Thymidine kinase-independent click chemistry DNADetect probes for DNA proliferation assessment in malaria parasites

Abstract

Metabolic chemical probes are small molecule reagents that utilise naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells however is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labelling of replicating asexual intraerythrocytic P. falciparum parasites, as determined by flow cytometry using copper catalysed azide-alkyne cycloaddition (CuAAC) to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.