Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Search for Novel Anti-Malarials


By: Carol A. Rouzer, VICB Communications
Published: January 12, 2015



A high-throughput screen of β-hematin formation identifies a structurally diverse group of inhibitors that are toxic to the malaria parasite in culture.


Despite major efforts to control the disease, malaria remains a major public health problem in the developing world causing nearly 1 million deaths per year predominantly among children in sub-Saharan Africa. The most virulent agent that causes malaria is the parasite, Plasmodium falciparum, which spends a portion of its life cycle within the circulating red blood cells of the infected host. Within the red blood cell, the parasite acquires needed nutrients by degrading hemoglobin, a process that releases free heme (ferroprotoporphyrin IX), which is rapidly oxidized to α-hematin (ferriprotoporphyrin IX). α-Hematin is toxic at high concentrations, so the parasite converts the accumulating porphyrin to hemozoin (β-hematin), a nontoxic polymer (Figure 1). Hemozoin formation is critical to the survival of the parasite, as indicated by the fact that some of the oldest anti-malarial drugs, such as chloroquine, work by blocking this process. Chloroquine and many related quinoline drugs are no longer effective in treating malaria due to acquired resistance by the parasite. However, the resistance results from the presence of an efflux pump that prevents the drug from reaching the site of hemozoin formation. Thus, inhibition of hemozoin formation remains a viable anti-malaria drug target if new compounds can be found that are not substrates for P. falciparum’s efflux pump. This led Vanderbilt Institute of Chemical Biology investigator David Wright and his laboratory to search for novel inhibitors of hemozoin formation [R.D. Sandlin, et al. (2014) Int. J. Parasitol. Drugs Drug Resist., 4, 316].



Figure 1. Figure 1. Structure of a portion of the hemozoin (β-hematin) polymer. The heme subunits first form dimers through a reciprocal bond between a propionate side chain of each α-hematin and the Fe(III) of its partner. Then, the dimers join through hydrogen bonds between the remaining propionate side chains. Image courtesy of Wikimedia Commons under the GNU Free Documentation License.


Although the process of hemozoin formation remains a mystery, strong evidence supports a role for neutral lipids as nucleating agents. Reproducing the exact mixture of mono- and diglycerides that appear to carry out this function in vivo, however, proved to be impractical for routine laboratory investigations, so the Wright lab went in search of a substitute. Their discovery that the nonionic detergent NP-40 efficiently initiates hemozoin formation in vitro provided the foundation for the development of a high-throughput screen which they used in their search for hemozoin formation inhibitors [R.D. Sandlin, et al. (2011) Antimicrob. Agents Chemother. 55, 3363].  For their assay, the investigators mix test compounds with heme, NP-40, and acetone and incubate for 6 hours to allow hemozoin formation to occur. Then, they add pyridine, which forms a complex with any remaining free α-hematin. The pyridine-hematin complex absorbs strongly at 405 nm, providing a measure of the amount of α-hematin that did not form hemozoin. Thus, high absorbance is a reflection of an effective inhibitor (Figure 2).


Figure 2.  Outline of the hemozoin (β-hematin) formation inhibition assay. Test compounds are first delivered to the plate (96-well plates are depicted here, but 384-well plates were used in the screen). Then, water, α-hematin (hematin), NP-40, and acetone are added to each well. Following incubation for 6 h at 37oC, pyridine is added, which forms a yellow complex with any remaining free α-hematin. Absorbance at 405 nm reveals the effectiveness of inhibition of hemozoin formation.


The investigators screened 144,300 compounds from the Vanderbilt Institute of Chemical Biology’s compound library at an initial concentration of 19.3 μM. The 729 compounds that exhibited ≥80% inhibition of hemozoin formation were designated as hits and subjected to further testing in a dose-response assay using a concentration range of 0.5 to 100 μM. The 527 compounds that exhibited an IC50 (concentration that inhibits hemozoin formation by 50%) of ≤27 μM in this assay were then evaluated in a screen that tested their ability to kill P. falciparum in cultured red blood cells. This screen identified 171 compounds that killed ≥90% of the parasites, and further dose-response testing of these compounds revealed 73 that exhibited IC50 values of ≤5 μM. Initial P. falciparum screens used a strain (D6) that is sensitive to many drugs, including chloroquine. Therefore, the investigators retested their most promising compounds against the drug resistant C235 strain, identifying 21 compounds that remained effective in this assay.


An encouraging outcome of these efforts was the wide structural diversity of the identified hemozoin formation inhibitors. Active compounds represented fourteen distinct scaffolds, including quinolines, phenylbenzamides, benzylethenes, and triaryl imidazoles, examples of which are shown in Figure 3. Thus, the efforts of the Wright lab provide an excellent foundation for the discovery of novel anti-malarial drugs that promise to be effective against resistant strains of P. falciparum. We look forward to hearing about the next steps in their efforts to exploit these most recent findings!


Figure 3.  Examples of novel hemozoin (β-hematin) formation inhibitors representative of the quinoline, benzylethene, phenylbenzamide, and triaryl imidazole scaffolds. Data are provided for IC50s in the β-hematin formation inhibition assay and the P. falciparum inhibition assays using drug sensitive (D6) and resistant (C235) strains of the parasite.














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