Toward Molecularly Targeted Therapies for Renal Disease
dc.contributor.author | Grimley, Edward | |
dc.date.accessioned | 2017-10-05T20:26:29Z | |
dc.date.available | NO_RESTRICTION | |
dc.date.available | 2017-10-05T20:26:29Z | |
dc.date.issued | 2017 | |
dc.date.submitted | ||
dc.identifier.uri | https://hdl.handle.net/2027.42/138499 | |
dc.description.abstract | Pax2 is a developmental control gene that is essential for organogenesis of the kidney and urogenital tract. Genes of this nature are generally suppressed in healthy adult tissues. However, developmental genes and pathways are frequently reactivated in renal diseases like cancer or polycystic kidney disease. Furthermore, following acute renal injury, regenerating renal epithelial cells require reactivation of embryonic genes to drive proliferation and differentiation of the new tubular cells. Throughout this thesis, knowledge of Pax genes in renal development and disease is leveraged to generate innovative therapeutic interventions for an assortment of kidney diseases. Using a novel reporter cell line, that responds to Pax2 to drive transcription of luciferase, I developed an assay compatible with high-throughput screening (HTS). An HTS campaign of 69,125 unique small-molecules yielded 48 molecules that negatively regulate Pax2 mediated transcription activation and two molecules that positively regulate this process. In collaboration with Dr. Liao from Dr. Nikolovska-Coleska’s lab, a virtual screening campaign based on homology modeling was developed to identify small-molecules with the potential for binding to the Pax2 DNA-binding domain. This campaign yielded 227 candidate molecules that were subsequently examined in the Pax2 reporter assay. Following confirmation, counterscreen, and titration assays, five molecules remained. Analogs of these molecules yielded a more potent compound, EG1. This molecule was further characterized and shown to inhibit Pax2 DNA-binding through a direct interaction with the paired domain. Furthermore, EG1 treatment recapitulates aspects of Pax2 loss of function in an embryonic organ culture model system. Embryonic kidneys grown in the presence of EG1 exhibit defects in branching morphogenesis and have gene expression changes consistent with loss of Pax2 activity. Embryonic lungs, which undergo a Pax independent branching morphogenesis, were unaffected by EG1. Similarly, Pax2 negative cancer cells were unaffected by EG1 treatment while Pax2 positive cancer cells exhibited a significant decrease in viability. These findings suggest that I have identified a bona fide small-molecule regulator of Pax2 transcription activation. Furthermore, my findings represent an important proof of concept, demonstrating that developmental regulatory transcription factors, like Pax2, can be targeted by small-molecules and that doing so may provide an avenue for developing novel therapeutic approaches. | |
dc.language.iso | en_US | |
dc.subject | Pax2 | |
dc.subject | paired domain | |
dc.subject | drug discovery | |
dc.subject | high-throughput screen | |
dc.subject | virtual screen | |
dc.subject | kidney disease | |
dc.title | Toward Molecularly Targeted Therapies for Renal Disease | |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Molecular & Cellular Pathology | |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | |
dc.contributor.committeemember | Dressler, Gregory R | |
dc.contributor.committeemember | Barolo, Scott E | |
dc.contributor.committeemember | Fearon, Eric R | |
dc.contributor.committeemember | Figueroa, Maria | |
dc.contributor.committeemember | Nikolovska-Coleska, Zaneta | |
dc.subject.hlbsecondlevel | Biological Chemistry | |
dc.subject.hlbsecondlevel | Genetics | |
dc.subject.hlbsecondlevel | Molecular, Cellular and Developmental Biology | |
dc.subject.hlbsecondlevel | Science (General) | |
dc.subject.hlbtoplevel | Science | |
dc.description.bitstreamurl | https://deepblue.lib.umich.edu/bitstream/2027.42/138499/1/egrimley_1.pdf | |
dc.identifier.orcid | 0000-0003-0438-506X | |
dc.identifier.name-orcid | Grimley, Edward; 0000-0003-0438-506X | en_US |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
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