Determining the protein target(s) and mechanism(s) of drug candidates found in phenotypic screens is critical to subsequent structure-activity-based development and optimization, but existing methods for target identification are limited. Here we present a method that applies directed evolution to a genetically engineered, drug sensitive Saccharomyces cerevisiae strain. Whole genome sequencing of yeast clones that have evolved drug resistance, in concert with in vitro cell free assays and computer modeling, can be a useful tool for target identification and binding site characterization.
To demonstrate the ease and utility of this method, we applied it to the identification of the molecular target and binding site of a range of cytotoxic molecular compounds with activity against eukaryotic pathogens and human cancers. These studies include known drug target combinations, as well as application to experimental compounds with unknown drug targets. As proof of concept, the method correctly identified the precise binding pocket of the protein synthesis inhibitor, cycloheximide, as the ribosomal protein Rpl28. We also correctly identified topoisomerase II inhibitor as the target of the human cancer chemotherapeutic, etoposide.
We next used the method to identify novel drug target combinations, which were then validated using a combination of genetic, biochemical, structural and chemical structure activity relationships (SAR)-based assays. We identified a p-type ATPase, ScPma1, as the target of the spiroindolone antimalarials, of which KAE609 is currently in stage 2b clinical trials. We determined that the pre- clinical phenyl-amino-methyl-quinolinols (PAMQ) antimalarials inhibit the cyclic AMP signaling pathway, a mechanism of action that is different from existing commercial antimicrobials. We also demonstrated that MMV001239, a compound with antitrypanosomal activity, targets ScErg11, the yeast homolog of the T, cruzi Cyp51p, and a protein crucial for ergosterol biosynthesis. Taken together, our approach expands on the number of tools available for analyzing compound-target interactions and can be applied to studies of other eukaryotic antimicrobials and chemotherapeutics.
|Advisor:||Winzeler, Elizabeth A.|
|Commitee:||McCammon, James A., Nizet, Victor, Rifkin, Scott, Spector, Deborah|
|School:||University of California, San Diego|
|School Location:||United States -- California|
|Source:||DAI-B 79/01(E), Dissertation Abstracts International|
|Subjects:||Biology, Genetics, Medicine|
|Keywords:||Chemogenomics, Drug discovery, Drug target, Malaria, Yeast|
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