Many drug discovery programs are limited by a lack of understanding about the protein target responsible for pharmacological activity. This is a major issue for drug candidates that were identified by phenotypic screening, and is also important for target-based drug discovery programs, where confirmation of on-target activity is often critical to progression into clinical trials. Recent advances in proteomics technologies have provided opportunities to identify targets of test compounds in a non-biased manner, and in combination with metabolomics approaches, can often identify and/or confirm relevant mechanistic targets within the cellular context. Our work has combined chemo-proteomics, thermal stability proteomics and limited proteolysis proteomics, with untargeted proteomics and metabolomics assays, to reveal the mechanisms of action for promising antimalarial drug candidates.
Untargeted proteomics, peptidomics and metabolomics, along with chemoproteomics and targeted thiol metabolomics revealed that haemoglobin digestion and redox pathways were primarily targeted by the current first-line artemisinin antimalarials. This approach also demonstrated mechanistic similarities between artemisinins and newer peroxide antimalarials under development.
Investigation of a new series of hydroxamic acid-based compounds, which display excellent antimalarial activity in vitro and in vivo, demonstrated specific inhibition of the P. falciparum M1 and M17 aminopeptidases. Thermal Stability Proteomics - which uses mass spectrometry to detect ligand-induced thermal stabilization of proteins in a parasite lysate – was applied to the potent inhibitor, MIPS-1778, and revealed both M1 and M17 as the primary targets among ~1400 proteins. Analysis of more selective inhibitors revealed M1 as the only aminopeptidase stabilized for MIPS-2673, and M17 was the only target identified for MIPS-2571, which is consistent with the Ki values from biochemical enzyme inhibition assays. Untargeted metabolomics analysis also confirmed the inhibition of aminopeptidases by these compounds, observed as unique signatures of peptide accumulation for each of the M1 and M17 inhibitors. Limited proteolysis mass spectrometry (LiP-MS) was also performed for MIPS-2673, confirming selective inhibition of the M1 aminopeptidase.
The combination of metabolomics and proteomics approaches revealed mechanisms of action for peroxide-based antimalarials that exhibit polypharmacology, as well as for aminopeptidase inhibitors that inhibit specific targets. This new knowledge will support the further development of novel drugs for malaria and inform strategies to overcome drug resistance.