Chemical genetic analysis of Alepidea amatymbica, a traditional medicine in Africa

Natural products are a reliable source of drug leads, with plant natural products contributingsome of modern medicine’s most important pharmaceuticals. Africa has a rich source ofmedicinal plants that have not yet been heavily investigated for their therapeutic potential. Onesuch plant is Alepidea amatymbica. The traditional use of A. amatymbica suggests a broadrange of bioactivity that has yet to be fully explored. In this thesis, the mechanism of action ofA. amatymbica extract and semi-purified compounds were explored using chemical genetics.

Using the genetic model Saccharomyces cerevisiae (Baker’s yeast), initial phenotypicscreening of the crude extract and the semi-purified compound B showed that A. amatymbicais a potential substrate of the pleiotropic drug response system. A genome-wide analysis, usingthe haploid deletion collection in the pdr1∆pdr3∆ background, revealed five genes that whendeleted showed significantly reduced growth in the presence of A. amatymbica. These wereDin7, Ura5, Eft2, Glo2 and Get5. The functions of the first four genes are mitochondrialgenome stability, de novo pyrimidine biosynthesis, ribosomal translocation and a functionrelating to glyoxalase system, respectively. The most sensitive gene, Get5, is a member of theguided-entry tail anchored protein (Get) complex involved in tail anchor (TA) proteinbiosynthesis. Upon further investigation, we found the entire Get family, as well as theirinteracting chaperones, to be bioactive in A. amatymbica. Further evaluation of the Getpathway was conducted by overexpressing Get3 in pdr1∆pdr3∆get1∆ and pdr1∆pdr3∆get2∆coincidently with A. amatymbica extract treatment, where we found that Get3 overexpressionconfers sensitivity to A. amatymbica. A proteomic analysis using a GFP library was conductedto investigate the mislocalization of TA protein when the cells were treated with A. amatymbicaextract, whereby changes in localization of ER proteins Erg9 and Cyb5 were detected.

Together, these results identified genes, proteins and pathways involved in buffering theactivity of A. amatymbica extract.