAK1 and STAT3 and of other recurrent mutations identified in RCDII
4
Gomes, Guilherme Wataru. "Expressão gênica dos transportadores de membrana ABCB1,ABCG2, SLC22A1 e SLCO1A2 em linhagens celulares tratadas com inibidor comercial da via JAK-STAT." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-16032016-095918/.
Abstract:
INTRODUÇÃO: A desregulação da via de sinalização JAK-STAT é uma característica marcante das neoplasias mieloproliferativas (NMPs), doenças clonais da célula tronco hematopoética, dentre as quais encontra-se a mielofibrose (MF). Diversos inibidores de JAK foram desenvolvidos para o tratamento da MF e encontram-se em diferentes fases de desenvolvimento clínico. Devido ao seu desenvolvimento recente, pouco se sabe a respeito do papel de transportadores de membrana na farmacocinética desses compostos. Essas proteínas realizam o influxo e efluxo celular de substratos endógenos e xenobióticos, e alterações na expressão desses transportadores podem influenciar a resposta a esses fármacos. OBJETIVO: Avaliar o efeito de um inibidor comercial da via JAK-STAT na expressão gênica dos transportadores de membrana ABCB1, ABCG2, SLC22A1 e SLCO1A2 em células HepG2, Caco-2 e HEL92.1.7. MÉTODOS: Linhagens de carcinoma hepatocelular (HepG2), adenocarcinoma colorretal (Caco-2) e eritroleucemia humana homozigotas para JAK2V617F (HEL92.1.7) foram cultivadas e tratadas o inibidor comercial da via JAK-STAT JAK Inhibitor I. Para determinar a concentração ideal para o tratamento com o inibidor, as células foram tratadas com diversas concentrações do inibidor de JAK por 24 horas e foram feitos testes de viabilidade celular e fragmentação do DNA. Com as condições de tratamento padronizadas, foi extraído o RNA total das células e sintetizado o cDNA, para análise das expressões de RNAm dos genes ABCB1, ABCG2, SLC22A1 e SLCO1A2 por PCR em tempo real. Foi também avaliada a expressão dos transportadores de efluxo ABCB1 e ABCG2 por citometria de fluxo, utilizando anticorpos primários direcionados a essas proteínas. RESULTADOS: Nas células HepG2, foi observado um aumento da expressão de RNAm de ABCB1 nas células tratadas com 4,00 µM do inibidor de JAK, quando comparado com o controle (células incubadas apenas com o veículo) (P=0,041). Não foi observada alteração da expressão de RNAm de ABCG2 e SLC22A1 com o tratamento com o inibidor de JAK nessa linhagem (P>0,05); a expressão de RNAm de SLCO1A2 não foi detectada nessa linhagem. Nas células Caco-2, a expressão de ABCB1, ABCG2, SLC22A1 e SLCO1A2 não se alterou com o tratamento com o inibidor de JAK nas concentrações utilizadas (0,25 µM a 1,00 µM) por 24 horas (P>0,05). Para as células HEL92.1.7, não foi observada diferença na expressão de RNAm de ABCB1, ABCG2 e SLC22A1 com o tratamento com 1,00 µM do inibidor de JAK por 24 horas em comparação ao controle (P>0,05); nessa linhagem, a expressão de RNAm de SLCO1A2 não foi detectada. A expressão proteica dos transportadores ABCB1 e ABCG2 não sofreu alteração com o tratamento com o inibidor de JAK nas condições utilizadas nas três linhagens celulares estudadas (P>0,05). CONCLUSÕES: Apenas as células HepG2 apresentaram um aumento da expressão de RNAm do transportador de efluxo ABCB1 em concentrações elevadas do inibidor de JAK, sugerindo que os inibidores de JAK podem modular a expressão do gene desse transportador no fígado. O tratamento com o inibidor da via JAK-STAT não foi associado com alterações na expressão proteica de ABCB1 e ABCG2 em todas as células estudadas.BACKGROUND: JAK-STAT pathway signaling disregulation is a hallmark of myeloproliferative neoplasms (MPN), hematopoietic stem cell clonal diseases, among which is myelofibrosis (MF). Several JAK inhibitors have been developed for MF treatment and are found in different stages of clinical development. Because the recent development of these compounds, the role of drug transporters in their pharmacokinetics is poorly understood. These proteins perform celular influx and effux of endogenous substrates and xenobiotics, and changes in the expression of these drugs transporters may affect the response to these drugs. AIM: To evaluate the effect of a JAK-STAT pathway commercial inhibitor in gene expression of drug transporters ABCB1, ABCG2, SLC22A1 and SLCO1A2 in HepG2, Caco-2 and HEL92.1.7 cells. METHODS: Hepatocellular carcinoma cell line HepG2, colorectal adenocarcinoma cell line Caco-2 and human erythroleukemia homozygous JAK2V617F cell line HEL92.1.7 were grown and treated with the JAK-STAT pathway inhibitor JAK Inhibitor I. In order to determine the optimal concentration for treatment with the inhibitor, cells were treated with several concentrations of JAK inhibitor by 24 hours, and cell viability and DNA fragmentation tests were performed. Once the treatment conditions were standardized, total RNA were obtained from the cells, and cDNA was synthesized in order to evaluate the mRNA expression of ABCB1, ABCG2, SLC22A1 and SLCO1A2 genes, performed by real time PCR. We also evaluate the expression of drug efflux transporters ABCB1 and ABCG2 by flow cytometry, using primary antibodies directed to these proteins. RESULTS: In HepG2 cells, it was observed an increase in ABCB1 mRNA expression in cells treated with 4,00 µM of JAK inhibitor, when compared with controls (cells exposed only to the vehicle) (P=0.041). There was no change in ABCB2 and SLC22A1 mRNA expression with the treatment with JAK inhibitor in this cell line (P>0.05); SLCO1A2 mRNA was not detected in this cell line. In Caco-2 cells, ABCB1, ABCG2, SLC22A1 and SLCO1A2 mRNA expression did not change with treatment with the JAK inhibitor at the concentrations used (0.25 µM to 1.00 µM) by 24 hours (P>0.05). In HEL92.1.7 cells, it was not observed differences in ABCB1, ABCG2 and SLC22A1 mRNA expression with the treatment with 1 µM of JAK inhibitor by 24 hours when compared with controls (P>0.05); in this cell line, SLCO1A2 mRNA was not detected. Protein expression of ABCB1 and ABCG2 drug transporters has not changed with treatment with the JAK inhibitor under the conditions used in the three cell lines studied. CONCLUSIONS: Only HepG2 cells presented an increase in mRNA expression of drug efflux transporter ABCB1 in presence of high levels of JAK inhibitor, suggesting that JAK inhibitors could modulate this transporter gene expression in liver. Treatment with JAK-STAT pathway inhibitor was not associated with changes in ABCB1 and ABCG2 protein expression in all cell lines studied.
5
Khatchaturyan, Levon [Verfasser], Udo [Akademischer Betreuer] Markert, Uta-Christina [Akademischer Betreuer] Hipler, and Ulrike [Akademischer Betreuer] Kämmerer. "Die Rolle von PIAS (Protein Inhibitors of Activated STATs) in der Regulation von STAT (Signal Transducer and Activator of Transcription) : vermittelten Funktionen trophoblastärer Zellen / Levon Khachaturyan. Gutachter: Udo Markert ; Uta-Christina Hipler ; Ulrike Kämmerer." Jena : Thüringer Universitäts- und Landesbibliothek Jena, 2012. http://d-nb.info/1020402113/34.
6
Aubert-Jürgens, Ana. "STAT3 inhibitors for cancer treatment." [S.l.] : [s.n.], 2005. http://elib.tu-darmstadt.de/diss/000563.
7
Hill, Jacqueline M. "Transition state analogues as inhibitors of metallo-proteases." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260112.
8
Fisher, Michael I. "Transition state analogue inhibitors of the aspartyl proteases." Thesis, University of Huddersfield, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363233.
9
Bhasin, Deepak. "Small Molecule Inhibitors asAnticancer Agents." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1305826098.
10
Haque, Mohammad Rashedul. "Novel STAT3 small-molecule inhibitors as potential anticancer agents." Thesis, University of London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.535504.
11
Singh, Danny Ravinder. "Phosphorus containing transition state analogue inhibitors of the aspartyl proteases." Thesis, University of Huddersfield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368303.
12
Chamberlain, Christopher Daniel. "Development and validation of assays used to evaluate STAT3 inhibitors." Thesis, King's College London (University of London), 2017. https://kclpure.kcl.ac.uk/portal/en/theses/development-and-validation-of-assays-used-to-evaluate-stat3-inhibitors(007f8426-c173-4802-955e-181bf9aec424).html.
Abstract:
Transcription factors are important control proteins in cells that bind to their cognate DNA sequences in the promoter regions of genes, either up-regulating or down-regulating protein expression. In many cancer types, transcription factors are up-regulated and promote the expression of genes important in survival and metastasis. For this reason, transcription factors are good targets for novel anticancer agents. The STAT family of transcription factors (seven are now acknowledged) recognize and bind to a ~10 base pair sequence of DNA in the promoter region of a number of genes, enhancing the expression of oncogenic proteins such as Survivin, Cyclin D1, Bcl-2 and VEGF. There are currently no small-molecule STAT3 inhibitors in clinical use, so there is a need for the development of assays that can be used to screen molecules to identify lead compounds. The main focus of this project has been to develop an in vitro homogenous time resolved FRET (HTRF) assay that can be used in low-, medium- and high-throughput modes for the discovery of novel inhibitors. The project started with the cloning, production and purification of recombinant STAT3βTC, which is a homodimeric protein. This was challenging and time-consuming as initial solubility and stability issues were encountered. However, experimental conditions were eventually established that allowed useful quantities (i.e.10 mg batches) of purified and stable protein to be obtained. As part of the optimization process, the STAT3βTC was re-cloned into a HIS-Tag vector which facilitated purification using affinity (Ni2+) chromatography along with size exclusion chromatography to produce pure monomeric STAT3βTC. This could be dimerised to provide pure STAT3βTC homodimer. The pure protein was used to develop a HTRF assay by first labelling the STAT3βTC with Europium. Next, the cognate DNA recognition sequence in the form of an 18-mer duplex oligonucleotide was biotinylated and joined to the second fluorophore label (D2) via a streptavidin linkage. The strength of the FRET signal between these two components could then be used to measure the interaction between them. As part of a multi-well system, this could then be used to screen for small molecules capable of disrupting the protein/DNA complex. The assay was validated using unphosphorylated STAT3 that does not form the biologically-relevant homodimer, and non-biotinylated DNA, which would not form the active FRET pair. Further validation of the assay was carried out using known STAT3 inhibitors such as the peptidomimetics PYLKTK and YLPQTV, and the small-molecule inhibitors STA-21 and Stattic. It was then used to screen a 40-membered library of novel SH2-targeted molecules produced in-house, in which it successfully identified six “hit” molecules with low micro molar activity. These were further evaluated by establishing IC50 values in a number of cell lines including MDA-MB231, HELA, A4 and NCI-H1975. These studies revealed a correlation between the FRET assay results and the cytotoxicity of the molecules in the STAT3-dependent cell lines. The molecules were also studied in cellular experiments to establish their effect on STAT3-regulated genes such as Cyclin D1 and Survivin, in which a correlation was also observed. As a result, these molecules are now in further development. Finally, the assay has been modified for high-throughput use in a 384-well system, and will be used for robotic screening in the future.
13
Ekegren, Jenny. "Design and Synthesis of Novel HIV-1 Protease Inhibitors Comprising a Tertiary Alcohol in the Transition-State Mimic." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6737.
14
Smith, Cressida Sally. "Design and synthesis of novel transition state isostere inhibitors of MurD." Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418195.
15
Wang, Xiaodong. "Design, Syntheses, and Bioactivities of Conformationally Locked Pin1 Ground State Inhibitors." Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/26625.
Abstract:
Pin1 (protein interacting with NIMA 1) is a peptidyl-prolyl isomerase involved in mitosis. As a potential anti-cancer drug target, Pin1 interacts and regulates the activity of an increasing number of cell cycle enzymes by an unknown mechanism. These cell cycle enzymes include Cdc25, Cdc27, Cyclin D1, Myt1, Wee1, NIMA, Cdc2, Plk1 and c-Myc. Recent research has revealed that Pin1 is overexpressed in a variety of cancer cell lines and Pin1 inhibitors inhibit proliferation activity of several cancer cells overexpressing Pin1. The most potent Pin1 inhibitors identified so far are in the micromolar range and no pharmacophore has been identified.
In order to assist the understanding of the biological function of Pin1 using molecular probes, two amide isosteres of Ser-trans-Pro and Ser-cis-Pro dipeptides were designed and stereoselectively synthesized. The conformationally locked Ser–trans–Pro mimic, Boc-SerΨ[(E)CH=C]Pro–OH, was synthesized through the use of an Ireland-Claisen [3,3]-sigmatropic rearrangement in nine steps with 13% overall yield from a serine derivative. The Ser-cis-Pro mimic, Boc-SerΨ[(Z)CH=C]Pro–OH, was synthesized through the use of a Still-Wittig [2,3]-sigmatropic rearrangement in 11 steps with an overall yield of 20% from the same starting material.
Conformationally locked peptidomimetics, including two exactly matched peptidomimetics, Ac–Phe–Phe–pSer–Ψ(E)CH=C]Pro–Arg–NH2 and Ac–Phe–Phe–pSer–Ψ[(Z)CH=C]Pro–Arg–NH2, were synthesized from these Ser-Pro isosteres using Fmoc SPPS. A protocol for in vitro Pin1 inhibition assay was established for measuring the inhibition constant for these peptidomimetics. A conformationally locked cis peptidomimetic inhibits Pin1 with a Ki of 1.7 μM, 23-fold more potent than its trans counterpart, illustrating the preference of Pin1 for a cis amide bond in its PPIase domain. The A2780 ovarian cancer cell antiproliferation activity of these peptidomimetics parallels their respective Pin1 inhibition data. This research provides a start toward more drug-like Pin1 inhibitor design. Gly–trans–Pro isosteres were synthesized using the Ireland-Claisen route. The construction of a non-peptidic (Z)-alkene library for Pin1 inhibition was attempted using the Ser-cis-Pro mimic, Boc—SerΨ[(Z)CH=C]Pro–OH as the core.Ph. D.
16
Cao, Yu. "The synthesis of organo-phosphorus transition-state analogue inhibitors of dihydroorotase." Master's thesis, Canberra, ACT : The Australian National University, 1994. http://hdl.handle.net/1885/141358.
17
Xu, Guoyan. "Pin1 Inhibitors: Towards Understanding the Enzymatic Mechanism." Diss., Virginia Tech, 2010. http://hdl.handle.net/10919/37823.
Abstract:
An important role of Pin1 is to catalyze the cis-trans isomerization of pSer/Thr-Pro bonds; as such, it plays an important role in many cellular events through the effects of conformational change on the function of its biological substrates, including Cdc25, c-Jun, and p53. The expression of Pin1 correlates with cyclin D1 levels, which contributes to cancer cell transformation. Overexpression of Pin1 promotes tumor growth, while its inhibition causes tumor cell apoptosis. Because Pin1 is overexpressed in many human cancer tissues, including breast, prostate, and lung cancer tissues, it plays an important role in oncogenesis, making its study vital for the development of anti-cancer agents.
Many inhibitors have been discovered for Pin1, including 1) several classes of designed inhibitors such as alkene isosteres, non-peptidic, small molecular Pin1 inhibitors, and indanyl ketones, and 2) several natural products such as juglone, pepticinnamin E analogues, PiB and its derivatives obtained from a library screen. These Pin1 inhibitors show promise in the development of novel diagnostic and therapeutic anticancer drugs due to their ability to block cell cycle progression. In order to develop potent Pin1 inhibitors, the concept of transition-state analogues was used for the design of three classes of compounds: ketoamide, ketone, and reduced amide analogues.
Specifically, a convergent synthesis of α-ketoamide inhibitors of Pin1 was developed. An α-hydroxyorthothioester derivative of Ser was reacted directly with an aminyl synthon. The reaction was catalyzed by HgO and HgCl2 to form an α-hydroxyamide. Hydrolysis and coupling were combined in one step in 80% yield. Two diastereomers of a phospho-Ser-Pro α-ketoamide analogue were synthesized. The resulting IC50 values of 100 µM and 200 µM were surprisingly weak for the Pin1 peptidyl-prolyl isomerase.
Diastereomeric ketones were synthesized by coupling cyclohexenyl lithium to the serine Weinreb amide, via the Michael addition of a carboxylate synthon. The IC50 values of the two ketone diastereomers were determined to be 260 μM and 61 μM, respectively.
Five reduced amide inhibitors for Pin1 were synthesized through a selective reduction using borane. The most potent inhibitor was found to be Fmocâ pSerâ Ψ[CH2N]-Proâ tryptamine, which had an IC50 value of 6.3 µM. This represents a 4.5-fold better inhibition for Pin1 than a comparable cis-amide alkene isostere. The co-crystal structure of Acâ pSerâ Ψ[CH2N]-Proâ tryptamine bound to Pin1 was determined to 1.76 à resolution.
Towards understanding the two proposed mechanisms of Pin1 catalysis, nucleophilic-additition mechanism and twisted-amide mechanism, three classes of Pin1 inhibitors (ketoamide, ketone, and reduced amide analogues) involving a total of nine compounds were synthesized and evaluated. The weak inhibitory activities of ketoamide and ketone analogues do not support the nucleophilic-addition mechanism, while the twisted-amide mechanism of Pin1 catalysis is promising based on the reduced amide inhibitors with good potencies.Ph. D.
18
Wang, Xinning [Verfasser]. "Tetrazole-containing STAT5 Inhibitors Derived from Furazan-based Phosphate Mimetics / Xinning Wang." Berlin : Freie Universität Berlin, 2020. http://d-nb.info/121464130X/34.
19
Masciocchi, D. "DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF POTENTIAL STAT3 INHIBITORS AS ANTICANCER AGENTS." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/170266.
Abstract:
Signal Transducers and Activators of Transcription factors (STATs) are a class of latent cytoplasmic proteins that regulate cell growth and survival by modulating the expression of specific target genes. One member of the STAT family, STAT3, has received particular attention since it has been found constitutively activated in a broad spectrum of cancer cell lines and human tumors. These compelling results, combined with a well-known selectivity of STAT3 inhibitors for tumor cells, validated STAT3 as a promising anticancer drug target. Although the experimental studies on STAT3 inhibitors seem very interesting, to date, there is no evidence in literature of any impending clinical development.
The aim of this research project was the identification of new small molecules able to inhibit STAT3 activity through the direct binding to STAT3 protein. Starting from the structures of molecules which were known in the literature for their STAT3 inhibitory activity, three classes of compounds were developed: the oxadiazoles, the pyridazinones and the ortho-quinone derivatives. The oxadiazoles were structurally related to AVS-0288, which was identified through a screening of a Korean chemical library for its ability to inhibit STAT3 activity, although the exact mechanism of action had not been clarified yet. These derivatives were designed using the most classical approaches for a ‘hit to lead generation’: bioisosterism, vinilogy, homology. The second series of molecules, the pyridazinones, derived from the natural compound Cryptotanshinone, a direct STAT3 inhibitor, and was designed with the support of molecular modeling studies. These latter studies suggested a structural similarity between Cryptotanshinone and a series of pyridazinone derivatives, previously investigated by our research group.
Moreover, in order to combine the structural characteristics of the two series of molecules above, chimeric compounds, on the basis of an accurate conformational analysis, were designed and synthesized. Finally, with the aim to understand the mode of interaction of the parent compound Cryptotanshinone with the SH2 domain of STAT3, docking studies and in vitro screening were carried out. These suggested a key role of the ortho-quinone moiety, since it resembled the phosphotyrosine of the natural peptide able to bind SH2 domain. Therefore, ortho-quinone compounds, derived from a simplification of Cryptotanshinone structure, were synthesized. However, due to the instability and high volatility of some of the designed compounds, only two representative terms have been prepared to date.
All synthesized molecules were evaluated by a cell-based preliminary screening, the dual-luciferase assay, which selected for compounds able to exert an inhibition on STAT3 activity. Although some of these assays showed interesting preliminary results, a deeper investigation was fundamental, in order to determine their target and/or mechanism of action. With this aim, an in vitro competitive binding assay, the AlphaScreen technology system, was performed to check the ability of these compounds to bind the SH2 domain of STAT3 and inhibit its dimerization, a crucial step for STAT3 activation.
This biological investigation led to the identification of the oxadiazole amidic derivative F2e, which was found to exert positive results both in the luciferase assay (20% inhibition at 5 μM) and in the AlphaScreen-based assay (IC50 = 17.7 μM), and moreover, its inhibitory activity was found to be dose-dependent. The effect of F2e on the tumor cell growth was also checked and shown to have a good profile of inhibitory activity on cell proliferation, with an GI50 around 2 μM for most of the screened tumor cell lines. Due to these encouraging results, F2e is considered the lead compound for the development of a new series of derivatives.
20
Misale, Antonio. "Synthesis of angucycline-based small molecules as potential STAT3 : STAT3 protein-protein interaction inhibitors for cancer therapy." Thesis, University of London, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555844.
Abstract:
Inhibition of the ST AT3: ST AT3 protein-protein interaction is an attractive approach for cancer therapy as it can lead to suppression of tumour cell growth and induce apoptosis. The racemic ochromycinone (STA21) is one of the few known small-molecule STAT3:STAT3 inhibitors. Our synthetic efforts focused on synthesis of the natural product YM-181741, which possesses at least three points for chemical variation to prepare compound libraries as potential STAT3:STAT3 inhibitors. Synthesis of the angucycline molecule was achieved employing an approach based on a Gold (lII)-catalysed intramolecular [4+2] benzannulation reaction. A facile and highly efficient route for the preparation of racemic form of the natural product was developed that offers a high degree of flexibility for modification of the scaffold at different stages of its synthesis. The enantioselective synthesis of (S)- YM181741 was successfully carried out through the (R)-diyne building block via an enantioselective copper-catalysed 1,4-conjugate addition reaction on a system bearing a v-coordinating group, in order to install the chirality on the diyne moiety. The optimised reaction conditions afforded the Michael adduct in good yield and high enantiomeric excess (up to 96% ee). Further chemical elaboration of the (±)- YM-181741 natural product was investigated in order to explore the necessary chemical diversity to assess a preliminary model of interaction between the SH2 domain of ST AT3 and the angucycline scaffold. The biological evaluation of the focused library allowed the identification of angucycline derivatives possessing high binding affinity for the SH2 region and the ability to inhibit STAT3 transcriptional activity.
21
Miller, David James. "Phosphinic acids as inhibitors of D-Ala-D-Ala adding enzyme." Thesis, University of Southampton, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242865.
22
Stephan-Queffeulou, Emilie. "Sélection de peptides inhibiteurs de l'activité des protéines STAT5." Compiègne, 2011. http://www.theses.fr/2011COMP1928.
Abstract:
L'activation constitutive des protéines STAT5A et STAT5B, retrouvée dans de nombreux cas d'hémopathies malignes et de tumeurs solides, entraîne une forte transcription de gènes prolifératifs et anti-apoptotiques, contribuant au développement tumoral. Ces protéines représentent donc des cibles prometteuses pour de nouveaux traitements anticancéreux. De plus, la recherche d'inhibiteurs des protéines STAT5 est importante car les traitements actuels contre les leucémies sont peu spécifiques, entraînant des effets secondaires. Ainsi, ce projet vise à sélectionner des molécules capables d'inhiber l'activité des protéines STAT5 à partir d'une banque peptidique exprimée sur bactériophage (phage-display). Les premiers travaux ont consisté à produire et à purifier la protéine recombinante STAT5B. La recherche de peptides interagissant avec la protéine recombinante cible a ensuite été réalisée par une procédure de sélection par affinité, conduisant à l'identification de deux séquences peptidiques différentes (PepA et PepM) interagissant avec la protéine lorsqu'elles sont présentées sur phage. L'affinité des peptides sous forme soluble a ensuite été mesurée par Biacore (technologie SPR). Ces résultats montrent que le peptide PepM présente une forte affinité pour STAT5B de l'ordre du nanomolaire. Des expériences de pull-down également montrent que PepM interagit avec la protéine STAT5 lorsqu'elle est sous forme active. Enfin, après avoir confirmé le succès de l'internalisation du peptide dans différentes lignées cellulaires, les résultats préliminaires montrent que cette molécule diminue la viabilité de cellules cancéreuses lorsqu'elles dépendent de l'activité des protéines STAT5Constitutive activation of STAT5 proteins has been demonstrated in numerous cases of malignant hemopathies and solid tumors. This phenomenon results in enhanced transcription of proliferative and anti-apoptotic genes, contributing to cancer development. Consequently, STAT5 proteins are attractive targets for innovative anticancer therapy. Developing STAT5 direct inhibitors is all the more important that current treatment against leukemias are few specific and cause secondary effects. This project aims at selecting STAT5 inhibiting molecules from a peptide library expressed on bacteriophage surface (phage display technology). First, recombinant STAT5B protein was produced, purified and used as a target during affinity-based selection. After a two-step selection, two peptides (PepA and PepM) were identified. The affinity of the two soluble peptides was measured by Biacore (Surface Plasmon Resonance technology) : PepM shows an nanomolar affinity towards STAT5B recombinant protein. This peptide interacts also with active STAT5 protein as demonstrated by pull down experiments. Finally, PepM effects on different cell lines were studied. This peptide penetrates in cells and preliminary results show that PepM diminishes STAT5 dependent cell viability
23
Hajiw, Martha. "Étude des conditions de dissociation des hydrates de gaz en présence de gaz acides." Thesis, Paris, ENMP, 2014. http://www.theses.fr/2014ENMP0042/document.
Abstract:
La demande en énergies fossiles a connu une forte croissance au cours du vingtième siècle et représente aujourd'hui 80% de la consommation énergétique mondiale. Pour répondre à la demande, les industries pétrolières et gazières s'orientent vers de nouvelles sources. 40% des réserves de gaz contiennent un pourcentage important (jusqu'à 20%) de gaz acides (dioxyde de carbone et sulfure d'hydrogène). La production de ces gaz à forte teneur en gaz acides représente un défi pour les industries, étant donné la toxicité du sulfure d'hydrogène et la forte probabilité de corrosion des pipelines en présence d'eau (naturellement produite avec le gaz naturel). D'autre part, l'utilisation des énergies fossiles conduit au changement climatique avec des émissions importantes de dioxyde de carbone dans l'atmosphère. Le captage et le stockage du CO2 semble être un procédé prometteur. De l'eau est souvent présente lors du transport du gaz naturel et du CO2 capturé. Lors des étapes de production et de transport, les conditions de température et de pression sont sujettes au changement. La condensation de l'eau (à l'origine de la corrosion et donc d'une rupture possible des pipelines) et à la formation de glace et/ou d'hydrates en sont les conséquences principales. Or la formation d'hydrates est un sérieux problème avec un risque de blocage des pipelines. Pour éviter la formation des hydrates, des inhibiteurs chimiques sont utilisés. Il est donc indispensable de bien connaitre les équilibres entre phases pour les différents mélanges considérés pour un fonctionnement et une production en toute sécuritéThe twentieth century has seen an important increase of the fossil energy demand, representing today 80% of world energy consumption. To meet the request, oil and gas companies are interested in new gas fields. 40% of these reserves are acid and sour gases, i.e. the percentage of carbon dioxide and hydrogen sulphide is significant, sometimes over 20% of CO2 or H2S. Natural gas production with high content of acid gases can be a challenge, due to their corrosiveness potential in pipelines in the presence of water and H2S toxicity. On another hand, as a result of world's dependence on fossil energies, the release of carbon into atmosphere is increasing and leads to climate changes. Carbon Capture and Storage (CCS) is one of the most promising ways to reduce CO2 emissions in the atmosphere. Whether in natural gas or carbon dioxide transport, water may be present. During production, transportation and processing, changes in temperature and pressure can lead to water condensation (cause of corrosion, and consequently a possible pipeline rupture), ice and/or gas hydrates formation. Hydrates are a serious flow assurance problem and may block pipelines. To avoid hydrates formation, chemical inhibitors are used. Therefore accurate knowledge of mixtures phase equilibria are important for safe operation of pipelines and production/processing facilities
24
Gonzalez, Palmén Lorena. "Homotrimeric dUTPases : Principles of Catalysis and Inhibitor Design." Doctoral thesis, Högskolan i Kalmar, Naturvetenskapliga institutionen, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-6119.
Abstract:
The ubiquitous enzyme dUTPase hydrolyzes dUTP into dUMP and pyrophosphate, preventing DNA fragmentation and cell death due to accumulation of dUTP. Inhibitors of dUTPase could serve as drugs in the treatment of cancers and infectious diseases. This thesis presents five studies. A mutational study on the Escherichia coli dUTPase (S72A) provides new insights about the catalytic principles of the homotrimeric dUTPases. A model is presented in which transition state formation is associated with a rotation of the conserved Ser72 side chain. The model can explain the strict order of deamination and hydrolysis catalyzed by the bifunctional dCTP deaminase:dUTPases. The S72A/D90N double mutant is currently investigated. Preliminary data indicate that this form preserves the binding properties of the S72A mutant but is completely inactive, making it attractive for structural studies. In the remaining studies we compare the binding of substrate analogues to the human, the E. coli and the equine infectious anemia virus (EIAV) homotrimeric dUTPases. One study concerns 2´,3´-dideoxy-UTP (ddUTP) and shows that removal of the 3´-hydroxyl group increases KM, ten times with the cellular dUTPases and fifty times with the viral dUTPase, but does not affect kcat with any of these enzymes. Another study concerns the inhibitory effects of 3´-azido-2´,3´-dideoxy-UTP. This derivative binds to the bacterial dUTPase but not to the other forms making it a potential lead for the development of antibacterial dUTPase inhibitors. Yet another study investigates two uracil derivatives. Both compounds are found to inhibit the human, the bacterial but not the viral dUTPase. The inhibition is shown to be competitive.
25
Al-Lami, Naeemah. "Synthesis of nitrogen-containing bicyclic sesquiterpenes as potential transition state inhibitors of aristolochene synthase." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/53674/.
Abstract:
Aristolochene synthase from Penicillium roqueforti(PR-AS) is sesquiterpene synthase that catalyses the Mg2+-dependent conversion of farnesyl diphosphate FDP to (+)-aristolochene. Through the use of site directed mutagenesis, fluorinated FDPs and an aza-analogue of the eudesmane cation, the reaction was previously shown to involve germacrene A and eudesmane cation as intermediates. The subsequent series of rearrangements that transform the eudesmane cation to (+)-aristolochene have not been investigated previously. To probe the carbocationic nature of these 1,2-hydride and methyl shifts, new aza-analogues were designed to mimic the geometric and electrostatic properties of postulated carbocation intermediates in the catalytic mechanism of PR-AS. Here is described the synthesis of both enantiomers of 10-aza-eremophilane in enantiomerically pure from the common precursor (4S)-limonene oxide and their analysis as inhibitors of PR-AS. The synthesis of (7R,4S,5S)-10-aza-eremophilane cation was accomplished in 8 steps, starting from a known keto ester that in turn was obtained by degradation of (-)-limonene oxide. An identical synthetic protocol was repeated from (4R)-limonene oxide to give the enantiomer of 10-aza-eremophilane cation. Inhibition studies with compound (7R,4S,5S)-10-aza-eremophilane indicated that this ammonium salt acted as a moderate competitive inhibitor of PR-AS (Ki = 38 μM), and showed that eremophilane cation is likely a true intermediate on the pathway from FDP to aristolochene during PR-AS catalysis. The inhibition potency of 10-aza-eremophilane was increased by the addition of diphosphate PPi (Ki = 2.9 μM). This synergetic kinetic effect suggests that the possible involvement of PPi as a stabilizing anion for the eremophilane carbocation in PR-AS biosynthesis. Inhibition studies of the enantiomer of (7R,4S,5S)-10-aza-eremophilane cation, (7S,4R,5R)-10-aza-eremophilane cation, which has incorrect stereocenteres, with PR-AS indicated that this ammonium salt was a poor inhibitor of PR-AS (Ki = 1.03 mM). The data obtained for this compound highlight the chiral environment of the active site of PR-AS, and more importantly supports the postulate that terpene synthases form a product-like contour at their active site that steers the carbocationic cascade catalyzed by PR-AS toward the production of a single enantiomer. iv In the second part of the present work, progress was made towards the stereoselective synthesis of 5-aza-eudesmane cation. This teriary amine is a structural mimic of the 5-eudesmane carbocation, another putative intermediate in the reaction cascade catalysed by PR-AS. However, this tertiary amine was not obtained with desired stereochemistry, nevertheless, two diastereoisomers of the desired compound were obtained.
26
Maughan, Michael A. T. "Cyclic imine sugars : towards the synthesis of transition-state mimics as potential glycosyltransferase inhibitors." Thesis, Durham University, 2003. http://etheses.dur.ac.uk/3694/.
Abstract:
This thesis describes the development of methodology for the synthesis of cyclic imine-sugars, and their use in the synthesis of aza-sugars as potential sugar-processing enzyme inhibitors. Our goals include the synthesis of cyclic imines of L-rhamnose, D-glucose, and L-idose stereochemistry, and the introduction of functionality via nucleophilic addition reactions. Work on the synthesis of cyclic imines commenced with the use of the simple model systems piperidme and 2-methylpiperidine. N-Chlorination of these systems was performed and the products converted into their cyclic imine derivatives through elimination of HCl. Nucleophilic addition reactions to these systems were attempted, but the low stability and reactivity of the imines led to the isolation of only one adduct. The synthesis of novel cyclic pyrrolidine imine-sugars of L-rhamnose stereochemistry was performed by a Staudinger aza-Wittig reaction. The aza-Wittig reaction of a known L-rhamnose derived azido-sugar gave a novel cyclic L-rhamnopyrrolidine aldimine A novel synthesis of this azido-sugar was also devised. Successful nucleophilic additions to the cyclic L-rhamnopyrrolidine aldimine were performed with a range of Grignard reagents giving novel protected aza-sugars in good yields and with excellent diastereoselectivities. A novel cyclic L-rhamnopyrrolidme ketimine was also synthesised via a Staudinger aza-Wittig from a novel azido-sugar, although time constraints prevented screening this system with nucleophiles. The synthesis of novel cyclic piperidine imine-sugars of D-glucose, and L-idose stereochemistry was performed, both via N-chlorination/elimination of the protected parent aza-sugars, and via the Staudinger aza-Wittig reaction of novel azido-sugars. The elimination of HCl from six-membered iV-chloro aza-sugars of D-glucose and L- idose stereochemistry was investigated, and methodology developed in the case of D- glucose system for the regioselective elimination of HCl to give either the aldimme or the ketimine derivative. Comparison of elimination reactions of the N-chloro aza- sugars of D-glucose, and L-idose stereochemistry allowed rationalisation of the observed regiocontrol. Screening of the cyclic imines of D-glucose and L-idose stereochemistry with nucleophiles, followed by deprotection of the adducts, allowed the synthesis of novelaza-sugars via late-stage introduction of functionality. Low yields were obtained for these additions, but diastereocontrol was generally good, and could be rationalised by accepted stereoelectronic and steric approach control factors. The formation of cyclic piperidine imines of L-idose, and D-glucose stereochemistry was also performed via the Staudinger aza-Wittig reaction. These systems were found to be identical to those synthesised by the N-chlorination/elimination protocol. We also performed the first synthesis of the enantiomer of the natural product (+)- adenophorine thereby allowing assignment of the absolute configuration of the natural product (+)-adenophorine. The key synthetic step in this synthesis was stereoselective reduction of a novel intermediate cyclic piperidine ketimine-sugar of L-idose stereochemistry.
27
Swedberg, Joakim Erik. "Rational design of serine protease inhibitors." Thesis, Queensland University of Technology, 2011. https://eprints.qut.edu.au/48131/1/Joakim_Swedberg_Thesis.pdf.
Abstract:
Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.
28
Mautsa, Nicodemus. "Structural and functional characterisation of the protein inhibitor of activated STAT3 (PIAS3)." Thesis, Rhodes University, 2011. http://hdl.handle.net/10962/d1004050.
Abstract:
The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction.
29
Ziegler, Inna. "Posttranslationale Modifikationen der IL-6-Typ-Zytokin-Rezeptoren gp130 und LIFR und ihr Einfluss auf die Assoziation mit Detergenz-resistenten Membranmikrodomänen (DRM)." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:100-opus-3124.
30
Frase, Hilary. "TOWARDS DEVELOPING SPECIFIC INHIBITORS OF THE ATP-DEPENDENT LON PROTEASE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1175637588.
31
Yu, Wenying. "Computational, Synthetic, Biochemical and Biological Studies and Characterization on STAT3 Inhibitors for Potential Anticancer Therapy." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1373328058.
32
Csatary, Erika Elizabeth. "Asymmetric Multicomponent Aza-Diels-Alder Reaction for Construction of Multicyclic Heterocycles and Development of XZH-5 Derivatives as Inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3)." Miami University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=miami1435110305.
33
Altundas, Abdullah Bilal. "Synthesis of XZH-5 Derivatives as Inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3) and Synthesis of π-Extended Tetraphenylporphyrins." Miami University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=miami1473201129.
34
Schust, Jochen. "Neue Ansätze zur Identifizierung niedermolekularer Inhibitoren der STAT3-Aktivierung und -Homodimerisierung." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=982197438.
35
Goodall, Scott. "Probing the structure of acetylcholinesterase inhibitors in their binding site using solid state nuclear magnetic resonance." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270619.
36
DAKA, PHILIAS. "ENAMINE-METAL LEWIS ACID BIFUNCTIONAL CATALYSTS FOR ASYMMETRIC ALDOL REACTIONS. DESIGN AND SYNTHESIS OF STAT3 INHIBITORS." Miami University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=miami1374852476.
37
Couto, Jason. "Biologic Activity of the Novel Small Molecule STAT3 Inhibitor Against Canine Osteosarcoma Cell Lines." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1373986927.
38
Feng, You. "Kinetic Mechanism and Inhibitory Study of Protein Arginine Methyltransferase 1." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/chemistry_diss/68.
Abstract:
Protein arginine methyltransferase 1 (PRMT1) is a key posttranslational modification enzyme that catalyzes the methylation of specific arginine residues in histone and nonhistone protein substrates, regulating diverse cellular processes such as transcriptional initiation, RNA splicing, DNA repair, and signal transduction. Recently the essential roles of PRMT1 in cancer and cardiovascular complications have intrigued much attention. Developing effective PRMT inhibitors therefore is of significant therapeutic value. The research on PRMT inhibitor development however is greatly hindered by poor understanding of the biochemical basis of protein arginine methylation and lack of effective assays for PRMT1 inhibitor screening.
Herein, we report our effort in the kinetic mechanism study as well as the fluorescent probe and inhibitor development for PRMT1. New fluorescent reporters were designed and applied to perform single-step analysis of substrate binding and methylation of PRMT1. Using these reporters, we performed transient-state fluorescence measurements to dissect the rate constants along the PRMT1 catalytic coordinate. The data give evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, we identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. On the other hand, we discovered a type of naphthyl-sulfo (NS) compounds that block PRMT1- mediated arginine methylation at micromolar potency through a unique mechanism: they directly target the substrates but not PRMT enzymes for the observed inhibition. We also found that suramin, an anti-parasite and anti-cancer drug bearing similar functional groups, effectively inhibited PRMT1 mediated methylation. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation. Addionally, to dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. While certain acetylations present a repressive impact on PRMT-1 mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases activity of PRMT1 but increases that of PRMT5. Furthermore, hyperacetylation increases the content of ordered secondary structures of H4 tail. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation, and unravel that complex intercommunications exist between different posttranslational marks in cis.
39
Wong, Ee Lin [Verfasser]. "The transcription factor STAT5 catalyzes Mannich ligation reactions yielding inhibitors of leukemic cell proliferation / Ee Lin Wong." Berlin : Freie Universität Berlin, 2019. http://d-nb.info/1201346711/34.
40
Torikoshi, Kazuo. "Protein inhibitor of activated STAT, PIASy regulates α-smooth muscle actin expression by interacting with E12 in mesangial cells." Kyoto University, 2013. http://hdl.handle.net/2433/174820.
41
Turner, Kimberly Ann. "Deliberate Memory in Three-Year-Old Children: Interrelations among Task Approaches, Working Memory, and Inhibitory Control." NCSU, 2008. http://www.lib.ncsu.edu/theses/available/etd-03242008-181800/.
Abstract:
Preschool children are capable of displaying strategies in memory tasks and demonstrating an early understanding of memorization (e. g., Wellman, 1988; Baker-Ward, Ornstein, & Holden, 1984). Questions remain, however, about the origins of strategic behavior in early childhood. A great deal of recent attention has been devoted to the interrelations among working memory and measures of executive functioning/inhibitory control in elementary-school children (e.g., Schneider, Schumann-Hengsteler, & Sodian, 2005). The goal of this investigation was to extend this work to preschool children in order to examine possible influences on the emergence of deliberate remembering. Specifically, interrelations among working memory, inhibitory control, and deliberate task approaches were examined in 168 three-year-olds who participated in a large-scale, broadly-focused investigation of development, the Durham Child Health and Development Study. Although predicted relations among multiple domains of cognitive functioning were not observed, important findings did emerge. Previous results examining the use of deliberate task approaches were replicated in a more diverse and younger sample. Support for the presence of deliberate remembering in young preschoolers was found in a significant positive relation between language ability and the extent of deliberate task approaches. Finally, an unexpected relation between deliberate task approaches and subsequent recall performance was found; this result is discussed in relation to Utilization Deficiency. Implications for understanding some of the contributors to the emergence of deliberate remembering are presented, and directions for future research are discussed.
42
Younis, Usir, and Usir Younis. "Inhalational Delivery of a JAK3 Inhibitor for the Novel Treatment of Asthma and the Investigation of Pharmaceutical Salts in HFA Propellant Systems." Diss., The University of Arizona, 2018. http://hdl.handle.net/10150/626756.
Abstract:
Asthma is a significant lung disease involving chronic inflammation and remodeling of the airways, resulting in reduced quality of life for those who suffer from the condition. Current therapeutic guidelines suggest the use of inhaled corticosteroids for long-term anti-inflammatory relief to manage moderate to severe chronic asthma; however, inhaled corticosteroids fail to provide prophylactic or reversal treatment of damaged airways incurred by chronic asthma as well as exhibiting adverse side effects (skeletal complications, diabetes, and weight gain).Therefore, there is a need for a new type of drug therapy to address these gaps in the treatment of chronic asthma. There is growing interest aimed towards the inhibition of the Janus Kinase and Signal Transducer and Activator of Transcription (JAK-STAT) pathway for the treatment of asthma. Despite the promising opportunity to investigate this new pathway towards this clinical application, no published work is available using an established and characterized JAK 1/3 inhibitor for the treatment of chronic asthma delivered via inhalation.
This work investigated tofacitinib citrate, a selective JAK 3 inhibitor, and its potential to be delivered locally to the lungs for the treatment of chronic asthma. Several preformulation studies were conducted to determine the basic physical and chemical properties of the compound and its free base, tofacitinib, for proper inhalational formulation development. The drug was delivered to BALB/c mice challenged with house dust mite (HDM) allergen via nebulization utilizing a nose-only chamber. After a three week dosing schedule, mice treated with tofacitinib citrate exhibited an increase in monocyte cell numbers with a simultaneous decrease in eosinophil cell count, gathered from BAL fluid. Further, the experimental groups treated with tofacitinib citrate had a decrease in total protein concentrations in comparison to the experimental groups that were only challenged with HDM or were both exposed to HDM and vehicle. These findings demonstrated that the proper formulation was developed for nebulized delivery of tofacitinib citrate, and that the compound was capable of reducing total protein concentrations and eosinophil cell recruitment, both recognized as biomarkers for an asthmatic response. Although significant work is still needed to be done, these data hold promise for the potential of a locally delivered JAK 3 inhibitor as a treatment for chronic asthma.
Further, the solubility of tofacitinib citrate and five other pharmaceutical salts were determined in HFA 134a, HFA 227, and DFP with varying cosolvent content (0-20% v/v ethanol). The experimental solubilities of the free acid and base compounds were larger than the solubilities of their respective salts in all three systems for tofacitinib, albuterol, and salicylic acid. Warfarin, phenytoin, and ciprofloxacin had similar solubilities with their respective salt forms. Solubilities also increased with increasing cosolvent concentration for all compounds investigated. The model propellant, DFP, provided a slightly stronger correlation of solubility values with HFA 134a in comparison to HFA 227. The observed solubility values were also compared to calculated values obtained from the ideal solubility model, where it was determined that the observed solubility was indeed also dependent on its surrounding solvent interactions and not solely on its ideal solubility (melting point). While some physical changes were observed for the pharmaceutical salts in HFA 134a and 227, more quantitative studies are needed for a larger database of compounds to better understand the factors that contribute to the solubility of pharmaceutical salts (and their correlation to DFP), in HFA-based systems. This information could potentially contribute to a predictive model, saving time and money during the process of pMDI formulation development.
43
Camerino, Eugene. "Trifluoromethyl ketones: Potential insecticides towards Anopheles gambiae." Thesis, Virginia Tech, 2013. http://hdl.handle.net/10919/54015.
Abstract:
Malaria continues to cause significant mortality in sub-Saharan Africa and elsewhere, and existing vector control measures are being threatened by growing resistance to pyrethroid insecticides. With the goal of developing new human-safe, resistance-breaking insecticides we have explored several classes of acetylcholinesterase inhibitors. In vitro assay studies have shown that trifluoromethyl ketones (TFK\'s) are potent inhibitors of An. gambiae AChE (AgAChE), that inhibit the enzyme by making a covalent adduct with the catalytic serine of the enzyme. However research in the Carlier group has shown that trifluoromethyl ketones bearing benzene and pyrazole cores have shown very little toxicity to An. gambiae, perhaps due to hydration and rapid clearance. Focus was directed towards synthesis of oximes, oxime ethers, and hydrazones as potential prodrugs to prevent immediate hydration and reach the central nervous system. The synthesis of various oximes, oxime ethers, and hydrazones has been shown to give cimpounds toxic to Anopheles gambiae within 3- to 4- fold of the toxicity of propoxur. However, thus far we have not been able to link the toxicity of these compounds to a cholinergic mechanism. Pre-incubation studies suggest that significant hydrolysis of these compounds to TFKs does not occur or 22 h at pH 7.7 or 5.5.
Future work will be directed towards TFKs that have better pharmacokinetic properties. Work will also be directed at synthesis of oxime and hydrazone TFK isosteres to determine the mechanism of action of these compounds.
Master of Science
44
Júnior, Paulo de Sousa Carvalho. "Pharmaceutical salts of the antidepressants Paroxetine and Fluoxetine, selective serotonin reuptake inhibitors: crystal engineering, solid-state characterization and thermodynamic aspects." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-27012017-084846/.
Abstract:
The development of new solid forms of active pharmaceutical ingredients (API) is relevant both from fundamental as well as industrial perspectives. To this end, Crystal Engineering plays an ever-increasing important role in pharmaceutical sciences. Among the crystal engineering strategy, salt formation is the most important and implemented approach. The salt forms of API could be used to modulate and tuned the solubility and stability of API to provide optimal practical uses. Herein, we report pharmaceutical salts of two Selective Serotonin Reuptake Inhibitor antidepressants used in the treatment of depression and anxiety disorders, Paroxetine (PRX) and Fluoxetine (FLX). For this purpose, salt formers, supramolecular synthesis and crystallization protocols have been driven by the systematization of structural and supramolecular data of molecules and analogues from the Cambridge Structural Database. Paroxetine bromide hemihydrate ((PRXBr)0.5H2O), Paroxetine Nitrate hydrate (PRXNO3H2O) and two polymorphs of Fluoxetine Nitrate (FLXNO3) have obtained. All were characterized by a combination of techniques including Single Crystal X-ray Diffraction, Differential Scanning Calorimetry (DSC), Thermogravimetry analysis (TGA), Hot Stage Microscopy, Fourier transform infrared spectroscopy (IR) and solubility measurements. Since the hydration/dehydration process in APIs induces phase transitions that compromise its efficiency, the structural characterization of (PRXBr)0.5H2O help to understand its reversible dehydration process. Also, this study has implication in the understating of dehydration of isostructural PRX hydrochloride salt. Additionally, the (PRXNO3)H2O have shown the conformational flexibility and supramolecular diversity of PRX. On the other hand, the chirality of FLX is related to two nitrate salt polymorphs. A racemate and a non-centrosymmetric structure with independent enantiomers in the asymmetric unit were obtained for FLXNO3. Their packing have shown the existence of different racemic motifs, resulting in different enantiomer orientations The rare occurrence of racemic systems in non-centrosymmetric space groups becomes this event a noteworthy case. By their physicochemical properties, the polymorphs were monotropically related. The scientific contributions of this thesis show the diversity of the solid forms and define candidates to new antidepressants APIs solid formulations.O desenvolvimento de novas formas sólidas de ingredientes farmacêuticos ativos (API) é relevante tanto numa perspectiva fundamental como industrial. Para tal, a Engenharia de cristais tem desempenhado um papel importante nas ciências farmacêuticas. Dentre as estratégias, a formação de sais é a abordagem mais importante e implementada. Os sais de APIs são capazes de modular e ajustar a solubilidade e a estabilidade, a fim de proporcionar uso prático. Nesta tese, são reportados sais de dois fármacos Inibidores Seletivos de Recaptação de Serotonina, consolidados no tratamento da depressão e distúrbios de ansiedade, a Paroxetina (PRX) e a Fluoxetina (FLX). Brometo de Paroxetina hemiidratado ((PRXBr)0.5H2O), Nitrato de Paroxetina hidratado (PRXNO3H2O) e polimorfos de Nitrato de Fluoxetina (FLXNO3), síntese e protocolos de cristalização foram cuidadosamente delineados, com base na sistematização de dados estruturais e supramoleculares das moléculas e seus análogos, depositados no Cambridge Structural Database. Todos os sais foram caracterizados por Difração de Raios-X por Monocristal, Calorimetria Explanatória Diferencial (DSC), Análise termogravimétrica (TGA), Termomicroscopia, Espectroscopia vibracional na região do infravermelho (IR) e solubilidade. Considerando que a hidratação/desidratação induz mudanças de fases que comprometem a eficiência do API, a caracterização do (PRXBr)0.5H2O auxiliou no entendimento do processo de desidratação reversível que ocorre para esse fármaco. Estas mudanças de fase resultam também em implicações sobre a compreensão do processo de desidratação do sal isoestrutural de cloreto de PRX hemiidratado. Além disso, por meio da elucidação estrutural do (PRXNO3)H2O, foi possível analisar a diversidade conformacional e supramolecular da PRX. Quanto à FLX, verificou-se que sua quiralidade está relacionada com seu polimorfismo. Um racemato e uma estrutura não centrossimétrica com dois enatiômeros independentes na unidade assimétrica foram obtidos para o FLXNO3. A comparação destas estruturas permitiu mostrar a existência de arranjos supramoleculares racêmicos, constituídos por diferentes orientações de enatiômeros. A rara ocorrência de sistemas racêmicos em grupos espaciais não-centrossimétricos tornou este evento um caso notável. A partir das propriedades físico-químicas, os polimorfos puderam ser monotropicamente relacionados. Os resultados desta tese trazem importantes contribuições científicas para diversidade de formas sólidas e também define novas formulações sólidas para utilização como antidepressivos.
45
Wooldridge, Lydia Katherine. "Supplementing Bovine Embryo Culture Media to Improve the Production and Quality of In Vitro Produced Bovine Embryos." Diss., Virginia Tech, 2020. http://hdl.handle.net/10919/105143.
Abstract:
Initial studies in this work explored the role of interleukin-6 (IL6) and leukemia inhibitory factor (LIF) in preimplantation bovine embryos. Neither cytokine affected the total percentage of embryos which developed to the blastocyst stage in vitro. However, supplementation of IL6 increased blastocyst inner cell mass (ICM) cell number without affecting trophectoderm (TE) cell number. Additionally, we found that IL6 activated signal transducer and activator of transcription 3 (STAT3) specifically within ICM cells. LIF, however, did not affect ICM cell number or activate STAT3 in ICM cells, and was not pursued further. This increase in ICM cell number by IL6 was largely comprised of hypoblast (GATA6+:NANOG-) cells, and most IL6-responsive cells in day 9 blastocysts were hypoblast cells (as measured by STAT3 activation). However, some epiblast (NANOG+) cells were also IL6-responsive, and IL6 appeared to initially slow epiblast differentiation. Finally, IL6-treated blastocysts also had increased transcripts of hypoblast/primitive endoderm (PE) markers. These results indicate that IL6 may improve pregnancy retention of IVP embryos by improving yolk sac development, but further work is needed to confirm this theory.
Activation of STAT3 by IL6 could be blocked with a chemical Janus kinase 2 (JAK2) inhibitor (AZD1480). JAK2 inhibition from day 5 to 8 resulted in blastocyst ICMs with fewer than 10% the normal cell number, regardless of IL6 supplementation. This indicates that STAT3 is critical for bovine ICM development. Further analysis revealed that inhibition of JAK2/STAT did not prevent ICM formation but disrupted its maintenance.
Additionally, we assessed the suitability of zinc sulfate and a bovine embryonic stem cell culture media (TeSR) for improving bovine embryo development in vitro. Zinc sulfate increased day 8 blastocyst total and ICM cell number. Therefore, zinc sulfate appears to improve blastocyst quality. The TeSR medium improved embryo development beyond day 8. In normal synthetic oviduct fluid, blastocysts degenerated after day 8, while blastocysts moved to TeSR had greatly increased cell numbers, and even exhibited PE migration out from the ICM, a phenomenon that has not been reported in vitro. This indicates that extended blastocyst culture is possible with TeSR media.Doctor of Philosophy
Bovine embryos have been produced in vitro for the purpose of being transferred to recipient cattle to produce a calf since the 1980s. This practice allows cattle breeders to increase the number of offspring from their best females each year, and also allows for more rapid progress in generational genetic improvement. However, only approximately 10% of bovine oocytes survive and produce a calf. This poor efficiency of bovine in vitro embryo production negatively impacts the procedure's widespread use. A significant portion of these embryo losses are likely a result of inadequate in vitro culture conditions, particularly of the embryo culture media, the fluid in which embryos are grown. This media is often called "synthetic oviduct fluid," or SOF, because it is designed to mimic the fluid present in the cow's oviduct, where the embryo would normally reside. However, SOF is much simpler in nature than actual cow oviduct fluid, and this leads to reduced embryonic survival of in vitro produced embryos. Unfortunately, we know very little of what molecules control and promote bovine embryo development. Therefore, one major goal of bovine embryo research is to identify these factors and add them to SOF. The goal of this work was to examine the ability of three molecules, interleukin-6 (IL6), leukemia inhibitory factor (LIF), and zinc sulfate, to increase the number and quality of blastocysts produced through in vitro culture techniques. Additionally, I tested the replacement of SOF with a complex cell culture media, known as TeSR. This medium is more complex than SOF, and therefore should better promote embryo development. This work revealed that IL6, but not LIF, improves in vitro produced (IVP) bovine blastocyst quality. Unfortunately, neither IL6 nor LIF affected the percentage of embryos which survived to the blastocyst stage. However, IL6, but not LIF, increased the number of cells in the inner cell mass (ICM) of the blastocysts. ICM cells are the portion of the embryo which will produce the future calf. IVP bovine embryos are known to have fewer cells than normal, in vivo derived, blastocysts, and this issue is believed to cause some embryonic death after embryo transfer. Therefore, treatment with IL6 may increase the percentage of embryos which will survive after transfer and produce a calf. We also found the addition of zinc sulfate to SOF to benefit embryo quality. None of the concentrations of zinc significantly improved the percentage of embryos which survived to the blastocyst stage, but 2 µM zinc did increase ICM cell number. Like IL6, this may improve embryo survival after transfer. The use of the TeSR media as a replacement for SOF had some benefits. Unfortunately, this media is unusable for producing embryos for transfer to recipients, as we discovered early embryos could not survive in the media. However, blastocyst-stage embryos thrived in it, and could be cultured in vitro for a longer period of time as a result. Therefore, this media will be a useful tool for studying bovine embryo development in vitro, however it is unlikely to benefit calf production. In summary, this work provides evidence that zinc sulfate and IL6 are beneficial additions to SOF. However, future work is needed to determine if embryos produced with these factors are more able to produce a calf. Additionally, we discovered that TeSR is a superior extended blastocyst culture medium.
46
Dimberg, Lina. "Apoptosis Regulation in Multiple Myeloma." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7099.
47
SANTORO, Marco. "THE ANTINEOPLASTIC ROLE OF STAT5 INHIBITION IN BCRABL1-POSITIVE CELLS EXPOSED TO PIMOZIDE ALONE AND IN COMBINATION WITH DASATINIB AND PONATINIB." Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/514733.
Abstract:
EVEN though the last decades have seen the success of the targeted treatments for BCRABL1-positive Chronic Myeloid Leukemia (CML), with the outstanding achievement of placing selected patients into the so called treatment-free remission, a minor part of these subjects face TKI resistance and/or intolerance. Resistance is a challenging point in the clinical management of CML, occurring in approximately 10–20% of CML cases, due to several mechanisms, among which point mutations of the BCR‐ABL kinase domain, BCRABL overexpression or alternative splicing, sub-efficient plasma concentration of the inhibitor and abnormal drug efflux/influx.
The Signal Transducer and Activators of Transcription (STAT) proteins are a family of transcription factors commonly involved in multiple intracellular tasks and pathways, among which survival and proliferation. The STAT family is made up of seven factors, each one with a specific role. In particular, STAT3 and STAT5 (A and B) are strictly involved in cell survival and proliferation and thus are commonly implied in the pathogenesis of many neoplasms. STAT5 is of peculiar interest for its critical role in cellular differentiation, adipogenesis, oncogenesis, immune function and is known to be constitutively activated after the BCRABL1 effect in CML cells. STAT5 expression has been strictly linked to BCRABL1 mutations and disease progression to accelerated and blast phase and may thus represent a significant target to overcome resistance to TKI in CML.
In 2011, Nelson et al. evidenced the effect of STAT5 inhibition exerted by a Pimozide, a commonly used neuroleptic drug. As far as we know, literature reports experiments involving STAT5 inhibitors in association with BCRABL inhibition only with first generation inhibitor imatinib. There is no news about the association between STAT5 inhibitors and newer generations of TKIs.
The aim of this study is to explore the antineoplastic role of the STAT5 inhibitor Pimozide in association with 2nd and 3rd generation TKIs, dasatinib and ponatinib respectively, and to identify the cytotoxic in vitro concentrations.
For the purpose of the study, K562 cell line was used to simulate the frame of a classical Chronic Myeloid Leukemia disease. The cytotoxic effect was evaluated by the Trypan blue dye exclusion test. K562 cell lines were exposed to pimozide alone and in association with ponatinib and dasatinib at different concentrations to explore the drugs association effect and the in vitro cytotoxic concentrations.
Pimozide showed a synergic effect when associated with ponatinib and dasatinib in survival inhibition of K562 cell lines. This results are of note and pave the way for a possible in vivo associations. Moreover, an extension of the present study is evaluating the cytotoxic effect on CML leukemic stem cells (LSC), aiming at revealing whether STAT5 inhibition may determine a more profound effect on CML disease and may overcome the LSC escape mechanisms on which TKIs are not active.
48
Ball, Sarah Lynnette. "Small molecule inhibitors, LLL12 and celecoxib, effectively inhibit STAT3 phosphorylation, decrease cellular viability and induce apoptosis in medulloblastoma and glioblastoma cell lines." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1298906960.
49
Albrengues, Jean. "Rôle de la cytokine Leukemia Inhibitory Factor (LIF) dans l'activation et le maintien des fibroblastes pro-invasifs lors de la carcinogénèse." Thesis, Nice, 2014. http://www.theses.fr/2014NICE4107/document.
Abstract:
Le stroma inflammatoire joue un rôle primordial lors de la carcinogénèse. Dans ce contexte, nous montrons que la cytokine LIF est à l'origine d'une population de fibroblastes capable de remodeler la matrice extracellulaire de manière à la rendre permissive à l'invasion collective des cellules tumorales. En effet, nous montrons que la production de LIF par les cellules tumorales et fibroblastiques, après une stimulation au TGFβ, va réguler les capacités contractiles et pro-invasives de ces dernières via la régulation du cytosquelette d'acto-myosine et de manière indépendante de l'expression de α-SMA. En effet, l'inhibition pharmacologique des kinases JAKs permet de bloquer l'environnement fibrotique des tumeurs et d'ainsi bloquer l'invasion des cellules tumorales in vitro et in vivo. Nous montrons ensuite que LIF est à l'origine d'un switch épigénétique responsable de l'activation constitutive de la voie de signalisation JAK1/STAT3. Ce processus, régulé par la forme acétylée de STAT3, et son interaction avec l'ADN methyltransférase DNMT3b permet l'hypermethylation du promoter de la phosphatase SHP1 et donc la phosphorylation constitutive de JAK1. Une fois mis en place, ce nouveau profil de méthylation est maintenu par DNMT1. La surexpression de LIF dans les carcinomes humains corréle avec un environnement fibrotique, la présence de nodules invasifs et un mauvais pronostic clinique. De même, il existe une forte corrélation négative entre l'acétylation de STAT3 et l'expression de SHP1 dans le stroma tumoral. Nos résultats montrent qu'inhiber l'activité des DNMT et des kinases JAK permet de reprogrammer les capacités pro-invasive des fibroblastes associés aux carcinomesSignaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. We identify LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin expression. We demonstrate that a pulse of transforming growth factor β (TGF-β) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion. Indeed, pharmacological inhibition of JAK activity by counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. We next unveil that LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signaling, which results in sustained pro-invasive activity of fibroblasts. The process is mediated by p300-histone acetyltransferase acetylation of STAT3, and DNA methyltransferase DNMT3b, which induce the hypermethylation of SHP1 phosphatase promoter and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signaling is maintained by DNMT1. Accordingly, carcinomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Moreover, we show that STAT3 acetylation and phosphorylation are inversely correlated with SHP1 expression in tumors stroma. Combined inhibition of DNMT activities and JAK signaling results in long-term reversion of CAF-associated pro-invasive activity and restoration of the wild-type fibroblast phenotype
50
Wu, Gengshu. "Lipoprotein lipase : mechanism for adaptation of activity to the nutritional state." Doctoral thesis, Umeå : Umeå univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-175.