Date of Award
Doctor of Philosophy (PhD)
Cell-permeable small molecules that can act on their targets on fast time scales are powerful probes of cell division mechanisms and intracellular transport processes. Phenotype-based screens with chemical libraries have been used to identify such inhibitors. However, probes for most proteins are still not available, and the requirements on compound collections to yield such probes are not well understood. Here I present two approaches to find and use such probes. First, in an attempt to find probes for cell division, I have shown that a small collection of 100 diaminopyrimidines yielded a range of cell division phenotypes, including changes in spindle geometry, chromosome positioning and mitotic index. Monopolar mitotic spindles were induced by 4 inhibitors including one that targets Polo-like kinases, evolutionarily conserved serine/threonine kinases. Using chemical inhibitors and high-resolution live-cell microscopy, I found that Polo-like kinase activity is needed for the assembly and maintenance of bipolar mitotic spindles. Inhibition of Polo-like kinase destabilized kinetochore microtubules while stabilizing other spindle microtubules, leading to monopolar spindles. Second, I used pigment organelles in Xenopus melanophore cells, a specialized intracellular transport system for which the motor proteins driving movement, and primary signals regulating movement, are known, to identify inhibitors of intracellular transport. Screening of an unbiased commercial library yielded a compound, 37P11, that could be characterized as possible inhibitor of a yet unknown element in the regulation of organelle movement. From a collection of diaminopyrimidines, one compound, DAP-29, was identified that potentially targets cytoplasmic dynein, an essential motor protein in many transport processes. Both compounds showed activity in assays in a variety of other intracellular transport systems, highlighting their potential use as biological probes. My findings indicate that development of new screening systems and further testing of compounds based on „privileged scaffolds‟, such as diaminopyrimidines, can lead to powerful new probes for cell division and intracellular transport.
Peters, Ulf, "Small Molecules as Probes for Cell Division and Intracellular Transport" (2008). Student Theses and Dissertations. 202.