Malaria was responsible for over 600,000 deaths in 2020, of which more than 90% was caused by the protozoan parasite Plasmodium falciparum. Artemisinin-based combination therapies (ACTs) target the blood stage of the parasite life cycle and serve as the frontline antimalarial treatment. However, the emergence of drug resistance threatens the efficacy of ACTs. Thus, there is an urgent need to better understand the mechanism of action of ACTs and to identify novel malaria drug targets. Potential, underexplored drug targets in P. falciparum are the enzymes that mediate ubiquitination, an essential post-translational modification. The covalent attachment of ubiquitin to substrate proteins is mediated by an enzymatic cascade consisting of a ubiquitin-activating (E1), conjugating (E2), and ligase (E3) enzyme. Interestingly, previous studies have shown that deletion of the E2 PfUbc13 increases parasite sensitivity to dihydroartemisinin, suggesting that PfUbc13 has a role in ACT antimalarial activity. PfUbc13 is an essential gene and a central mediator of Lys63-linked polyubiquitin (K63-Ub) chains, also implicating this specific modification in ACT activity. Hence, targeting PfUbc13 and its interactome serves as a novel avenue for combating malaria.
The compound NSC697923, a known covalent inhibitor of HsUbc13, was identified as a covalent inhibitor of PfUbc13. Similar degrees of thermal destabilization were observed when recombinant HsUbc13 and PfUbc13 were treated with NSC697923, whereas no thermal shift was observed with the mutant protein PfUbc13-C86S. Using high-resolution mass spectrometry, the mass of the PfUbc13 covalently modified Cys86-containing tryptic peptide (929.4952 kDa) was detected. Furthermore, dose-dependent NSC697923 inhibition of PfUbc13~Ub conjugate formation was observed in Ub transfer assays monitored by anti-Ub western blots. The Ki was determined to be 0.36 μM using a fluorescence polarization assay. The NSC697923 EC50 values against the parasite blood, liver, and sexual stages were determined to be 6.7, 7.8, <10 μM, respectively. Additionally, 64 putative PfUbc13 substrate proteins modified with K63-Ub were identified through differential bottom-up proteomics. Overall, this work has identified the first-known chemical probe of PfUbc13 and provided insight into the PfUbc13 interactome, broadly informing future investigations into the role of PfUbc13 and K63-Ub in the mechanism of ACTs and furthering malaria drug discovery efforts.