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Mass spectrometry-based systems approach for identification of inhibitors of Plasmodium falciparum fatty acid synthase - PubMed

Mass spectrometry-based systems approach for identification of inhibitors of Plasmodium falciparum fatty acid synthase

Shilpi Sharma et al. Antimicrob Agents Chemother. 2007 Jul.

Abstract

The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (beta-ketoacyl-ACP synthase), FabG (beta-ketoacyl-ACP reductase), FabZ (beta-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (-)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite.

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Figures

FIG. 1.
FIG. 1.

Schematic representation of the elongation module of FAS.

FIG. 2.
FIG. 2.

(a) CS-PAGE gel showing the migration of various acyl-PfACPs synthesized using apo-PfACP, respective acyl-CoAs, and the AcpS enzyme. A 13%, 2.5 M urea sample containing native PAGE gel was run at 20 mA at 4°C. Lane 1, malonyl-PfACP, which runs as a doublet; lane 2, C4-PfACP; lane 3, C6-PfACP; lane 4, C8-PfACP; lane 5, C10-PfACP; lane 6, apo-PfACP; lane 7, C12-PfACP; lane 8, C14-PfACP; lane 9, C16-PfACP; lane 10, C16:1-PfACP. As can be seen from the figure, acyl-PfACPs of various chain lengths do not show significant mobility differences, unlike E. coli acyl-ACPs. (b) A 12% sodium dodecyl sulfate-PAGE gel showing the purities of all the enzymes used in the reconstitution experiment along with the E. coli AcpS used for the synthesis of P. falciparum acyl-ACPs. Lane 1, 108-kDa PfFabB/F; lane 2, 36-kDa PfFabI; lane 3, molecular mass marker from MBI Fermentas; lane 4, 29-kDa PfFabG; lane 5, 17-kDa PfFabZ; and lane 6, 14-kDa AcpS.

FIG. 3.
FIG. 3.

(a) MALDI-TOF spectra showing in vitro reconstitution of the fatty acid synthesis cycle of Plasmodium falciparum. C6-PfACP (9,856 Da) + malonyl-PfACP → C8-PfACP (9,884 Da) + C10-PfACP (9,912 Da) + C12-PfACP (9,940 Da) + C14-PfACP (9,968 Da) + holo PfACP (9,758 Da). The peak at 9,420 Da corresponds to apo-ACP. (b) MALDI-TOF spectra showing in vitro reconstitution of the fatty acid synthesis cycle of Plasmodium falciparum. C12-PfACP (9,941 Da) + malonyl-PfACP → C14-PfACP (9,969 Da) + PfACP (9,758 Da). The peak at 9,420 Da corresponds to apo-ACP.

FIG. 4.
FIG. 4.

Inhibition of PfFAS by cerulenin. The boxed area shows the presence of only the substrate peak (C6-ACP, 9,856 Da) with time and no sign of the appearance of any of the product peaks (β-keto C8-ACP [9,898 Da], β-hydroxy C8-ACP [9,900 Da], octenoyl-ACP [9,882 Da], C8-ACP [9,884 Da]), indicating inhibition of the first condensation step by cerulenin. The 9,758-Da peak corresponds to holo-ACP, and the 9,420-Da peak corresponds to apo-ACP.

FIG. 5.
FIG. 5.

Inhibition of PfFAS by (−)-catechin gallate. The boxed area shows the conversion of the substrate peak, C6-PfACP (9,856 Da) to β-keto C8-PfACP (9,898 Da) (a product of PfFabB/F) but no sign of β-hydroxy C8-PfACP (9,900 Da) (a product of PfFabG), octenoyl-ACP (9,882 Da), or C8-ACP (9,884 Da), indicating inhibition of the PfFabG step. The 9,758-Da peak corresponds to holo-ACP, and the 9,420-Da peak corresponds to apo-ACP.

FIG. 6.
FIG. 6.

Inhibition of PfFAS by NAS-21 and NAS-91. The boxed area shows the conversion of the substrate peak C6-PfACP (9,856 Da) to β-hydroxy C8-PfACP (9,900 Da) (a product of PfFabG) but no sign of octenoyl-ACP (9,882 Da) or C8-ACP (9,884 Da), indicating inhibition of the PfFabZ step. The 9,758-Da peak corresponds to holo-ACP, and the 9,420-Da peak corresponds to apo-ACP.

FIG. 7.
FIG. 7.

Inhibition of PfFAS reaction with triclosan, using C10-ACP as the primer. The reaction was initiated by the addition of 5 μg PfFabB/F, and the first aliquot was taken out at 0 min (A). After 10 min of incubation, another aliquot was taken out (B) and 2 μg PfFabG was added to the same reaction mixture. After incubation for 10 min, another aliquot was taken out (C) and 3 μg PfFabZ was added to the reaction mixture. The mixture was incubated for another 10 min before an aliquot was taken out (D). Finally 2 μg PfFabI plus triclosan was added to the mixture and was incubated for 10 min before termination of the reaction (E). (F) The reaction in which PfFabI was added without triclosan. All the aliquots were snap frozen and analyzed by MALDI-TOF MS. Results are shown for C10-PfACP (9,913 Da), β-keto C12-PfACP (9,955 Da), β-hydroxy C12-ACP (9,957 Da), C12-PfACP (9,941 Da), and C14-PfACP (9,969 Da). The molecular weights of acylated-ACPs were calculated keeping holo ACP (9,758 Da) as the standard.

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