Leveraging a Fluorescent Fatty Acid Probe to Discover Cell-Permeable Inhibitors of Plasmodium falciparum Glycerolipid Biosynthesis

Abstract

A sensitive and quantitative fluorescence-based approach is presented for characterizing fatty acid acquisition and lipid biosynthesis by asexually replicating, intraerythrocytic Plasmodium falciparum. We show that a BODIPY-containing, green-fluorescent fatty acid analog is efficiently and rapidly incorporated into parasite neutral lipids and phospholipids. Prelabeling with a red-fluorescent ceramide analog permits normalization and enables reliable quantitation of glycerolipid labeling. Inhibition of lipid labeling by competition with natural fatty acids and by acyl-coenzyme A synthetase and diacylglycerol acyltransferase inhibitors demonstrates that the fluorescent fatty acid probe is acquired, activated, and transferred to lipids through physiologically-relevant pathways. To assess its utility in discovering small molecules that block parasite lipid biosynthesis, the lipid labeling assay was used to screen a panel of mammalian lipase inhibitors and a selection of compounds from the "Malaria Box" anti-malarial collection. Several compounds were identified that inhibited the incorporation of the fluorescent fatty acid probe into lipids in cultured parasites at low micromolar concentrations. Two contrasting profiles of suppression of neutral lipid and phospholipid synthesis were observed, which implies the inhibition of distinct pathways. IMPORTANCE The human malaria parasite Plasmodium falciparum relies on fatty acid scavenging to supply this essential precursor of lipid synthesis during its asexual replication cycle in human erythrocytes. This dependence on host fatty acids represents a potential vulnerability that can be exploited to develop new anti-malarial therapies. The quantitative experimental approach described here provides a platform for simultaneously interrogating multiple facets of lipid metabolism- fatty acid uptake, fatty acyl-CoA synthesis, and neutral lipid and phospholipid biosynthesis- and of identifying cell-permeable inhibitors that are active in situ.

Keywords: Malaria Box; Plasmodium falciparum; fatty acid; fluorescent probe; malaria; neutral lipid; phospholipid.

FIG 1

Labeling of parasite lipids with fluorescent fatty acid analogs. (A) Comparison of labeling of neutral lipids (NL) and polar lipids (PL) by five fluorescent fatty acids. P, parasite lipids; S, fluorescent fatty acid standard. Major lipid species are indicated with arrowheads. o, origin. (B) Time course of C4,C9-FA incorporation into parasite neutral and polar lipids. Presumed identities of major lipids species are indicated. FA, C4,C9-FA; *, residual DAG fluorescence; BTC, BODIPY-TR-ceramide fluorescence. (C) Live-cell images of two infected erythrocytes (center and lower left) and one uninfected erythrocyte (upper right) after labeling with BTC and C4,C9-FA. DNA was stained with Hoechst 33342. Arrow, perinuclear ER fluorescence; arrowheads, punctate fluorescence. (D) Merge of the C4,C9-FA and DNA panels from the lower left parasite in (C). The contrast of the C4,C9-FA panel was adjusted to emphasize the circumnuclear fluorescence, leading to saturation of pixels associated with the punctate fluorescence. (E) HCS LipidTox Red staining of neutral lipids in a parasite labeled with C4,C9-FA identifies the fluorescent punctum as a lipid droplet (arrowhead). Scale bar, 3 μm in C and E, 1.8 μm in D.

FIG 2

Standard protocol for C4,C9-FA labeling of parasite lipids in cultures of synchronized trophozoites. RPMI-Alb, incomplete RPMI; BSA, fatty acid-free bovine serum albumin.

FIG 3

Assay validation. (A) Treatment with the diacylglycerol acyltransferase inhibitor A-922500 (10 μM) identifies TAG species that likely contain one (“TAG”) and two (“TAG2”) C4,C9-FA acyl chains. (B) Effects of equimolar mixtures of palmitate and oleate (“P + O,” concentration of each is indicated in μM) or triacsin C (μM) on C4,C9-FA incorporation into parasite neutral and polar lipids. FA, C4,C9-FA; o, origin. (C) Plots of BTC-normalized fluorescence intensities for the images in (B), expressed as a ratio of those for a no-additive control. Lines are nonlinear regression fits to a four-parameter sigmoidal curve. Data are representative of two independent experiments that yielded similar results.

FIG 4

Effects of ATFK and AKU-002 on C4,C9-FA lipid labeling. (A) Compound structures. (B) Concentration dependence of inhibition of C4,C9-FA incorporation into DAG, TAG, PC and PE. BTC-normalized fluorescence values are expressed as a fraction of the no-inhibitor control. Data points are means from three independent experiments with standard deviation represented with bars. (C) Live-cell images of parasites labeled with C4,C9-FA in the presence of DMSO vehicle, 10 μM ATFK, or 10 μM AKU-002. DNA was stained with Hoechst 33342 and is pseudocolored red. Scale bar, 3 μm.

FIG 5

Identification of an inhibitor of P. falciparum lipid synthesis from the Malaria Box. (A) Screen of 20 pools representing 71 Malaria Box compounds for inhibition of parasite neutral lipid synthesis. C, no inhibitor control; A, ATFK (10 μM) positive control for inhibition. o, origin; FA, C4,C9-FA. The red asterisk indicates inhibition by pool 11. (B) Identification of the active compound in pool 11. a, b, c, d represent the four compounds in the pool; compound c is MMV665915.