The Malaria Box, assembled by the Medicines for Malaria Endeavor, is a set of 400 structurally diverse, commercially available compounds with demonstrated activity against blood-stage antimalarials identified from the high-throughput screening efforts of St. Box compounds were tested for activity against the formation of -hematin, a synthetic form of the heme detoxification biomineral, hemozoin. Further, the mechanism of action of these compounds within the malaria parasite was explored. Ten of the Malaria Box compounds exhibited significant inhibition of -hematin formation. In this assay, doseCresponse data revealed IC50 values ranging from 8.7 to 22.7?M for these hits, each of which is more potent than chloroquine (a known inhibitor of hemozoin formation). The antimalarial activity of these ten hits was confirmed in cultures of the chloroquine sensitive D6 strain of the parasite resulting in IC50 values of 135C2165?nM, followed by testing in the multidrug resistant strain, C235. Cultures of (D6) were then examined for their heme distribution following treatment with nine of the commercially available confirmed compounds, seven of which disrupted the hemozoin pathway. was first reported, and since then the malaria parasite continues to rapidly develop resistance to current replacement therapeutics, including sulfadoxine-pyrimethamine and artemisinin combination therapies (Abdul-Ghani et?al., 2013; Ashley et?al., 2014; Wongsrichanalai et?al., 2002). While pharmaceutical companies have lacked interest in developing new drugs for malaria, the advent of public-private partnerships (PPP’s) has facilitated collaborative efforts between pharmaceutical companies with nonprofit organizations and universities (Nwaka and ZAP70 Ridley, 2003). An exemplar PPP, Medicines for Malaria Endeavor (MMV), buy Etidronate (Didronel) was established in 1999 to enable the discovery of new, effective and affordable antimalarial drugs. Notably, MMV supported the high-throughput screening (HTS) efforts of St. Jude Children’s Research Hospital, Novartis and GlaxoSmithKline (GSK) to screen over 4 million compounds for antimalarial activity (Guiguemde et?al., 2010; Plouffe et?al., 2008; Gamo et?al., 2010). Of these, over 20,000 compounds have been identified with potent antimalarial activity. Perhaps the most impressive aspect of this collaborative discovery effort is that the structures of these chemical starting points have been deposited in the ChEMBL neglected tropical diseases archive, an Open Access screening repository that allows researchers from around the world to access this data free of charge (https://www.ebi.ac.uk/chemblntd). To encourage the broader investigation of these compounds, MMV announced free access to the compounds of the Malaria Box C a set of 400 compounds selected from the 20,000 hits that are representative of the breadth of chemical diversity and predicted to be pharmacologically valid. While these compounds are potent antimalarials, all possible drug targets have not been explored. In this report, the Malaria Box compounds have been tested for inhibitory activity against the formation of -hematin, the synthetic form of the heme-detoxification biomineral, hemozoin, followed by target buy Etidronate (Didronel) validation in a parasite culture. During the intraerythrocytic stage of the life cycle, the malaria parasite catabolizes buy Etidronate (Didronel) host hemoglobin as its primary source of nutrition. This process occurs within the parasite’s digestive food vacuole, an acidic organelle (pH 4.8) (Hayward et?al., 2006). During the process of hemoglobin degradation, toxic free heme is usually released. Lacking the enzyme heme oxygenase used for heme-detoxification by most organisms, the parasite coverts the free heme into a non-toxic, insoluble crystal called hemozoin. Since the parasite catabolizes up to 80% of the erythrocyte’s hemoglobin content, local concentrations of free heme could potentially reach 200C500?mM if hemozoin crystallization did not occur (Scholar and Pratt, 2000). Hemozoin formation is usually mediated by neutral lipid bodies concentrated within the digestive food vacuole that serve as a reservoir for free heme (Hoang et?al., 2010b; Pisciotta et?al., 2007). These lipids were extracted from the parasite and shown to consist of a specific blend of mono- and di-acylglycerols (Pisciotta et?al., 2007). Synthetic neutral lipid droplets (SNLDs) composed of the biologically relevant blend of neutral lipids were shown to be a kinetically qualified site for the formation of -hematin, with a crystallization half-life of <5?min (Hoang et?al., 2010a, 2010b). In addition to accumulation of free heme within the SNLDs, molecular dynamic simulations have exhibited that this lipophilic environment of the lipid body would serve to stabilize the hemozoin precursor dimer and that formation of hemozoin would be favored at the lipid/aqueous interface (de Villiers et?al., 2007). These observations would suggest that inhibitors of hemozoin formation would interact with free heme either at the lipid/aqueous interface or within the neutral lipid body. One of the most successful antimalarial.