Heme binding contributes to antimalarial activity of bis-quaternary ammoniums

Abstract

Quaternary ammonium compounds have received recent attention due to their potent in vivo antimalarial activity based on their ability to inhibit de novo phosphatidylcholine synthesis. Here we show that in addition to this, heme binding significantly contributes to the antimalarial activity of these compounds. For the study, we used a recently synthesized bis-quaternary ammonium compound, T16 (1,12-dodecanemethylene bis[4-methyl-5-ethylthiazolium] diodide), which exhibits potent antimalarial activity (50% inhibitory concentration, approximately 25 nM). Accumulation assays reveal that this compound is readily concentrated several hundredfold (cellular accumulation ratio, approximately 500) into parasitized erythrocytes. Approximately 80% of the drug was shown to be distributed within the parasite, approximately 50% of which was located in the parasite food vacuoles. T16 uptake was affected by anion substitution (permeation increasing in the order Cl(-) < Br(-) = NO(3)(-) < I(-) < SCN(-)) and was sensitive to furosemide-properties similar to substrates of the induced new permeability pathway in infected erythrocytes. Scatchard plot analysis of in situ T16 binding revealed high-affinity and low-affinity binding sites. The high-affinity binding site K(d) was similar to that measured in vitro for T16 and ferriprotoporphyrin IX (FPIX) binding. Significantly, the capacity but not the K(d) of the high-affinity binding site was decreased by reducing the concentration of parasite FPIX. Decreasing the parasite FPIX pool also caused a marked antagonism of T16 antimalarial activity. In addition, T16 was also observed to associate with parasite hemozoin. Binding of T16 to FPIX in the digestive food vacuole is shown to be critical for drug accumulation and antimalarial activity. These data provide additional new mechanisms of antimalarial activity for this promising new class of antimalarial compounds.

FIG. 1.

Chemical structure of T16 (1,12-dodecanemethylene bis[4-methyl-5-ethylthiazolium] diodide) and [³H]T16.

FIG. 2.

Time course of 50 nM [³H]T16 uptake into uninfected (•) and P. falciparum-infected (○) erythrocytes suspended in HEPES-buffered RPMI 1640 medium (pH 7.4). Data are the means ± standard error of three independent experiments.

FIG. 3.

Effect of anion substitution from the suspending solution on the uptake (increase relative to chloride [n-fold]) of 50 nM [³H]T16 into P. falciparum-infected erythrocytes. Data represent the means ± standard error of three independent experiments.

FIG. 4.

Scatchard plot of the total binding of [³H]T16 onto P. falciparum-infected erythrocytes. Shown is a single representative plot from five similar experiments. The curved line suggests the presence of at least two components—a high-affinity T16 binding site and a low-affinity T16 binding site.

FIG. 5.

UV and visible spectral scans of (A) 3 μM FPIX (λ_max, 395 nm in aqueous HEPES [pH 7.0]) and (B) 3 μM PIX (λ_max, 410 nm in aqueous HEPES [pH 7.0]) in the presence (dashed line) or absence (solid line) of 3 μM T16. Data are representative of three independent experiments.

FIG. 6.

Nonlinear-regression analysis of [³H]T16 binding to FPIX-loaded ghost erythrocyte membrane. Data points are from two independent experiments performed in triplicate.

FIG. 7.

Scatchard plot of the high-affinity binding of [³H]T16 to P. falciparum-infected erythrocytes in the presence (•) and absence (○) of 10 μM Ro 40-4388. Data points are means of duplicate observations from three separate experiments.

FIG. 8.

Isobole plot of T16 in vitro antimalarial activity in combination with Ro 40-4388.

FIG. 9.

(A) Binding of [³H]T16 to FPIX and hemozoin in vitro and subsequent separation by discontinuous sucrose gradient. (B) Binding of [³H]T16 to freed (saponin treated) Plasmodium falciparum trophozoites and subsequent separation by discontinuous sucrose gradient. Control experiments indicate that free [³H]T16 and [³H]T16-FPIX complexes were restricted to the 0.2 M fraction and that [³H]T16-hemozoin complexes were restricted to the 2 M fraction.