Discovering Drug Targets in Trypanosoma brucei by Thermal Proteome Profiling

Kinetoplastid pathogens (such as Trypanosoma brucei, Trypanosoma cruzi, and Leishmania species) infect millions of people yearly causing a significant health burden worldwide. To better treat these diseases, improved therapeutics are needed. However, many candidates in drug discovery pipelines have an unknown mode-of-action (MOA), limiting their development. Hence, there is a great need for methods to identify the MOA of these drug leads. We have adapted a Proteome Integral Solubility Alteration assay, a variation of thermal proteome profiling, to identify drug targets in Trypanosoma brucei. This assay identifies likely drug targets by detecting proteins with altered stability due to drug treatment (see figure). To validate this approach for identifying a drug's MOA, we used eflornithine that is a known ornithine decarboxylase (ODC) suicide inhibitor. We initially identified approximately 16,300 peptides and 1,400 protein groups per sample, indicating we obtained about 17% proteome coverage. Off-line fractionation was optimized to increase peptide and proteome coverage. Criteria used to classify proteins as potential drug targets include 3 or more peptides per protein with p-values < 0.05 (2-sided Student's T-test) in the same direction when comparing drug to vehicle-control treatments. Along with ODC being identified as an eflornithine target, other potential drug targets identified include aspartate aminotransferase and tryparedoxin peroxidase. These may be additional direct or indirect drug targets as eflornithine is an amino acid analog and trypanothione synthesis is downstream of ODC. We are applying this methodology to identify the MOA for hit compounds identified in phenotypic screening assays. We expect this assay will improve drug development pipelines for Trypanosoma brucei and related kinetoplastid diseases.