Elsevier

Phytomedicine

Volume 15, Issue 12, December 2008, Pages 1125-1129
Phytomedicine

Short communication
Inhibiting enoyl-ACP reductase (FabI) across pathogenic microorganisms by linear sesquiterpene lactones from Anthemis auriculata

https://doi.org/10.1016/j.phymed.2008.02.018Get rights and content

Abstract

Enoyl-ACP reductase (FabI) is a key enzyme of the type II fatty acid biosynthesis (FAS-II) pathway and a validated antimicrobial target. In the current study, three linear sesquiterpene lactones obtained from Anthemis auriculata, namely anthecotulide (1), 4-hydroxyanthecotulide (2) and 4-acetoxyanthecotulide (3) were evaluated for specific inhibitory effects against the FabI enzyme from three pathogenic microorganisms, Plasmodium falciparum (PfFabI), Mycobacterium tuberculosis (MtFabI) and Escherichia coli (EcFabI). In addition, the compounds were also tested against two elongation enzymes from the plasmodial FAS-II system, β-ketoacyl-ACP reductase (PfFabG) and β-hydroxyacyl-ACP deydratase (PfFabZ). The compounds showed clear differentiation in inhibition of FabI enzymes from different microorganisms. Anthecotulide (1) was most active against MtFabI (IC50 4.5 μg/ml), whereas the oxygenated derivatives thereof (compounds 2 and 3) specifically inhibited plasmodial FAS-II enzymes, PfFabI and PfFabG (IC50 values 20–75 μg/ml). All compounds were inactive towards EcFabI. In whole cell assays, all three compounds exhibited antimalarial and antibacterial activities.

Introduction

Fatty acid (FA) biosynthesis is a metabolic pathway by which acetyl-CoA is converted into short to long chain FAs. In plants, bacteria and some Apicomlexan parasites such as Plasmodium falciparum, it is carried out by the type II fatty acid synthase (FAS-II), a multienzyme complex that differs totally from the multifunctional human FAS-I (Smith et al., 2003). FA biosynthesis includes seven enzymatic steps, three of which are involved in the initiation phase and four in the elongation cycle. The formation of FAs is vital to survival, therefore the inhibition of individual enzymes of the pathway (e.g. FabG, FabZ; FabI and FabB/F) is an established approach in antimicrobial drug discovery (Heath et al., 2002; Zhang et al., 2006; Goodman and McFadden, 2007). Bacterial FAS-II pathway is located in the cytoplasm, whereas in P. falciparum the FAS-II system (PfFAS-II) takes place in the apicoplast, a previously discovered chloroplast-like organelle (McFadden et al., 1996), which is thought to have arisen from an ancient cyanobacterial endosymbiont through the process of secondary endosymbiosis (Williams and Keeling, 2003). Because of its cyanobacterial origin, there are significant similarities in the organization and overall structures of the bacterial and plasmodial FAS-II enzymes (Muench et al., 2007). Consistent with this, the inhibitors often exhibit cross activity towards FAS-II enzymes of different origin. For example, triclosan, a very potent inhibitor of Escherichia coli FabI enzyme (EcFAbI) (Sivaraman et al., 2003) also inhibits PfFabI with a similar potency and mechanism (Surolia and Surolia, 2001; Kapoor et al., 2004). We have been engaged for some time in the discovery of natural products targeting PfFAS-II enzymes (Tasdemir et al., 2005, Tasdemir et al., 2006; Karioti et al., 2007), particularly PfFabI, a crucial enzyme catalyzing the final reduction of the FA intermediates in each elongation cycle. In order to establish species selectivity, we have recently started testing our natural PfFabI inhibitors toward FabI homologous of various pathogenic organisms. We recently reported the first marine natural products inhibiting FabI of P. falciparum (PfFabI), the causative agent of human malaria, Mycobacterium tuberculosis (MtFabI), the causative agent of pulmonary tuberculosis and E. coli (EcFabI), the causative agent of gastroenteritis, neonatal meningitis and urinary tract infections (Tasdemir et al., 2007).

The genus Anthemis (Asteraceae) comprises about 130 species predominately distributed around the Mediterranean area, several of which are used as aromatic and herbal medicines, insecticides and dyes (Fernandes, 1976; Mabberley, 1997). Anthemis auriculata Boiss. is an endemic plant of the South Balkan peninsula and Turkey (Fernandes, 1976). In a recent communication, we reported three linear sesquiterpene lactones (SLs) anthecotulide (1), 4-hydroxyanthecotulide (2) and 4-acetoxyanthecotulide (3) with antibacterial activity from Greek A. auriculata (Theodori et al., 2006). We also described the isolation and structure elucidation of anthecularin (4), a minor, cyclic SL with a novel ring system from the same plant that exhibited dual PfFAS-II enzyme inhibitory activity (Karioti et al., 2007). This bioactivity encouraged us to test the related irregular SLs 13 against PfFabI, as well as MtFabI and EcFabI. In addition, the compounds were screened toward two other recombinant enzymes, β-ketoacyl-ACP reductase (PfFabG) and β-hydroxyacyl-ACP deydratase (PfFabZ) of the plasmodial FAS-II system. In vitro antimalarial, antimycobacterial and antibacterial activities of the compounds 13 are also reported.

Section snippets

Plant material, isolation and characterization of compounds 13

A. auriculata was collected in Central Greece in 2001 and compounds 13 were purified from the organic (EtOAc) extract of the aerial parts of the plant as described (Theodori et al., 2006). Their structures (Fig. 1) were determined by spectroscopic means. The purity of compounds (>95%) was confirmed by 1H and 13C NMR.

PfFabI enzyme assay

The PfFabI assay was carried out as reported (Tasdemir et al., 2007). Briefly, the compounds were dissolved in DMSO and tested at up to 100 μg/ml concentration in the presence of 1 

Results

Our earlier phytochemical studies on A. auriculata (Theodori et al., 2006) afforded the irregular, linear SL anthecotulide (1), as well as two new derivatives thereof, 4-hydroxyanthecotulide (2) and 4-acetoxyanthecotulide (3). Further investigation of the same plant yielded anthecularin (4), a minor, tricyclic SL, which is possibly derived from compound 2 as discussed (Karioti et al., 2007). Anthecularin (4) showed moderate antimalarial activity by inhibiting PfFabI and PfFabG, two key

Discussions

In this study, we have investigated the efficacy of three related linear SLs against FabI enzymes from a range of microbial species, in order to establish the species specificity of these natural compounds. A further aim was to establish a structure-activity-relationship (SAR) among the anthecotulide derivatives (13). The compounds were also tested versus two other enzymes involved in the plasmodial FAS-II system, i.e. β-ketoacyl-ACP reductase (PfFabG) and β-hydroxyacyl-ACP deydratase (PfFabZ)

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