Malaria continues to be a significant global health challenge and resistance has emerged to all approved antimalarials. New medicines that act by novel mechanisms are urgently needed to overcome resistance, but drug target deconvolution remains a major barrier. To address this, we have developed a multi-omics pipeline to elucidate drug mechanisms and molecular targets on a system-wide scale.
A previous high throughput screen against malaria parasites identified cyclopropyl amide compounds with promising antimalarial activity and an unknown mode of action that we selected for multi-omic-based target deconvolution. We initially profiled the functional response to sublethal cyclopropyl amide treatment over 5 h by untargeted metabolomics. MMV1804508 and MMV1804743 (exemplars from this series) resulted in specific accumulation of metabolites upstream of the enzyme DHODH (N-carbamoyl-L-aspartate and dihydroorotate) and depletion of the downstream pyrimidine nucleotide products. This signature was absent for an inactive cyclopropyl compound and is consistent with functional loss of DHODH, an essential enzyme for parasite de novo pyrimidine synthesis.
To identify the direct target in parasites we applied solvent proteome profiling (SPP) on the latest Orbitrap Astral MS technology. Amongst an unprecedented ~4,500 proteins reproducibly detected from a parasite lysate, MMV1804508 selectively stabilised 8 of the 12 proteins in the parasite cytochrome bc1 complex (p<0.05), which is required to recycle the cofactor ubiquinone for the activity of DHODH. Combined, our multi-omics analysis revealed that cyclopropyl amides deprive the parasite of essential pyrimidine nucleotides by targeting the cytochrome bc1 complex to indirectly inhibit DHODH function. We further verified this mechanism via cross resistance studies in several cytochrome b and DHODH mutant parasite lines and showed that MMV1804508 inhibits cytochrome bc1 activity from a P. falciparum mitochondrial extract.
Our study demonstrates the power of MS-based multi-omics approaches to resolve complex drug mechanisms and facilitate the development of next-generation medicines.