Physiologic Targets and Modes of Action for CBL0137, a Lead for Human African Trypanosomiasis Drug Development
Molecular and Cellular Biology
CBL0137 is a lead drug for human African trypanosomiasis, caused by Trypanosoma brucei. Herein, we use a four-step strategy to (a) identify physiologic targets and (b) determine modes of molecular action of CBL0137 in the trypanosome. First, we identified fourteen CBL0137-binding proteins using affinity chromatography. Second, we developed hypotheses of molecular modes of action, using predicted functions of CBL0137-binding proteins as guides. Third, we documented effects of CBL0137 on molecular pathways in the trypanosome. Fourth, we identified physiologic targets of the drug, by knocking down genes encoding CBL0137-binding proteins and comparing their molecular effects to those obtained when trypanosomes were treated with CBL0137. CBL0137-binding proteins included glycolysis enzymes (aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, phosphoglycerate kinase), and DNA-binding proteins (UMSBP2, RPA1, RPA2). In chemical biology studies CBL0137 did not reduce ATP level in the trypanosome, ruling out glycolysis enzymes as crucial targets for the drug. Thus, many CBL0137-binding proteins are not physiological targets of the drug. Instead, CBL0137 inhibited (i) nucleus mitosis, (ii) nuclear DNA replication, and (iii) polypeptide synthesis as the first carbazole inhibitor of eukaryote translation. RNAi against RPA1 inhibited both DNA synthesis and mitosis, whereas RPA2 knockdown inhibited mitosis, consistent with both proteins being physiologic targets of CBL0137. Principles used here to distinguish drug-binding proteins from physiological targets of CBL0137 can be deployed with different drugs in other biological systems. To distinguish drug-binding proteins from physiologic targets in the African trypanosome we devised and executed a multi-disciplinary approach involving biochemical, genetic, cell, and chemical biology experiments. The strategy we employed can be used for drugs in other biological systems.
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