abstract: Quiescin Sulfhydryl Oxidase 1 (QSOX1) generates disulfide bonds in its client substrates via oxidation of free thiols. Localized to the Golgi and secreted, QSOX1 helps to fold proteins into their active form. Early work with QSOX1 in cancer began with the identification of a peptide from the long form of QSOX1 in plasma from patients with pancreatic ductal adenocarcinoma. Subsequent work confirmed the overexpression of QSOX1 in numerous cancers in addition to pancreatic, including those originating in the breast, lung, brain, and kidney. For my work, I decided to answer the question, “How does inhibition of QSOX1 effect the cancer phenotype?” To answer this I sought to fulfill the following goals A) determine the overexpression parameters of QSOX1 in cancer, B) identify QSOX1 small molecule inhibitors and their effect on the cancer phenotype, and C) determine potential biological effects of QSOX1 in cancer. Antibodies raised against rQSOX1 or a peptide from QSOX1-L were used to probe cancer cells of various origins for QSOX1 expression. High-throughput screening was utilized to identify 3-methoxy-n-[4(1pyrrolidinyl)phenyl]benzamide (SBI-183) as a lead inhibitor of QSOX1 enzymatic activity. Characterization of SBI-183 activity on various tumor cell lines revealed inhibition of viability and invasion in vitro, and inhibition of growth, invasion, and metastasis in vivo, a phenotype that was consistent with QSOX1 shKnockdown cells. Subsequent work identified 3,4,5-trimethoxy-N-[4-(1-pyrrolidinyl)phenyl]benzamide (SPX-009) as an SBI-183 analog with stronger inhibition of QSOX1 enzymatic activity, resulting in a more potent reduction in tumor invasion in vitro. Additional work with QSOX1 shKnockdown and Knockout (KO) cell lines confirmed current literature that QSOX1 is biologically active in modulation of the ECM. These results provide evidence for the master regulatory role of QSOX1 in cancer, making it an attractive chemotherapeutic target. Additionally, the small molecules identified here may prove to be useful probes in further elucidation of QSOX1 tumor biology and biomarker discovery. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2020

<p>A successful flaviviral life cycle involves several coordinated events between viral proteins and host factors. The polyprotein processing at the surface of the ER membrane results in the formation of several replication proteins that bring about changes in the ER membrane making it permissive for viral genome amplification. Non-structural proteins 4A (NS4A) and non-structural protein 4B (NS4B) are two of the most important integral membrane proteins of DENV that are essential part of the viral replicase complex. The cleavage at NS4A-2K-NS4B is temporally and spatially regulated. The cleavage at the N-terminal of 2K is carried out by viral NS2B/3 protease while host signalase cleaves on the C-terminal side at the ER lumen to give rise to a mature NS4B protein. This thesis primarily focuses on demonstrating the function of 2K as an independent peptide rather than simply a signal sequence, and the role 2K plays, when present as 2K-NS4B vs NS4B. Moreover, this thesis has attempted to explore the function of transmembrane domains (TMDs) in replication separating them from their membrane anchor function. This thesis will also describe the development of a ZIKV replicon and its use in screening small molecule inhibitors in the last chapter.</p><p>In Chapter 2 of the thesis, we established 2K as an independent, information carrying peptide rather than just a signal peptide. A strategy involving chimeric virus generation and mutational analysis supported the notion that 2K is rather unique and important for viral replication and infectious particle production. Using an interserotypic 2K chimeric virus, it was established that the 2Ks of DENV are serotype specific, however, they are interchangeable with a huge fitness cost in infectious particle production. We further showed that individual amino acid residues towards then end of h-region and C-terminus of the 2K peptide affect viral replication and infectious particle production. Moreover, it was shown that the 2K peptide consists of a highly conserved ‘DNQL’ region at its N-terminal that plays an important role in viral replication.</p><p>Chapter 3 details the mechanistic aspect of the effects observed in interserotypic 2K chimeric viruses. The interserotypic chimeric viruses were comparable to wild type in replication, however, they were deficient in infectious particle production early in the life cycle. The major change to be noted in the chimeric viruses was the absence of signalase cleavage at the 2K-NS4B junction. We demonstrated that in a virus infected system, 2K-NS4B and NS4B populations are always present which led us to look for any specific functions of the cleaved vs uncleaved 2K-NS4B protein. Using a transcomplementation system where NS4B was presented in the absence of 2K, we showed that particle production can be rescued in the interserotypic 2K chimeric viruses. It was further concluded using NS4B truncations that the property of NS4B to rescue particle production was concentrated in the ER luminal loop. Further, alanine scanning mutagenesis of the conserved residues of ER loop resulted in pinpointing T198 and its involvement in the early stages of viral packaging.</p><p>Chapter 4 examined the role of TMDs of NS4A and NS4B and attempted to define their roles separately from their membrane anchoring functions. Several interserotypic TMD chimeric viruses were generated to address the function of these domains. We concluded that TMD1 and TMD3 of NS4A could be replaced with partial success across the DENV serotypes, whereas, TMD2 was serotype specific. The specificity of TMD2 of NS4A is not contributed by a single amino acid and should be a function of the secondary structure formed by TMD2 as it sits on the inner leaflet of the ER membrane. We demonstrated the variable roles different TMDs of NS4B play in viral replication using a similar strategy of reverse genetics of chimeric viruses. TMD1 of NS4B was replaceable with no to minimal effect, whereas, the remaining four showed variable effect upon substitution. More importantly, we demonstrated how the reorientation of TMD5 of NS4B post NS2B/3 cleavage might vary in different serotypes of DENV using revertant virus obtained from the TMD5 interserotypic chimera. Analysis of interserotypic cytosolic and ER luminal loop chimeras of NS4B pointed to functional conservation of the cytosolic loop between DENV-2 and DENV-3, whereas, the remaining cytosolic loops and the ER loops showed variable level of defects upon substitution, suggesting their functions in serotype-dependent manner.</p><p>Chapter 5 describes the construction and characterization of a ZIKV replicon system and use of it to screen several small molecule inhibitors of the flaviviruses MTase. Several small molecule inhibitors of flavivirus N-7-MTase were designed/synthesized in Dr. Arun K Ghosh’s lab which would target the extra pocket unique to the flavivirus SAM-binding site. We analyzed the docking of a set of these compounds into MTase domain of NS5 of ZIKV, DENV and YFV and screened them for their ability to inhibit replication of ZIKV, DENV and YFV. A huge variation in the activity profile of these compounds were observed against different flaviviruses even though these compounds were targeted against the highly conserved MTase domain of flavivirus NS5. GRL-002- and GRL-004-16-MT specifically inhibited ZIKV replication with low micromolar IC₅₀ value, while these compounds showed little to no effect on DENV and YFV.<b> </b>On the other hand, compounds GRL-007-, GRL-0012- and GRL-0015-16-MT demonstrated a dual inhibitory effect against DENV and YFV albeit the CC₅₀ values of the GRL-012 and GRL-015 were concerning. Compounds GRL-007-16-MT showed broad spectrum activity against ZIKV, DENV and YFV even though it was slightly cytotoxic to Vero cells. Moreover, GRL-002-16 was inhibitory to YFV while ineffective against DENV, whereas, GRL-016-16 had the opposite effect. Our results reveal the differential efficacies of the small molecule inhibitors targeting N-7-MTase. The experimental data suggests these compounds have different cytotoxicities in different cell lines and the compounds act in a virus-specific way. Nonetheless, we were able to shortlist some potent compounds for future modifications.</p>

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No description available.

Four known small molecule uracil-DNA glycosylase (UNG) inhibitors were synthesized and tested against human melanoma cells, IgR3 and MM200. They were found to be effective against cell proliferation at micromolar concentrations and to operate through a nonapoptotic mechanism. Thus, small molecules that target UNG may be useful as potential chemotherapeutic agents against human melanoma.

An alternative molecular recognition approach was developed for sensing small molecule analytes using the differential binding of an allosteric transcription factor (TF, specifically TetR) to its cognate DNA as the molecular recognition element coupled with Förster resonance energy transfer (FRET) to yield an internally calibrated optical signal transduction mechanism. Sensors were evaluated comprising Cy5-modified DNA (FRET acceptor) with either a tdTomato-TetR fusion protein (FP-TF) or quantum dot-TetR conjugate (QD-TF) as the FRET donor by measuring the ratio of acceptor and donor fluorescence intensities (FA/FD) with titrations of a derivative of the antibiotic tetracycline, anhydrous tetracycline (aTc). A proof-of-concept FRET-based biosensor was successfully demonstrated through the modulation of FA/FD signal intensities based on varying analyte concentrations. Sensor design parameters affecting overall signal-to-noise ratio and sensitivity of the sensors are also identified. / 2020-06-03T00:00:00Z

Cette thèse décrit la synthèse de carbénoïdes métalliques et leur application vers l’activation de petites molécules et la réduction catalytique du CO2 par BH3.Le premier chapitre décrit la synthèse d’un nouveau ligand asymétrique Ph2P(S)CH2P(BH3)Ph2. Puis, les complexes monoanioniques et dianioniques de lithium et de magnésium ont pu être synthétisés. Leurs structures ont été résolves par diffraction des rayons X et calculées par DFT.Dans le deuxième chapitre, l'oxydation des complexes dianioniques Ph2P(S)CLi2P(BH3)Ph2 avec C2Cl6 et Ph2P(S)CMgP(BH3)Ph2 avec CBr4, a conduit à une insertion intramoléculaire B-H sans précédent de BH3 sur le carbone central via une espèce carbénoïde. Ensuite, le carbénoïde Ph2P(S)C(Cl)P(S)Ph2Li,fais une insertion intermoléculaire B-H avec BH3. Le mécanisme d’insertion B-H a été étudié par spectroscopie RMN et rationalisé par DFT, impliquant un état de transition avec concomitance de la rupture des liaisons B-H et C-Cl et formation des liaisons Li-Cl et C-H. Cela explique la facilité de l’insertion. Aussi, la tentative d'isolement du carbénoïde de magnésium intermédiaire a été décrite.La synthèse et la réactivité du carbénoïde bis(iminophosphoranyl) de lithium [Ph2P(NMes)C(Cl)P(NMes)Ph2Li] (Mes = 1,3,5-Me3C6H2 ) sont présentées dans le troisième chapitre. La réaction avec BH3 conduit à des espèces de boronium ClC{PPh2NMes}2BH2 et LiBH4.Le quatrième chapitre décrit l'utilisation du composé boronium comme l'un des meilleurs catalyseurs pour la réduction de CO2 par BH3. Un intermédiaire ClC{PPh2NMes}2BH{OC(O)H}, a pu être isolé et un mécanisme de cette transformation a été proposé.Enfin, au chapitre cinq, la synthèse d'une nouvelle espèce carbénoïde de lithium asymétrique (Ph2P(S)C(Cl)P(NMes)Ph2Li a été décrite. La réactivité avec BH3 a été étudiée et a donné une insertion B-H. / The thesis describes the synthesis of metal carbenoids and their application towards small molecule activation and the catalytic reduction of CO2 with borane.Chapter 1 describes the synthesis of a novel unsymmetric phosphonium-stabilized methane ligand, Ph2P(S)CH2P(BH3)Ph2. The monoanionic and dianionic lithium and magnesium derivatives were subsequently synthesized. Their electronic structures were elucidated by X-ray crystallography and DFT calculations.Chapter 2 reports the mild oxidation of the dianionic complexes Ph2P(S)CLi2P(BH3)Ph2,and Ph2P(S)CMgP(BH3)Ph2, with C2Cl6 and CBr4 respectively, which underwent an unprecedented intramolecular B-H insertion with BH3 into the central carbon via a carbenoid species, which readily dimerizes. In addition, the carbenoid Ph2P(S)C(Cl)P(S)Ph2Li, underwent a similar intermolecular B-H insertion with BH3 to form Ph2P(S)CBH2(H)P(S)Ph2. The mechanism of the B-H bond insertion was studied by NMR spectroscopy and DFT calculations, which shows the concerted bond breaking of a B-H and C-Cl bond and the bond forming of a Li-Cl and C-H bond. This accounts for the low energy required for the B-H insertion reaction. Moreover, the attempted isolation of the magnesium carbenoid intermediate was described.The synthesis and reactivity of bis(iminophosphoranyl)carbenoid Ph2P(NMes)C(Cl)P(NMes)Ph2Li (Mes = 1,3,5-Me3C6H2 )was presented in Chapter and reacted with BH3 yielding the boronium species. ClC(PPh2NMes)2BH2,Chapter 4 describes the application of the boronium species as one of the best catalyst for CO2 reduction by BH3. An intermediate ClC{PPh2NMes}2BH{OC(O)H}, was isolated and a mechanism of this transformation was proposed.Lastly, in chapter 5, the step wise synthesis of a novel unsymmetric carbenoid Ph2P(S)C(Cl)P(NMes)Ph2Li was described. The reactivity with BH3 was probed and showed a B-H insertion reaction.

Indiana University-Purdue University Indianapolis (IUPUI) / The human aldehyde dehydrogenase superfamily consists of 19 distinct genetic loci that play key roles in both health and disease. Aldehyde dehydrogenases are primarily involved in the metabolism of reactive aldehyde substrates; the ALDH1A subfamily, in particular, metabolizes retinaldehyde and is involved in a pathway regulating tissue differentiation, cell proliferation, and apoptosis. Recently, ALDH1 isoenzymes have been implicated as significant elements in cancer progression. ALDH1 activity has been used as a marker of cancer stem cells, a subpopulation of cancer stem cells with high drug resistance, proliferative potential, and ability to differentiate into multiple cell types. In accordance with this, ALDH1 activity and expression has been shown to correlate with lower survival, increased chemoresistance, and increased chance of relapse in multiple solid cancer types, including breast, ovarian, lung, and colorectal. Despite the clear relevance of ALDH1 enzymes in cancer, the specific roles of individual isoenzymes are unclear. Isoenzyme-selective small molecule modulators of the ALDH1A subfamily would allow the probing of the function of individual isoenzymes in healthy and disease states. Two ALDH1A1 inhibitors, CM38 and C10, were previously identified in a high-throughput screen. In this study, CM38, an ALDH1A1-selective inhibitor, and CM10, an ALDH1A inhibitor, were characterized using kinetic assays, structural biology, and cell culture experiments. A structure-activity relationship was built for each series, and an X-ray crystallography structure was used to determine the binding mode. These approaches allowed the investigation of the ALDH1A active site and identification of structural features that can be used to design and improve selective modulators of this subfamily. CM38 and CM10 were also tested in a breast cancer cell line to determine their efficacy in a cellular environment. While the CM38 series showed warning signs of potential off-target toxicity, members of the CM10 compound series showed excellent initial characteristics as potential chemical tools. The results of this study may be useful in the design of new chemical tools to delineate the functions of individual ALDH1 isoenzymes in cancer biology, as well as in the development of drugs to selectively target cancer stem cells.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20930号 / 理博第4382号 / 新制||理||1630(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21690号 / 医科博第94号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 川口 義弥, 教授 上杉 志成, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM