A chemical proteomic approach in antimarial drugs discovery

We have previously identified new compounds with high in vitro efficacies against the intraerythrocytic stages of Plasmodium falciparum (P.f.) (IC50 <10 nM), but the molecular target is unknown and preliminary results indicate that indeed multiple targets may exists.(1-4) Proteomics offers a unique tool for target identification and several proteomic approaches are available. One of the most interesting is the so called “chemical proteomics”, which couples affinity purification methods with mass spectrometry and therefore permits to increase selectivity and sensitivity. Several limitations can affect the results of chemical proteomics approaches. For example, it is necessary to use tag compounds that present high activity and affinity for the target protein. Moreover, considering that LC-MS analysis is a very sensitive methodology used for identification of the target protein, non-specific protein binding could cause high background noise, therefore greatly complicating the proteomic analysis. Chemical proteomics applied to antimalarial drug discovery is a difficult task and several attempts have been made. The main problem derives from the Plasmodium which is a very small organism living inside the red blood cells and therefore only a very limited amount of protein is available and a noteworthy interference is expected from the host proteins. We present here a chemical proteomics study using a new selective approach (Figure1) that overcomes non-specific protein binding using a cleavable linker that allows the selective release of the tagged protein from the affinity beads. Furthermore, the cleavable linker containing the affinity tag (Biotin) is introduced through a click reaction after incubation with the lysate, reducing therefore the perturbation in the structure of the starting inhibitor. Preliminary results indicate that one or two specific proteins are involved in the mechanism of action of this new class of P.f. inhibitors.