Identification and characterization of small molecule inhibitors of the hedgehog pathway acting downstream of smoothened
- Ari Joseph Firestone.
- Nov. 2011.
- Physical description
- online resource (xvii, 156 pages) : illustrations (some color)
- Firestone, Ari Joseph.
- Chen, James Kenneth. thesis advisor (primary).
- Meyer, Tobias. thesis advisor.
- Nelson, W. J. (W. James) thesis advisor.
- Scott, Matthew P. thesis advisor.
- Stanford University. Department of Chemical and Systems Biology.
- Stanford University. Committee on Graduate Studies. degree grantor.
- Includes bibliographical references (p. 242-256) and other references. 138 refs.
- Proper activation of Hh signaling is a critical mediator of metazoan development whereas aberrant activation, in post-embryonic tissues, is associated with initiation or progression of numerous common tumors. In chapter 1 of this dissertation I review the history, development, and status of extant small-molecule Hh inhibitors. I discuss limitations in the current approaches utilized in the discovery of these compounds, and the successes and challenges associated with tackling this pathway with targeted therapeutics. In chapter 2, I detail our progress using high-throughput screening to discover new small-molecule inhibitors of Hh signaling by specifically targeting elements of the pathway downstream of the transmembrane protein Smoothened (Smo), the target of the vast majority of existing inhibitors. These efforts resulted in the identification and preliminary characterization of four small molecules, each of which displays a distinct and novel combination of effects on various downstream signaling processes including formation of the primary cilia, the Hh-dependent regulation of Gli proteolytic-processing, and activation of Gli transcription factors. These compounds therefore represent valuable probes for further dissection of mechanisms of Hh signaling downstream of Smo and possible leads in the development of mechanistically novel Hh targeted chemotherapeutics. The third chapter continues with an in depth study of the Hh antagonist capable of preventing formation of primary cilia. We name this compound and its structurally and functionally related congeners ciliobrevins. We observe that acute treatment of cells with ciliobrevins leads to an accumulation of proteins in the distal tips of primary cilia, a phenotype that is consistent with inhibition of dynein-dependent retrograde protein trafficking. Ciliobrevin treatment also causes defects in mitotic spindle formation and the subcellular movement of organelles that are reminiscent of those observed under dynein loss of function conditions. Finally we show that ciliobrevins inhibit dynein-dependent microtubule gliding and dynein ATPase activities in vitro, demonstrating that this motor protein represents the critical physiological target of these small-molecule Hh inhibtors. This dissertation is concluded by a broader review of the use and development of small-molecules to control additional developmental signaling pathways and processes such as differentiation and self-renewal. Alterations in fate choice are central to a plethora of human diseases and chemically manipulating these will be a critical aspect in the advancement of medical technology from the treatment of tumors, to the advent of regenerative therapies.
- Publication date
- Submitted to the Department of Chemical and Systems Biology and the Committee on Graduate Studies of Stanford University.
- Thesis (Ph.D.)--Stanford University, 2011.