Summary /

ML238 showed an LC50 value of 34.06 μM in HEPG2 (Figure 1), while showing subnanomolar EC50 values in both 3D7 and Dd2 lines of P. falciparum (Table 1). This corresponds to a safety window greater than 30,000-fold. In addition, we have also screened the probe and several of its structural analogs for toxicity in kidney epithelial cells, dermal fibroblasts, lung epithelial cancer cells, and erythrocyte lysis (Table 2). The probe and selected analogs show excellent safety window.

Figure 1. Dose-response curves for the probe (ML238) obtained using Luciferase assays against P. falciparum 3D7 (A) and Dd2 (B), and using a SYBR-green based assay in 3D7 (C) and Dd2 (D), low nanomolar to subnanomolar activity of the probe. The probe (ML238) only shows cytotoxicity in HEPG2 (E) at high micromolar, resulting in a large safety window.

Table 1. Summary of Data Obtained in the Flow Cyclometry Assay Against P. falciparum after ML238 (and analogs) treatment.

Table 2. Summary of Cytotoxicity Data in Kidney Epithelial Cells, Dermal Fibroblasts, Lung Epithelial Cancer Cells (A549) and Erythrocytes.

Summary /

Delayed death inhibitors specifically target the parasite apicoplast; an organelle of cyanobacterial origin that employs prokaryotic-derived transcription and translation machinery (Lim 2010). During asexual bloodstage development, this organelle adopts an elongated and branched structure as the parasite matures into its trophozoite form, then segregates into individual organelles that parse with the individual daughter merozoites prior to their release from the infected erythrocyte. To verify if the compounds do interfere with the apicoplast, a whole-cell imaging screen with a recombinant parasite line that has GFP-labeled apicoplasts was used. This line, ACPL-GFP, has already been developed and benefits from chromosomal integration of the GFP fusion, producing excellent signal homogeneity. Antibiotics (such as zithromycin and doxycycline) that are known to interfere with the apicoplast have been observed to  disrupt the morphology of the apicoplast in this GFP-expressing line (Figure 1) (Dahl 2007). Azithromycin was used as a positive control, while parasites treated with drug vehicle (medium containing 0.05 – 0.1% DMSO) served as a negative control. ML238 appeared to disrupt the apicoplast. The apicoplast morphology resembles the phenotype seen with chloroquine treatment; it is much smaller than in the untreated parasites but still present. Mitochondrial staining was generally diminished, though not entirely absent, in parasites treated with ML238 and some of its analogs. The mode of action of ML238 is still unknown. But its structure, very different from known antimalarial drugs and probes, suggests that its mode of action could involve a new target. In this context, target identification will help understanding the mechanism of action of the probe and will be a priority in the near future.

Figure 1. Parasites expressing GFP targeted to the apicoplast, 3D7 ACP(L)-GFP, were treated with the indicated compounds in 200 μL of culture media at a 1% hematocrit. At 96 hours, 30 μl of culture was harvested and stained with Hoechst (1 μg/ml) and MitoTracker Red (20 nM) for 30 minutes at 37 ºC. Stained parasites were imaged on a Nikon Ti inverted microscope with a 100X objective. The nuclei appear blue, mitochondria appear red, and the apicoplast appears green. The periphery of the infected erythrocyte is indicated with a yellow arrow. Drug concentrations: chloroquine, 16 nM; azithromycin, 80 nM; BRD-K81592585-001-02-2, 400 nM; ML238 (BRD-K45399554-001-01-6), 0.64 nM; BRDK91442916-001-01-0, 16 nM; BRD-K05563014-001-02-7, 400 nM; BRD-K06180729-001-01-7, 50 μM; BRD-K95212245-001-01-5, 0.64 nM.