Synthesis, characterization, and cellular localization of a fluorescent probe of the antimalarial 8-aminoquinoline primaquine

Abstract

Primaquine (PQ) is the only commercially available drug that clears dormant liver stages of malaria and blocks transmission to mosquito vectors. Although an old drug, much remains to be known about the mechanism(s) of action. Herein we develop a fluorescent tagged PQ to discover cellular localization in the human malaria parasite, Plasmodium falciparum. Successful synthesis and characterization of a primaquine-coumarin fluorescent probe (PQCP) demonstrated potency equivalent to the parent drug and the probe was not cytotoxic to HepG2 carcinoma cells. Cellular localization was found primarily in the cytosol of the asexual erythrocytic and gametocyte stages of parasite development.

Pan-active imidazolopiperazine antimalarials target thePlasmodium falciparumintracellular secretory pathway

“…Based on results with these two reporter systems, we speculated that there was an impairment of protein secretion that occurs before signal peptide cleavage, leading to degradation of the uncleaved products or potentially a block in synthesis. Although this could be a result of reduced total protein synthesis, we observed continued incorporation of 35 S methionine after treatment with GNF179, in contrast with cycloheximide. Thus, if protein synthesis was inhibited, it was likely to only be for a subset of proteins.…”

“…To gain further insight into the compound’s function we next examined subcellular localization using a fluorescently-conjugated form of GNF179 in P. falciparum , as recently performed for primaquine 35 . We first generated both NBD- or Coumarin-1-conjugated forms of GNF179 (Figure 3a), leveraging an existing reaction series 36 .…”

“…HSP70 serves as a loading control. (c) 35 S incorporation of newly synthesized amino acids at different concentrations of KAF156, chloroquine (negative control for inhibition) and cycloheximide (positive control for inhibition). Counts were normalized to data obtained from no-drug controls (NDC).…”

“…The effect of drug treatment on parasite translation was evaluated by quantifying the incorporation of 35 S-labeled amino acids into newly synthesized protein by adapting a published protocol 76 . Briefly, synchronized, trophozoite-stage parasites (5% parasitemia, 26-30 hpi) were first washed in methionine-free media three times prior to drug treatment.…”

“…Briefly, synchronized, trophozoite-stage parasites (5% parasitemia, 26-30 hpi) were first washed in methionine-free media three times prior to drug treatment. Parasites were then incubated in various compound concentrations in 24-well plates for 1 hour at 37°C, at a final hematocrit of 5%, and a final concentration of 125 µMci/mL of EasyTag™ EXPRESS 35 S Protein Labeling Mix (Perkin Elmer, USA). Concentrations used for incubation correspond to 100-, 10-, 1-, 0.1- and 0.01-times the IC50 values of KAF156 (15 nM), chloroquine (85 nM) and cycloheximide (750 nM).…”