Anti-malarial drug targets: screening for inhibitors of 2C-methyl-D-erythritol 4-phosphate synthase (IspC protein) in Mediterranean plants.

Abstract

The recently discovered non-mevalonate pathway of isoprenoid biosynthesis serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa, most notably Plasmodium, but is absent in mammalian cells. It is therefore an attractive target for anti-infective chemotherapy. The first committed step of the non-mevalonate pathway is catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase (IspC). Using photometric and NMR spectroscopic assays, we screened extracts of Mediterranean plants for inhibitors of the enzyme. Strongest inhibitory activity was found in leaf extracts of Cercis siliquastrum.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Terpene; Deoxyxylulose inhibitors of 2C-methyl-D-erythritol 4-phosphate sythase (IspCprotein); Antibiotic; Malaria; Cercis siliquastrum

Introduction

On a worldwide basis, infections by microorganisms, viruses and parasites are among the most important factors of morbidity and mortality (Binder et al., 1999; Levin et al., 1999; Cohen, 2000; Falkow and Kennedy, 2001). In light of the rapid development of resistance against all anti-infective agents, there is an urgent need for the rapid development and deployment of novel drugs.

Any molecular target for anti-infective agents must be serving essential functions in the infectious agent. It is also desirable that the target should be essential in a wide variety of infectious organisms. On the other hand, it would be preferable if the target function were absent in the host in order to reduce the risks of toxicity. These requirements are all fulfilled by plasmodial enzymes of the fatty acid biosynthetic pathway and of the recently discovered non-mevalonate pathway of isoprenoid precursors (Ralph et al., 2004).

Classical work by Bloch, Cornforth and Lynen on mammalian and yeast cells in the 1950s culminated in the elucidation of the mevalonate pathway for the biosynthesis of the universal isoprenoid precursors, isopentenyl diphosphate (IPP) (7) and dimethylallyl diphosphate (DMAPP) (8) via acetyl-CoA (11), 3-hydroxy-3-methylglutaryl-CoA (10) and 5-diphospho-mevalonate (9) (for review, see Qureshi and Porter, 1981; Bach, 1995; Bloch, 1992; Bochar et al., 1999) (Fig. 1). For several decades, this pathway was believed to be the universal source of IPP and DMAPP in all taxonomic kingdoms. However, it is now clear that the mevalonate pathway is the unique source for isoprenoids in animals and certain eubacteria, whereas many eubacteria including most human pathogens use exclusively the non-mevalonate pathway [for review, see Boucher and Doolittle, 2000; Eisenreich et al., 2004) (Fig. 1). Notable exceptions are Gram-positive cocci including Staphylococcus and Streptococcus spp. (Wilding et al., 2000). It is also important to note that apicoplast protozoa including Plasmodium depend exclusively on the non-mevalonate pathway (Gardner et al., 2002) which has already been shown to be a valid anti-malarial target (Jomaa et al., 1999; Missinou et al., 2002). Specifically, the isoprenoid side chains of essential quinones can only be made via the non-mevalonate pathway in all intraerythrocytic stages of Plasmodium falciparum (Cassera et al., 2004).

[FIGURE 1 OMITTED]

The non-mevalonate pathway starts with the condensation of D-glyceraldehyde 3-phosphate (2) and pyruvate (1) affording 1-deoxy-D-xylulose 5-phosphate (3) and carbon dioxide (Sprenger et al., 1997; Lois et al., 1998). A skeletal rearrangement of 3 affords the branched carbohydrate, 2C-methyl-D-erythrose 4-phosphate, which is then reduced under formation of 2C-methyl-D-erythritol 4-phosphate (4) (Takahashi et al., 1998). These consecutive reactions are both catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase (IspC protein). In subsequent steps, the catalytic action of IspDEFGH enzymes finally affords a mixture of IPP and DMAPP (Fig. 1; for review, see Eisenreich et al., 2004).

In the 1980s, the antibiotic compound, fosmidomycin, was discovered in extracts of Streptomyces lavendulae (Okuhara et al., 1980; Kuemmerle et al., 1985), but was subsequently abandoned. However, the compound attracted renewed interest, when it was shown that fosmidomycin acts as a potent inhibitor of IspC protein in the non-mevalonate biosynthetic pathway (Kuzuyama et al., 1998). A structure of the IspC protein complexed with fosmidomycin confirmed that the inhibitor acts as a substrate analogue that strongly binds to the active center of the enzyme (Steinbacher et al., 2003) (Fig. 2B). Indeed, the compound was able to cure malaria in laboratory studies with mice (Missinou et al., 2002) and in a clinical trial with 20 patients (Lell et al., 2003). Clinical studies using fosmidomycin in combination with the established anti-malarial antibiotic, clindamycin, showed that the mixture is highly efficient in the treatment of uncomplicated malaria with the exception of children < 3 years (Wiesner et al., 2003; Borrmann et al., 2006). In light of this recent result, improved inhibitors against IspC protein are desirable.

In this manuscript, we report on functional assays to test plant extracts for inhibitory activity against IspC protein. In a pilot screen using extracts from Mediterranean plants, we found strong inhibition of the IspC protein from Escherichia coli in leaf extracts of Cercis siliquastrum.

[FIGURE 2 OMITTED]

Materials and methods

Materials

The preparation of unlabeled 1-deoxy-D-xylulose 5-phosphate (3) and [3,4,5-[.sup.13.C.sub.3]]-3 has been described elsewhere (Hecht et al., 2001b; Illarionova et al., 2006). Recombinant 2C-methyl-D-erythritol 4-phosphate synthase (IspC protein) from E. coli was prepared according to published procedures (Hecht et al., 2001c). Fosmidomycin was obtained from Molecular Probes (Invitrogen).

Plant accession

Plants were collected at the Botanical Garden of Tel-Aviv University, in the Negev desert and in the Gaza Strip during 2003. When available in sufficient quantity, flower, fruit, inflorescence, seeds, leaves, and stems were obtained separately. Immediately after harvesting, the plant material was submerged in liquid nitrogen and brought to the laboratory for storage at -80[degrees]C until extracted.

Preparation of plant extracts

Aliquots (1 g) of frozen plant material were ground in a pre-chilled mortar containing liquid nitrogen. Two ml of 50% methanol/water (v/v) were added, and the slurry was mixed and kept on ice for 15 min. The mixture was centrifuged (11,000 rpm, 5 min, room temperature). The supernatant was stored at -80[degrees]C for analysis.

Photometric assay of IspC activity

Assay mixtures contained 100mM Tris hydrochloride, pH 8.0, 2mM EDTA, 16.7mM magnesium chloride, 2.5 mM NADPH, 2.4 [micro]g of bovine serum albumin, and 1.23 [micro]g of IspC protein in a volume of 120 [micro]l. Plant extract (0.02-20 [micro]l) was added, and the reaction was started by the addition of 60 [micro]l of a solution containing 100mM Tris hydrochloride, pH 8.0, and 8 mM 3. In photometric assays to estimate the IC-50 value of the positive reference compound, fosmidomycin, 0.03-20 [micro]l of 60 [micro]M fosmidomycin were added instead of plant extract. The mixtures were incubated in a thermostatted cuvette at 24[degrees]C, and absorbance at 340 nm was recorded.

NMR assay of IspC activity

Assay mixtures contained 75 mM Tris hydrochloride, pH 8.0, 10mM magnesium chloride, 1.2 mM EDTA, 6mM NADPH, 6mM [3,4,5-[.sup.13.C.sub.3]]-3, 7.2 [micro]g of bovine serum albumin, 3.7 [micro]g of IspC protein, and 20% [D.sub.2]O (v/v) in a total volume of 600 [micro]l. Plant extract (6 [micro]l) or 12 [micro]l of a solution of 50 [micro]M fosmidomycin (final concentration in the assay, 1 [micro]M) was added. The mixtures were incubated at 37[degrees]C for 45min, and the reaction was terminated by the addition of EDTA to a final concentration of 20 mM. Without any further work-up, [.sup.13.C] NMR spectra were recorded using a DRX 500 spectrometer from Bruker Instuments, Karlsruhe, Germany. The [.sup.13.C] NMR signals of [3,4,5-[.sup.13.C.sub.3]]-3 and [1,3,4-[.sup.13.C.sub.3]]-4 have been assigned earlier (Hecht et al., 2001b, c).

Results and discussion

The evolution of 2C-methyl-D-erythritol 4-phosphate synthase (IspC protein) has been relatively conservative. A sequence of the IspC protein from E. coli and the catalytic domain of the P. falciparum protein indicates 145 identical (36%) and 216 similar (54%) amino acid residues (Fig. 2A). Virtually all residues directly involved in the binding of the substrate and NADPH and in the coordination of the essential divalent metal ions (cf. Fig. 2B) (Hecht et al., 2001b) are identical (Fig. 2A). The protein of E. coli, which can be easily obtained by recombinant overexpression appears as a valid model for the screening of enzyme inhibitors that may serve as lead compounds for the development of novel anti-bacterial and anti-malarial agents.

The conversion of 1-deoxy-D-xylulose 5-phosphate (3) into 2C-methyl-D-erythritol 4-phosphate (4) by IspC protein is accompanied by the dehydrogenation of stoichiometric amounts of NADPH, which can be monitored photometrically. On this basis, the effects of potential inhibitory compounds or compound mixtures can be tested in a rapid screening process involving ordered compound libraries (Illarionova et al., 2006) or plant extracts (this study). To validate the assay, a solution of the known IspC inhihibitor, fosmidomycin, was used as a positive control. The robustness of the method was also controlled in assays using different concentrations of fosmidomycin in five replicates for each concentration. The statistical analysis showed an excellent R2 value of 0.9986 (Fig. 3A). Under the given experimental conditions (for details, see Methods), an IC-50 value of 370 nM can be estimated for fosmidomycin.

Plants growing under extreme conditions are considered to produce unique natural metabolites as a metabolic answer to stress factors (e.g., heat, dryness, and salinity). With this in mind, we initiated a study to search for inhibitors of the IspC protein in Mediterranean plants including many desert plants. Specifically, we collected about 200 Mediterranean plant species. All specimens were taxonomically assigned. Plant tissue was extracted as described under Methods in a standardized manner (for [.sup.1.H] NMR fingerprints, see Supplemental Material), and the extracts were screened photometrically for IspC activity. Plant extracts that showed significant enzyme inhibition were again assayed at progressively higher dilution.

[FIGURE 3 OMITTED]

The cutoff for significant inhibitory activity was arbitrarily set to less than 20% residual enzyme activity observed after 4-fold dilution of the plant extract (reflecting about 5 [micro]l of a methanolic extract using 1 g of plant material and 2 ml of solvent). This level of inhibition was observed with 12 extracts of the plants under study (Table 1). The highest level of inhibition was observed with a leaf extract from Cercis siliquas-trum. Specifically, the addition of 0.2 [micro]l of plant extract to an assay volume of 200 [micro]l reduced the reaction rate to less than 25% (Fig. 3B). An apparent IC-50 value that equals the IC-50 value of 370 nM for fosmidomycin was obtained with C. siliquastrum extract after 380-fold dilution.

To verify the photometric read-out, extracts of C. siliquastrum were re-assayed by NMR spectroscopy. In order to enhance the sensitivity and selectivity of that assay, the enzyme substrate was multiply labeled with [.sup.13.C] ([3,4,5-[.sup.13.C.sub.3]]-3). As a consequence of the specific [.sup.13]C-labeling, [.sup.13.C]-signals of C-3, C-4 and C-5 of the substrate and of C-1, C-3 and C-4 of the product, [1,3,4-[.sup.13.C.sub.3]]-4, were detected with high selectivity in the [.sup.13.C] NMR spectrum of the assay without any prior work-up. Moreover, the multiplet signatures resulting from [.sup.13.C] coupling of the multiply [.sup.13.C]-labeled substrate and product were highly characteristic and enabled the unequivocal signal assignment. Enzyme, co-substrate and EDTA present in the analyzed mixture all had natural [.sup.13.C] abundance (i.e., with 1.1% [.sup.13.C]), and the intensities of their [.sup.13.C] NMR signals were therefore approximately two orders of magnitude below those of the signals of the [.sup.13.C]-labeled compounds. With typical measuring times (approximately 45min), only signals from [.sup.13.C]-labelled positions were detected (cf. Fig. 4). All [.sup.13.C] NMR signals and [.sup.13.C][.sup.13.C] coupling constants of [3,4,5-[.sup.13.C.sub.3]]-3 and [1,3,4-[.sup.13.C.sub.3]]-4 have been reported earlier (Hecht et al., 2001a, b). The signals of [3,4,5-[.sup.13.C.sub.3]]-3 in an assay mixture without protein are shown in Fig. 4A. The signals detected in a standard assay without an inhibitory agent are shown in Fig. 4B. It is immediately obvious that the signals due to [3,4,5-[.sup.13.C.sub.3]]-3 disappeared and that three new signals due to [1,3,4-[.sup.13.C.sub.3]]-4 appeared since the substrate was completely converted into the product. The [.sup.13.C] NMR signals of the same assay mixture in the presence of 6 [micro]l of an extract from C. siliquastrum is shown in Fig. 4C. Signals due to [1,3,4-[.sup.13.C.sub.3]]-4 were not detected showing that the enzyme reaction is completely inhibited. Fig. 4D displays the NMR signals in the presence of the positive reference compound, fosmidomycin at a concentration of 1 [micro]M. Only minor signals due to [1,3,4-[.sup.13.C.sub.3]]-4 were observed demonstrating the validity of the approach.

[FIGURE 4 OMITTED]

The surprisingly high number of plant extracts that showed strong inhibition (12 out of approximately 200 extracts) suggests that Mediterranean plants may represent a rich source for natural inhibitors of IspC protein from E. coli and its orthologous proteins. The nature of the inhibitory principle(s) in the extract of C. siliquastrum and the other plants listed in Table 1 is still unknown. Notably, the plants shown to be active belong to different families and it is therefore difficult to identify a common principle under the aspects of phylogenetic relationships. However, on the basis of the limited phytochemical analyses of C. siliquastrum (Salatino et al., 2000; Torck et al., 1971), A. andrachne (Aburjai et al., 1999; Sakar et al., 1991) and some other plants listed in Table 1 (Danne et al., 1994; Fecka et al., 2001; Fayad and Al-Showiman, 1990; Meselhy et al., 1994), flavonoids or galloyl triterpenes appear to be major components in the plants tested positive. In this context, it is interesting to note that flavonoids were repeatedly reported to act as inhibitors of plasmodial growth under in vitro as well as under in vivo conditions (van Baren et al., 2006; Beldjoudi et al., 2003; Andrade-Neto et al., 2004; Andayi et al., 2006; Weniger et al., 2006).

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft. We also thank Mrs. Ulrike Stier (Bad Soden) and the Hans Fischer Gesellschaft e. V. for generous sponsoring of this research work.

Appendix A. Supplementary materials

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.phymed.2006.12.018.

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J. Kaiser (a,1), M. Yassin (b,1), S. Prakash (c,1), N. Safi (b), M. Agami (d), S. Lauw (a), E. Ostrozhenkova (a), A. Bacher (a), F. Rohdich (a), W. Eisenreich (a,*), J. Safi (b,**), A. Golan-Goldhirsh (c,**)

(a) Lehrstuhl fur Organische Chemie und Biochemie, Technische Universitat Munchen, Lichtenbergstr. 4, D-85747 Garching, Germany

(b) Environmental Protection and Research Institute, Gaza, Palestinian National Authority 

(c) Albert Katz Department of Dryland Biotechnologies, Ben Gurion University of the Negev, Jacob Blaustein Institutes for Desert Research, Sede Boqer Campus, Israel

(d) Institute for Cereal Crops Improvement, Faculty of Life Sciences, Tel Aviv University, Israel

Received 28 August 2006; accepted 21 November 2006

* Corresponding author. Tel.: [phone]; fax: [phone].

** Also to be corresponded to.

E-mail address: [e-mail] (W. Eisenreich).

(1) These authors contributed equally to this work.

Table 1. Plants with strong inhibitory activity against IspC protein
from E. coli

                     Relative IspC activity (%) after addition of
                     certain volumes ([micro]l) of plant extract
                     to the assay (a)
Species/family       5 [micro]l  2 [micro]l  0.5 [micro]l  0.2 [micro]l

Gymnocarpos           9          17          60            83
  decandrum
  (Caryophyllaceae)
Helianthemum          4          16          21            49
  ventosum
  (Cistaceae)
Helianthemum          8          11          45            58
  vesicarium
  (Cistaceae)
Zizphus              18          28          65            66
  spina-christi
  (L.) Desf.
  (Rhamnaceae)
Phoenix               3          10          72            81
  dactylifera L.
  (Palmae)
Geranium molle L.    14          20          17            58
  (Geraniaceae)
Erodium gruinum      16          13          47            63
  (L.) L'Her.
  (Geraniaceae)
Cistus               12          16          37            60
  salviifolius L.
  (Cistaceae)
Sarcopoterium        19          31          40            56
  spinosum (L.)
  Spach
  (Rosaceae)
Fumana thymifolia    15          34          80            89
  (L.) Webb
  (Cistaceae)
Arbutus              11          12          24            34
  andrachne L.
  (Ericaceae)
Cercis                4           8          10            26
  siliquastrum L.
  (Caesalpiniaceae)

(a) The accuracy of the photometric test was tested with the positive
reference compound, fosmidomycin (see Fig. 3A). On this basis, the
standard deviations can be estimated to be below 5%.