The cellular thermal shift assay (CETSA) provides a means of understanding the extent to which a small molecule ligand associates with a protein target of therapeutic interest, thereby inferring targe...

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is widely valued for its speed and sensitivity in biomolecular analysis, yet the inherently nonquantitative nature hampers its use in many applications including high-throughput screening. Here, we introduce an iodo-based labeling strategy that enables accurate quantification of peptides and peptide libraries using high-resolution MALDI FT-ICR MS. The peptides are coupled at the N-terminus with benzoic acid (BA) or 4-iodobenzoic acid (IBA) to generate the analyte and its internal standard, respectively, differing only by a single iodine substitution. This new labeling strategy was first validated using a simple four-peptide mixture, and subsequently applied to quantitatively evaluate glycine-zipper peptide libraries containing up to 125 members for the discovery of bacterial-binding peptides. Screening of these libraries against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis revealed peptides with strong and selective interactions with the bacteria. This universally applicable, cost-effective, and straightforward approach for peptide quantification significantly enhances the reliability and accuracy of high-throughput peptide screening via MALDI FT-ICR MS.

The high burden of malaria and growing resistance to frontline antimalarials demand new drug target combinations with reduced propensities for conferring parasite resistance. An attractive approach for circumventing antimalarial drug resistance is target repurposing, in which known drugs that act through protein targets of human origin that are also active against the human malaria parasite Plasmodium falciparum are exploited to identify novel antimalarial drug targets. Here, we show that the human checkpoint kinase 1 (Chk1) inhibitor CHIR-124 is active in vitro against both drug-sensitive and drug-resistant asexual blood stage parasites and competitively binds to several Plasmodium kinases. The compound also shows moderate activity against both the liver and gametocyte forms of the parasite. Further target investigation of CHIR-124 via conditional knockdown experiments confirmed that P. falciparum Aurora-related kinase 1 (PfArk1) is implicated in its parasiticidal activity. Notably, CHIR-124 also inhibits β-hematin (synthetic hemozoin) formation and causes a dose-dependent increase in free heme that correlates with inhibition of parasite growth. These findings suggest that polypharmacology is involved in the activity of CHIR-124 against P. falciparum via the dual inhibition of Plasmodium PfArk1 and hemozoin formation, both essential for parasite proliferation. This is further supported by in vitro drug combination experiments, morphological studies, and resistance generation attempts. This study validates the feasibility of dual Plasmodium kinase/hemozoin formation inhibitors active against resistant strains with decreased resistance risks in the fight against malaria.