Summary Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to highly specialized cell compartments and structures. These adaptations drive their recognition, nondestructive penetration, and elaborate reengineering of the host’s cells to promote their growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty. Consequently, half of apicomplexan proteins are unique and uncharacterized. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii . This provides unprecedented comprehensive molecular definition of these unicellular eukaryotes and their specialized compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.

Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to novel, highly specialized cell compartments and structures. These adaptations drive their recognition and nondestructive penetration of host's cells and the elaborate reengineering of these cells to promote growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty that defines these new cell organizations and their functions. Consequently, half of apicomplexan proteins are unique and uncharacterized, and these cells are, therefore, very poorly understood. Here, we determine the steadystate subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition to these cells and their novel compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECHsalicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.