Oral Presentation 24th Annual Lorne Proteomics Symposium 2019

Combining chemical biology tools with metabolomics to identify small molecule targets of peroxide antimalarials (#24)

Carlo Giannangelo 1 , Dovile Anderson 1 , Ghizal Siddiqui 1 , Jonathan Vennerstrom 2 , Susan Charman 1 , Darren Creek 1
  1. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic, Australia
  2. University of Nebraska, Lincoln, Nebraska, USA

 

Malaria causes 445 000 deaths annually and threatens approximately 40% of the world population. The malaria parasite has developed resistance to most approved antimalarials, and treatment currently relies on peroxide antimalarials. New synthetic peroxides (OZs) are now in clinical trials and early clinical usage, but their mechanism(s) of action remain poorly defined. It is proposed that iron-mediated peroxide cleavage generates free radicals that alkylate a range of targets within the parasite. The aims of this study were to use metabolomics to reveal the small molecule targets of peroxide antimalarials, and to develop a Click Chemistry-based pull-down approach to enhance the sensitivity of their detection.

 The untargeted metabolomics analysis of OZ-treated parasites, based on methanol extraction and HILIC LC-MS, demonstrated depletion of a subset of small peptides that indicated disruption of haemoglobin metabolism, but did not reveal any alkylated metabolites. A second untargeted metabolomics analysis using acidic acetone extraction and reversed-phase LC-MS revealed several novel metabolite features in the treated parasites, which were identified as covalent adducts of OZ-derived free radicals with haem, in addition to several novel degradation products arising from this OZ-haem adduct.

 A novel chemical biology method was then developed to improve the sensitivity of detection for alkylated metabolites. Azide-modified OZ analogues were incubated with parasites and enriched with photocleavable Click Chemistry beads, followed by metabolomics analysis with high resolution LC-MS. Our novel enrichment approach successfully pulled-down azide-OZ analogues and their metabolites from a parasite extract using copper-free Click Chemistry. Photocleavage with UV irradiation then allowed release of the ‘clicked’ compounds for detection by LC-MS and identification by the IDEOM software with minor modifications. In conclusion, we have developed a novel approach for the identification of drug-derived metabolites and demonstrated that haem is the major small molecule target of OZ-derived radicals in the malaria parasite.