Lysine targeting covalent inhibitors of malarial kinase <i>Pf</i> CLK3

Malaria continues to devastate tropical regions of the world, with resistance to frontline drugs on the rise. Kinase inhibition has emerged as a promising novel mechanism of action in the fight against malaria. We previously reported the development of TCMDC-135051 (1), a highly potent, multi-stage inhibitor of <i>Plasmodium falciparum</i> CLK3 (<i>Pf</i>CLK3). Building on this work, we subsequently developed the first covalent kinase inhibitor for malaria, selectively targeting a unique cysteine residue. Despite their high potency and selectivity, covalent inhibitors that target cysteine residues are particularly vulnerable to resistance arising from single point mutations of the nucleophilic residue. This work presents a novel strategy targeting the essential kinase catalytic lysine residue which has the potential to evade this resistance mechanism. Using structure based drug design, analogues of TCMDC-135051 (1) targeting Lys394 of <i>Pf</i>CLK3 were developed. Four compounds, all harbouring benzaldehyde-based warheads, covalently engaged Lys394 as determined by protein mass spectrometry. These analogues were highly potent against recombinant protein, with good parasiticidal potency and cytotoxicity profiles. These molecules 4, 5, 8, 9 are the first lysine-targeting covalent inhibitors reported for malaria and offer a promising general strategy for future antimalarial drug discovery.