MMV-390048

The limited understanding of the mechanism of action (MoA) of several antimalarials and the rise of drug resistance toward existing malaria therapies emphasizes the need for new strategies to uncover the molecular target of compounds in Plasmodium falciparum. Integral solvent-induced protein precipitation (iSPP) is a quantitative mass spectrometry-based (LC-MS/MS) proteomics technique. The iSPP leverages the change in solvent-induced denaturation of the drug-bound protein relative to its unbound state, allowing identification of the direct drug-protein target without the need to modify the drug. Here, we demonstrate proof-of-concept of iSPP in P. falciparum (Pf) lysate. At first, we profiled the solvent-induced denaturation behavior of the Pf proteome, generating denaturation curves and determining the melting concentration (C_M) of 2712 proteins. We then assessed the extent of stabilization of three antimalarial target proteins in multiple organic solvent gradients, allowing for a rational selection of an optimal solvent gradient. Subsequently, we validated iSPP by successfully showing target-engagement of several standard antimalarials. The iSPP assay allows the testing of multiple conditions within reasonable LC-MS/MS measurement time. Furthermore, it requires a minimal amount of protein input, reducing culturing time and simplifying protein extraction. We envision that iSPP will be useful as a complementary tool for MoA studies for next-generation antimalarials.

Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase β (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.