<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>
High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis