An emerging technique for the identification of protein targets for novel drug candidates is Thermal Stability Proteomics (TSP). Thermal Stability Proteomics takes advantage of the thermal stabilisation that occurs when a protein is bound by a compound. This allows target proteins to be identified from a complex proteome following a thermal challenge, and is particularly useful for compounds that act by an unknown mode of action. Our TSP methodology takes advantage of a Data Independent Acquisition Mass Spectrometry (DIA-MS) approach to protein analysis, which utilises a comprehensive spectral library of over 3,000 P. falciparum proteins to perform a highly reproducible, label-free quantification of both high and low abundance parasite proteins, more efficiently than traditional label-based proteomics methods.
We’ve applied our TSP-DIA-MS approach to antimalarial compounds. First, we validated our method with the well-studied antimalarial pyrimethamine, identifying stabilisation of its known target, dihydrofolate reductase thymidylate-synthase, from a detected proteome of 1428 proteins. Subsequently, this approach was applied to a novel class of hydroxamic acid aminopeptidase inhibitors, designed to target the M1 and M17 aminopeptidases. Stabilisation of both the M1 and M17 aminopeptidases was observed amongst a shortlist of proteins. Finally, our TSP-DIA-MS approach was applied to a novel class of aminobenzimidazoles, which possess an unknown mechanism of action. From two TSP experiments, we identified a shortlist of stabilised proteins which warrant further investigation. This study demonstrates the potential of TSP-DIA-MS as an efficient method for the identification of binding targets for novel antimalarial compounds, and how it may applicable for other target identification programs.