Structural Elucidation of the Specificity of the Antibacterial Agent Triclosan for Malarial Enoyl Acyl Carrier Protein Reductase

Abstract

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD ؉ , and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for C␣s, 0.30 Å). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.Treatment of Plasmodium falciparum malaria has depended for decades on the use of the aminoquinoline chloroquine or the synergistic antifolate combination pyrimethamine-sulfadoxine. These drugs were characterized by their potency against the P. falciparum asexual intra-erythrocytic stages (responsible for malaria pathogenesis), their affordability and their safety. The occurrence and spread of drug-resistant strains of P. falciparum have largely contributed to a recent resurgence of malaria, which claims 1 to 3 million lives annually and which is endemic in inter-tropical areas representing 40% of the world's population (1). The current situation of antimalarial chemotherapy and resistance, in conjunction with the reappearance of malaria in formerly well-controlled areas, reinforces the need for new, highly potent antimalarials.Recent investigations into Apicomplexan parasites, including Plasmodium and Toxoplasma, have uncovered the existence of a unique organelle, the apicoplast (2-4). The finding that ciprofloxacin-mediated inhibition of plastid replication in Toxoplasma gondii tachyzoites blocked parasite propagation provided evidence for the indispensable nature of this nonphotosynthetic plastid organelle (5). Studies of plastid inhibitors and apicoplast mis-segregation mutants confirmed the essential requirement of this organelle for normal parasite development and indicated that inhibition of apicoplast function or loss of this organelle resulted in parasite death following reinvasion of host cells (5-7). This organelle appears to derive ultimately from a cyanobacterial endosymbiont (4,8,9) and as such was postulated to contain prokaryotic-type metabolic pathways, of significant interest from the perspective of developing antiparasitic drugs (10). Recent studies indicate that these pathways include fatty acid and isoprenoid bios...

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

“…More recently, triclosan has been shown to act as a slow-tight binding inhibitor of the P. falciparum enoyl-ACP reductase enzyme activity with an overall inhibition constant value of 96 pM (Kapoor et al 2004). Determination of the three-dimensional structure of malarial enoyl reductase-triclosan NAD + ternary complex has provided a structural framework that sheds light on the mode of binding of triclosan (Perozzo et al 2002). Accordingly, P. falciparum enoyl-ACP reductase is a validated target for antimalarial agent development and to be used in drug screening efforts.…”

Design, development, synthesis, and docking analysis of 2′‐substituted triclosan analogs as inhibitors for Plasmodium falciparum Enoyl‐ACP reductase

“…The crystal structures of PfENR (PDB Code: 1NHG) by Perozzo et al were used for docking studies (15). The structure co-ordinates were converted into mol2 format with MMFF94 charges assigned using the molecular operating environment (MOE) suite of programs (25).…”

“…In continuation of earlier work by several groups of investigators, this study further explores the B-ring of triclosan, focusing specifically on its 2 0 -position and the intricate molecular interactions using docking analysis (6,7,15,19). The chloro group at 2 0 -position of triclosan is shown to have close proximity to Ala 217 and pyrophosphate moiety of NAD 1 .…”

“…PfENR is known to be an important target for the development of antimicrobial and antiparasitic molecules (11)(12)(13)(14)(15). We have reported earlier that triclosan, a microbicide and a common additive in many household items, strongly inhibits PfENR with high potency and also exhibits slow-tight binding mechanism in presence of catechins (16,17).…”

Synthesis and in vitro antimycobacterial activity of B-ring modified diaryl ether InhA inhibitors

“…[12][13][14][15][16][17][18][19][20] In this study we describe two classes of molecules in which alterations have been made to the diphenyl ether 'B' ring. In one series of compounds we have replaced the B ring with isosteric heterocycles that incorporate nitrogen atoms within the ring, thereby causing little steric perturbation to the overall structure of the molecule (Scheme 2).…”