The DNA damage response is a cell-wide response that coordinates repair and cell-cycle progression. Crucial to fidelity of genetic propagation, survival, and apoptosis, dysfunctions in the response are at the root of genome instability syndromes and cancer predisposition in mammalian cells. Within the response lie hubs of coordination, called checkpoints, whose members and organization are ubiquitous amongst eukaryotes. The high conservation of these checkpoints enable the study of their dynamics by proxy via simpler model organisms. We use the budding yeast, Saccharomyces cerevisiae, to study the replication and DNA damage checkpoints --- both implicated in DNA damage repair. Using a yEGFP reporter driven by the RNR3 promoter and reporter-based synthetic genetic array analysis, we created a detector of potential checkpoint activation in response to two doses of MMS, 0.015% and 0.060% (v/v). The high-throughput screens and differential epistasis miniarray analyses (EMAPs) yield unanticipated involvement of oxidative stress response, ribosomal biogenesis, and chromatin remodelling genes.

In dieser Arbeit wurden verschiedene Ansätze verfolgt, um selektive Kinaseinhibitoren für verschiedene Ziele zu entwickeln. Ein chemisch-genetisches Verfahren, das als "analog-sensitiv" bezeichnet wird, wurde eingesetzt, um isozymselektive Inhibitoren zu erzeugen, die auf die Gatekeeper-Mutante der IP6Ks abzielen. Diese Kinasen sind für die Synthese von PP-InsPs verantwortlich, dicht geladenen und allgegenwärtigen sekundären Botenstoffen mit einer Vielzahl von Funktionen. Ein Hochdurchsatz-Screening lieferte die vielversprechende Leitverbindung FMP-201300, die wünschenswerte Eigenschaften und ein günstiges Inhibitionsprofil aufwies. Interessanterweise ergaben kinetische Messungen einen allosterischen Wirkmechanismus, während HDX-MS-Analysen auf eine Bindung von FMP-201300 in der Nähe der ATP-Stelle hindeuteten, was möglicherweise die durch die Gatekeeper-Mutation bewirkte Steigerung der Wirksamkeit erklärt. Parallel dazu wurde Photopharmakologie eingesetzt, um die Aktivität von TgCDPK1 mit hoher zeitlicher und räumlicher Präzision reversibel zu steuern. Bekannte Inhibitoren wurden in Arylazopyrazole mit guten photochemischen und photophysikalischen Eigenschaften umgewandelt. Während die Optimierung der photostationären Verteilungen eine Herausforderung blieb, ermöglichten die ausgezeichneten bioaktiven Unterschiede zwischen den beiden isomeren Formen die reversible Kontrolle der Kinaseaktivität in situ durch abwechselnde Bestrahlung mit Licht verschiedener Wellenlängen. Obwohl diese photoschaltbaren Inhibitoren auch auf CDPK1-Orthologe anderer Parasiten abzielten, führte eine sequentielle Erhöhung der Gatekeeper-Größe zu einem deutlichen Rückgang der Wirksamkeit. Wichtig ist, dass das Wachstum von T. gondii in vivo gehemmt werden konnte, mit reduzierter Wirksamkeit nach UV-Bestrahlung. / The development of potent and highly selective kinase inhibitors is a long-standing pursuit in pharmacological research. These efforts continue to provide invaluable tools to decipher the intricate cellular functions of these ubiquitous enzymes. Kinases are involved in a cornucopia of signaling events, many of which are related to widespread diseases such as diabetes and cancer. In this work, different approaches were taken to obtain selective kinase inhibitors for diverse targets. A chemical genetics method termed ‘analog-sensitive’ was employed to generate isozyme-selective inhibitors targeting the gatekeeper mutant of mammalian IP6Ks. These kinases are responsible for the synthesis of PP-InsPs, densely-charged and highly pervasive second messengers with a multitude of functions. High-throughput screening yielded the promising lead compound FMP-201300, which exhibited desirable properties and a favorable inhibitory profile. Intriguingly, kinetic measurements revealed an allosteric mode of action, while HDX-MS analysis suggested a binding of FMP-201300 adjacent to the ATP-site, potentially explaining its increase in potency conferred by the gatekeeper mutation. In a parallel effort, photopharmacology was used to reversibly control the activity of TgCDPK1 with high temporal and spatial precision. Known inhibitors were converted into arylazopyrazoles with good photochemical and photophysical properties. While the optimization of the photostationary distributions remained a challenge, excellent bioactive discrepancies between the two isomeric forms enabled the reversible control over kinase activity in situ by alternating irradiation with light of different wavelengths. Although these photoswitchable inhibitors also targeted CDPK1 orthologs from other parasites, sequential increase of the gatekeeper size led to a significant decline in potency. Importantly, the growth of T. gondii could be inhibited in vivo with reduced potency after UV irradiation.

For all living systems, there is a requirement to recycle and regulate proteins. In eukaryotic organisms this is accomplished by the proteasome. The p97 ATPase is another highly conserved and essential complex present throughout the eukaryotic cell. In Paper I we utilized UFD fluorescent substrates to address the role of p97 and cofactors in soluble proteasome degradation. Results using RNAi and Drosophila p97 mutants propose p97 to function upstream of the proteasome on cytosolic proteasome targets as an important unfoldase together with its Ufd1/Npl4 cofactors. The results implicate p97 to be important for degradation of proteasome substrates lacking natural extended peptide regions. In Paper II we focused on identifying transcription factors essential for production of proteasomal subunits and associated proteins in Drosophila S2 cells. We utilized an RNA library targeting 993 known or candidate transcription factors and monitored RNAi depleted Drosophila S2 cells expressing the UFD reporter UbG76VGFP. We identified a range of potential candidates and focused on the bZIP transcription factor Cnc-C. RNAi and qrt-PCR experiments implicated Cnc-C to be involved in transcription of proteasomal subunits. In Paper III we applied our knowledge gained from Paper I about p97 dependent substrates and set up a high-throughput microscopy screening method to potentially find inhibitors specifically targeting the p97 proteasomal sub-pathway. Utilizing UFD substrates with and without C-terminal peptide tails we determined if compounds inhibited the core proteasomal machinery or the p97 pathway specifically. Through a primary and secondary round of screening we identified several new compounds inhibiting the ubiquitin-proteasome pathway though none from our initial screening had specificity for p97. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

<p>  G Protein Coupled Receptor (GPCR) signaling plays a key role in intercellular communication and regulates many physiological processes relevant to disease. Approximately 30-40% of all FDA approved drugs target GPCR pathways, but limitations and off-target side effects remain obstacles. Regulator of G protein Signaling (RGS) proteins negatively modulate GPCR signaling by accelerating deactivation of the Gα subunit and thus represent a novel alternative to current approaches. While research on RGS proteins and how they are regulated has expanded rapidly, there are still gaps in knowledge for some members of the RGS family. One example is RGS2, which is selective for Gαq signaling. Lowered RGS2 levels are implicated in numerous diseases, and while the E3 ligase responsible for facilitating degradation of RGS2 has been identified more work needs to be done to viably drug it to enhance RGS2 protein levels. In this thesis, I explore how FBXO44, an E3 ligase substrate recognition component responsible for RGS2 degradation, interacts with RGS2 to explore approaches to inhibit degradation.</p> <p><br></p> <p>While the FBXO44-RGS2 interaction has been demonstrated previously, the degron sequence of RGS2 remained unknown. We hypothesized that FBXO44 binds RGS2 at its Nterminus and investigated this using N-terminally truncated RGS2 constructs. Our results indicated that FBXO44 binds between residues 5 and 16 of RGS2, as removal of these stabilized RGS2 against proteasomal degradation. Based on these results we designed a peptide microarray to identify important residues and properties for FBXO44 in vitro and found that Cys13 is essential for FBXO44 binding.</p> <p><br></p> <p>We also developed and optimized a high-throughput split luciferase screen to identify potential inhibitors of the FBXO44-RGS2 interaction. After forming a cell-line stably expressing tagged FBXO44 and RGS2 and optimizing assay condition, we achieved a robust assay for screening as determined by Z’-factor. <br>  </p>

No description available.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / ALDH isoenzymes are known to impact the sensitivity of certain neoplastic cells toward cyclophosphamides and its analogs. Despite its bone marrow toxicity, cyclophos-phamide is still used to treat various recalcitrant forms of cancer. When activated, cyclo-phosphamide forms aldophosphamide that can spontaneously form the toxic phospho-ramide mustard, an alkylating agent unless detoxified by ALDH isozymes to the carbox-yphosphamide metabolite. Prior work has demonstrated that the ALDH1A1 and ALDH3A1 isoenzymes can convert aldophosphamide to carboxyphosphamide. This has also been verified by over expression and siRNA knockdown studies. Selective small molecule inhibitors for these ALDH isoenzymes are not currently available. We hypothe-sized that novel and selective small molecule inhibitors of ALDH3A1 would enhance cancer cells’ sensitivity toward cyclophosphamide. If successful, this approach can widen the therapeutic treatment window for cyclophosphamides; permitting lower effective dos-ing regimens with reduced toxicity. An esterase based absorbance assay was optimized in a high throughput setting and 101, 000 compounds were screened and two new selective inhibitors for ALDH3A1, which have IC50 values of 0.2 µM (CB7) and 16 µM (CB29) were discovered. These two compounds compete for aldehyde binding, which was vali-dated both by kinetic and crystallographic studies. Structure activity relationship dataset has helped us determine the basis of potency and selectivity of these compounds towards ALDH3A1 activity. Our data is further supported by mafosfamide (an analog of cyclo-phosphamide) chemosensitivity data, performed on lung adenocarcinoma (A549) and gli-oblastoma (SF767) cell lines. Overall, I have identified two compounds, which inhibit ALDH3A1’s dehydrogenase activity selectively and increases sensitization of ALDH3A1 positive cells to aldophosphamide and its analogs. This may have the potential in improving chemotherapeutic efficacy of cyclophosphamide as well as to help us understand better the role of ALDH3A1 in cells. Future work will focus on testing these compounds on other cancer cell lines that involve ALDH3A1 expression as a mode of chemoresistance.