Figure 2

Examples of 8-day Radical Cure assay improvements used to characterize hits with activity against mature hypnozoites. (A) EC₅₀’s shown are for inhibition of hypnozoites. Possible hit MMV676121 was first identified from a run of the original dose response assay, which included a concentration gradient from 10 μM to 46 nM (v1). A CC₂₀ could not be calculated for inhibition of hepatic nuclei but was estimated to be > 2 μM. Bars represent SEM of 6 independent experiments, each with a single well at each concentration. MMV676121 was retested in the refined (v2) assay dose response format, which includes an expanded concentration gradient from 50 μM to 280 pM. Bars represent SEM of two independent experiments, each containing two wells at each concentration. Plotting inhibition of hepatic nuclei on the hypnozoite inhibition chart shows no selectivity window for this compound. The two assay versions were also used to identify and then further define the selective activity of two P. falciparum stage V gametocyte inhibitors, MMV021036 and MMV018983, against mature hypnozoites. Bars in the v1 charts represent SEM of three (for MMV021036) or four (for MMV018983) independent experiments, each with a single well at each concentration; bars in the v2 charts represent SEM of two independent experiments, each containing two wells at each concentration. (B) Radical Cure activity from a v2 assay of maduramicin against hypnozoites. Maduramicin is potently active against hypnozoites (EC₅₀ 22 nM) and toxic against host hepatocytes (EC₅₀ 4.28 μM), leading to a calculated selectivity of > 190. Bars represent SEM of two independent experiments, each containing two wells at each concentration. (C) Radical Cure activity from a v2 assay of P. falciparum stage V gametocyte inhibitor MMV019204, with activity against hypnozoites (EC₅₀ 1.01 μM) and toxicity against host hepatocytes (EC₅₀ 4.71 μM), leading to a calculated selectivity of 4.7. Black marker indicates hypnozoite inhibition data marked as an outlier. Bars represent SEM of two independent experiments, each containing two wells at each concentration. (D) Structures of the three P. falciparum stage V gametocyte inhibitors with activity against mature hypnozoites. (E) Plots of all EC₅₀’s for the control and reference compounds of Prophylactic and Radical Cure dose response assays used to generate data for this report. Dose response assays were performed at two sites (SMRU in Thailand and IPC in Cambodia) over 3 years using three different lots of human hepatocytes (UBV, PDC, and BGW) infected with sporozoites from a unique patient isolate of P. vivax for each run. For visualization, data was plotted at 3.3 μM for all runs in which activity was greater than the maximum concentration tested (10 μM for atovaquone or 3.3 μM for PI4Ki and monensin). Bar represents geometric mean.