Using Unbiased Chemical Proteomics Approaches to Explore the Target Landscape of the Resistance Refractory 7-Azaindole MMV022224 in <i> <i>Plasmodium falciparum</i> </i>

High Resolution Image Download MS PowerPoint Slide Current standard of care, artemisinin-based therapies for malaria, are threatened by emerging drug resistance. Developing antimalarials with novel mechanisms of action and low propensity for resistance is of the highest priority. Here, we explore the target landscape of MMV022224, a promising antimalarial that is active against multiple stages of Plasmodium falciparum and refractory to resistance generation. Using two orthogonal chemical proteomics approaches, chemical pulldown and thermal proteome profiling, we demonstrate that MMV022224 binds selectively and with high affinity to the genetically essential P. falciparum protein kinase 6 ( Pf PK6), as well as to several additional Plasmodium kinases. Enzymatic studies verify that MMV022224 inhibits Pf PK6; however, Pf PK6 knockdown does not affect parasite compound susceptibility, confirming that Pf PK6 inhibition is not the sole driver of antimalarial activity and that MMV022224 may act through broader, kinase-focused polypharmacology. Employing the same chemical proteomics strategies, we demonstrate that the structurally related azaindole, TCMDC-135051, is a selective inhibitor of the cyclin-dependent kinase Pf CLK3. Collectively, these studies demonstrate the value of chemical proteomics for antimalarial drug target deconvolution.