'FAS't inhibition of malaria

Abstract

Malaria, a tropical disease caused by Plasmodium sp., has been haunting mankind for ages. Unsuccessful attempts to develop a vaccine, the emergence of resistance against the existing drugs and the increasing mortality rate all call for immediate strategies to treat it. Intense attempts are underway to develop potent analogues of the current antimalarials, as well as a search for novel drug targets in the parasite. The indispensability of apicoplast (plastid) to the survival of the parasite has attracted a lot of attention in the recent past. The present review describes the origin and the essentiality of this relict organelle to the parasite. We also show that among the apicoplast specific pathways, the fatty acid biosynthesis system is an attractive target, because its inhibition decimates the parasite swiftly unlike the 'delayed death' phenotype exhibited by the inhibition of the other apicoplast processes. As the enzymes of the fatty acid biosynthesis system are present as discrete entities, unlike those of the host, they are amenable to inhibition without impairing the operation of the host-specific pathway. The present review describes the role of these enzymes, the status of their molecular characterization and the current advancements in the area of developing inhibitors against each of the enzymes of the pathway.

Scheme 1. Type II fatty acid biosynthesis system

Acetyl-CoA (1) is converted into malonyl-CoA (2) by ACC and then to malonyl-ACP (3) by FabD. The resulting malonyl-ACP condenses with another molecule of acetyl-CoA to form β-oxoacyl-ACP (4) catalysed by β-oxoacyl ACP synthase III (FabH). This is then converted into β-hydroxyacyl-ACP (5) by β-oxoacyl ACP reductase (FabG) and then dehydrated by β-hydroxyacyl ACP dehydratases (FabZ/FabA). The synthesis of unsaturated fatty acids branches out at this step catalysed first by FabA and then by FabB. The dehydrated product, enoyl-ACP (6) is then reduced by enoyl-ACP reductase (FabI) to form butyryl-ACP (7). This product re-enters the FAS cycle and the growing chain is elongated by two carbon units per cycle. The condensing enzymes involved in elongation are FabB and FabF.

Figure 1. Structures of the inhibitors of different enzymes of the Type II fatty acid biosynthetic pathway

Figure 2. NAS-91 and NAS-21 docked with homology-modelled FabZ from P. falciparum

Pfal FabZ being a dimer, has two active sites, which, hence, can house either two molecules of NAS-21 or NAS-91, or one of each, simultaneously. In this Figure, however, for clarity, only one of the active sites has been demonstrated. While NAS-21 occludes the entry to the active site, NAS-91 sits in the active site and inhibits FabZ.

Figure 3. Superposition of the binary complex of FabI–NADH and the ternary complex of FabI–NAD⁺–triclosan

Superposition of the binary complex of FabI–NADH (black) and the ternary complex of FabI–NAD⁺–triclosan (grey). Movement of residues 318–324 of the substrate-binding loop and the nicotinamide ring of NAD⁺ result in increased van der Waal's contacts, explaining the increased affinity of NAD⁺ and triclosan for FabI in the presence of triclosan and NAD⁺ respectively. TCL, triclosan.

Figure 4. Pictorial representation of the origin of the apicoplast

(A) The photosynthetic cyanobacterium (green) is engulfed by a primary eukaryote (light blue) and some of the genes from the primary endosymbiont are transferred to the host nucleus (dark blue). Subsequently, this eukaryote containing the endosymbiont is engulfed further by a secondary eukaryote (buff). Here again, some genes are transferred to the host nucleus (red) reducing the apicoplast genome to the bare minimum. (B) During evolution, most of the genes were transferred from the plastid to the nucleus. Some of these genes code for proteins destined to the apicoplast (APM, apicoplast membrane). These proteins carry a signal peptide (SP; shown in pink) followed by a transit peptide (TP; shown in blue). The SP directs the protein into the endoplasmic reticulum where it is cleaved by a signal peptidase I. The resultant protein carries the TP, which directs its entry into the apicoplast. The TP is then cleaved by the plastid peptidase. Thus the nuclear-encoded proteins enter the apicoplast by a bipartite targeting signal.

Figure 5. Phylogenetic relationship of FabI of P. falciparum with those of other organisms

The values shown represent those with confidence levels above 50%.

Figure 6. Giemsa-stained blood smears of the Plasmodium culture

Cultures were treated with various reagents as indicated and were incubated for 48 or 96 hours.

Figure 7. Death kinetics invoked by apicoplast-targeting drugs: triclosan and clindamycin

(A) Chloroquine (Q), triclosan (T) and CER (results not shown) ablate the parasites within the first cycle of asexual reproduction, unlike clindamycin (C) and chloramphenicol (H) which invoke parasite death only after 84 h (towards the end of the second cycle). Parasitaemia remained high in the untreated culture (N, no drug control). Parasite growth was monitored by making Giemsa-stained smears and counting number of infected cells per total number of cells, as well as by hypoxanthine incorporation (results not shown). Results are means±S.E.M. The statistical significance of changes in parasitaemia was confirmed using the two-tailed Student's t test (P<0.05). (B) Cartoon depicting the effect of the action of clindamycin and triclosan on the culture of P. falciparum. In the case of clindamycin, the effect is felt only in the second asexual cycle leading to the delayed-death phenotype, whereas triclosan effects immediate death by inhibiting the first asexual cycle itself.