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• W2000596554 abstract "The emergence of drug-resistant forms of Plasmodium falciparum emphasizes the need to develop new antimalarials. In this context, the fatty acid biosynthesis (FAS) pathway of the malarial parasite has recently received a lot of attention. Due to differences in the fatty acid biosynthesis systems of Plasmodium and man, this pathway is a good target for the development of new and selective therapeutic drugs directed against malaria. In continuation of these efforts we report cloning and overexpression of P. falciparum β-hydroxyacyl-acyl carrier protein (ACP) dehydratase (PffabZ) gene that codes for a 17-kDa protein. The enzyme catalyzes the dehydration of β-hydroxyacyl-ACP to trans-2-acyl-ACP, the third step in the elongation phase of the FAS cycle. It has a K m of 199 μM and k cat/K m of 80.4 m-1 s-1 for the substrate analog β-hydroxybutyryl-CoA but utilizes crotonoyl-CoA, the product of the reaction, more efficiently (K m = 86 μM, k cat/K m = 220 m-1 s-1). More importantly, we also identify inhibitors (NAS-91 and NAS-21) for the enzyme. Both the inhibitors prevented the binding of crotonoyl-CoA to PfFabZ in a competitive fashion. Indeed these inhibitors compromised the growth of P. falciparum in cultures and inhibited the parasite fatty acid synthesis pathway both in cell-free extracts as well as in situ. We modeled the structure of PfFabZ using Escherichia coli β-hydroxydecanoyl thioester dehydratase (EcFabA) as a template. We also modeled the inhibitor complexes of PfFabZ to elucidate the mode of binding of these compounds to FabZ. The discovery of the inhibitors of FabZ, reported for the first time against any member of this family of enzymes, essential to the type II FAS pathway opens up new avenues for treating a number of infectious diseases including malaria. The emergence of drug-resistant forms of Plasmodium falciparum emphasizes the need to develop new antimalarials. In this context, the fatty acid biosynthesis (FAS) pathway of the malarial parasite has recently received a lot of attention. Due to differences in the fatty acid biosynthesis systems of Plasmodium and man, this pathway is a good target for the development of new and selective therapeutic drugs directed against malaria. In continuation of these efforts we report cloning and overexpression of P. falciparum β-hydroxyacyl-acyl carrier protein (ACP) dehydratase (PffabZ) gene that codes for a 17-kDa protein. The enzyme catalyzes the dehydration of β-hydroxyacyl-ACP to trans-2-acyl-ACP, the third step in the elongation phase of the FAS cycle. It has a K m of 199 μM and k cat/K m of 80.4 m-1 s-1 for the substrate analog β-hydroxybutyryl-CoA but utilizes crotonoyl-CoA, the product of the reaction, more efficiently (K m = 86 μM, k cat/K m = 220 m-1 s-1). More importantly, we also identify inhibitors (NAS-91 and NAS-21) for the enzyme. Both the inhibitors prevented the binding of crotonoyl-CoA to PfFabZ in a competitive fashion. Indeed these inhibitors compromised the growth of P. falciparum in cultures and inhibited the parasite fatty acid synthesis pathway both in cell-free extracts as well as in situ. We modeled the structure of PfFabZ using Escherichia coli β-hydroxydecanoyl thioester dehydratase (EcFabA) as a template. We also modeled the inhibitor complexes of PfFabZ to elucidate the mode of binding of these compounds to FabZ. The discovery of the inhibitors of FabZ, reported for the first time against any member of this family of enzymes, essential to the type II FAS pathway opens up new avenues for treating a number of infectious diseases including malaria. Malaria continues to exact the highest mortality and morbidity rate next only to tuberculosis. “The scourge of the tropics,” malaria is endemic to around 100 countries in the world. Approximately 500 million cases of malaria are reported every year, and around 3000 children die of malaria every day (1World Health OrganizationWorld Health Rep. 1999; : 49-63Google Scholar). Emerging resistance to chloroquine and other currently prescribed drugs limits treatment of malaria today, in particular cerebral malaria, caused by Plasmodium falciparum (2Sherman I.W. Malaria. American Society for, Washington, D. C.logy Press1998Google Scholar, 3Asindi A.A. Ekanem E.E. Ibia E.O. Nawangawa M.A. Trop. Geogr. Med. 1993; 45: 110-113PubMed Google Scholar). The situation definitely warrants express remedial actions: extensive research on P. falciparum to identify drug targets and, ultimately, the development of a new armamentarium of antimalarials. Our recent demonstration of the occurrence of the type II fatty acid synthesis (FAS) 1The abbreviations used are: FAS, fatty acid synthesis; FabZ, β-hydroxyacyl-ACP dehydratase; ACP, acyl carrier protein; PfFabZ, Plasmodium falciparum β-hydroxyacyl-ACP dehydratase; PfFabI, P. falciparum enoyl-ACP reductase; FabA, β-hydroxydecanoyl thioester dehydratase; RT, reverse transcriptase; Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high pressure liquid chromatography; EcFabA, Escherichia coli β-hydroxydecanoyl thioester dehydratase.1The abbreviations used are: FAS, fatty acid synthesis; FabZ, β-hydroxyacyl-ACP dehydratase; ACP, acyl carrier protein; PfFabZ, Plasmodium falciparum β-hydroxyacyl-ACP dehydratase; PfFabI, P. falciparum enoyl-ACP reductase; FabA, β-hydroxydecanoyl thioester dehydratase; RT, reverse transcriptase; Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high pressure liquid chromatography; EcFabA, Escherichia coli β-hydroxydecanoyl thioester dehydratase. pathway in the malaria parasite and its inhibition by triclosan, an inhibitor of the rate-limiting enzyme of type II FAS, enoyl-acyl carrier protein (ACP) reductase, proved the pivotal role played by this pathway in the survival of the malarial parasite (4Surolia N. Surolia A. Nat. Med. 2001; 7: 167-173Crossref PubMed Scopus (402) Google Scholar, 5Kapoor M. Dar M.J. Surolia A. Surolia N. Biochem. Biophys. Res. Commun. 2001; 289: 832-837Crossref PubMed Scopus (71) Google Scholar). The essential role of fatty acids and lipids in cell growth and differentiation and the different type (type I) of fatty acid biosynthetic pathway occurring in the human host, which is distinct from type II FAS of the malaria parasite, makes this pathway an attractive drug target for treating malaria (6Surolia N. RamachandraRao S.R. Surolia A. Bioessays. 2002; 24: 192-196Crossref PubMed Scopus (43) Google Scholar, 7Ramya T.N.C. Surolia N. Surolia A. Curr. Sci. 2002; 83: 101-108Google Scholar). The type II fatty acid biosynthesis pathway, found in most bacteria and plants, is typified by the existence of distinct enzymes encoded by unique genes for catalyzing each of the four individual chemical reactions required to complete successive cycles of fatty acid elongation (Refs. 4Surolia N. Surolia A. Nat. Med. 2001; 7: 167-173Crossref PubMed Scopus (402) Google Scholar, 8Weeks G. Wakil S.J. J. Biol. Chem. 1968; 243: 1180-1189Abstract Full Text PDF PubMed Google Scholar, and 9Marrakchi H. Zhang Y.-M. Rock C.O. Biochem. Soc. Trans. 2002; 30: 1050-1055Crossref PubMed Scopus (99) Google Scholar and Scheme I). This is in contrast to the type I FAS characterized by a multifunctional enzyme catalyzing all the steps of the pathway (10Rock C.O. Cronan J.E. Biochim. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (289) Google Scholar). The third step of the elongation cycle, the dehydration of β-hydroxyacyl-ACP to trans-2-acyl-ACP, is carried out by FabA or FabZ (11Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). While FabZ only catalyzes the dehydration reaction, FabA is a bifunctional enzyme and carries out an additional isomerization reaction of trans-2-acyl-ACP to cis-3-acyl-ACP, a reaction essential to unsaturated fatty acid synthesis (12Brock D.J.H. Kass L.R. Bloch K.J. J. Biol. Chem. 1967; 242: 4432-4440Abstract Full Text PDF PubMed Google Scholar). In Streptococcus pneumoniae, instead of FabA, a new enzyme, FabM (trans-2,cis-3-decenoyl-ACP isomerase), catalyzes the isomerization step of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP following the dehydration step by FabZ (13Marrakchi H. Choi K.-H. Rock C.O. J. Biol. Chem. 2002; 277: 44809-44816Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). FabZ is the primary dehydratase that participates in the elongation cycles of saturated as well as unsaturated fatty acid biosynthesis, whereas FabA is more active in the dehydration of β-hydroxydecanoyl-ACP (11Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 14Hoang T.T. Schweizer H.P. Am. Soc. Microbiol. 1997; 179: 5326-5332Google Scholar). This dichotomy allows the synthesis of unsaturated as well as saturated fatty acids, which eventually dictate the composition of the cell membranes. Dehydratase activity is crucial for the supply of trans-2-acyl-ACP to FabI, which pulls each cycle of elongation to completion (11Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 15Heath R.J. Rock C.O. J. Biol. Chem. 1995; 270: 26538-26542Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). FabZ thus presents itself as a suitable, yet unexplored target for the design of antimalarials. Here we report the cloning, expression, and characterization of P. falciparum FabZ (PfFabZ), the gene sequence of which has been deposited earlier by us in a public data base (GenBank™ accession number AY118082). FabZ was expressed as a soluble, active protein, and its purification was achieved by a single step purification protocol. We report its molecular and enzymatic properties. Further we have identified two lead compounds with inhibitory activity toward P. falciparum FabZ, which represents a significant advance in this area as no inhibitors of FabZ from any source are known to date. Crotonoyl-CoA, β-hydroxybutyryl-CoA, imidazole, kanamycin, and SDS-PAGE reagents were obtained from Sigma. Cesium carbonate, 2-bromo-4-chlorophenol, N-methylpyrrolidinone, and cuprous chloride were purchased from Aldrich. Media components were obtained from Hi-media (Delhi, India). All other chemicals used were of analytical grade. [1,2-14C]Acetic acid, sodium salt (specific activity, 60 mCi/mmol), and [2-14C]malonyl-CoA (specific activity, 54.2 mCi/mmol) were obtained from PerkinElmer Life Sciences. Escherichia coli DH5α cells were used during the cloning of the gene. pET-28a(+) vector (Novagen) and BL21(DE3) cells (Novagen) were used for the expression of FabZ. Total RNA was isolated from 10 ml of packed erythrocytes (infected with P. falciparum,10–12% parasitemia) after saponin lysis by a single step method of RNA isolation (16Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar). The isolated RNA was treated with RQ1 RNase-free DNase (Promega, 1 unit/μg of RNA) for 45 min at 37 °C and repurified by phenol-chloroform extraction and ethanol precipitation. RT-PCR was performed using a one step RT-PCR kit (Qiagen, Valencia, CA). PCR was performed with the primers (forward, 5′-GGAATTCCATATGAATTTAACCTTTCCTAATTATG-3′, and reverse, 5′-CGGGATCCTTATTTCGATAAGGCAAACGTCATTTC-3′ with NdeI and BamHI sites underlined, respectively). PCR conditions used were: 1 × (94 °C 5 min), 30 × (94 °C 1 min, 50 °C 1 min, 72 °C 1 min), 1 × (72 °C 10 min). The primers were designed using the GenBank™ accession number AF237572 to clone the mature protein (without the leader peptide and transit sequence, required for targeting of the protein to apicoplast). The 465-bp RT-PCR product was excised from a 1.2% agarose gel, purified using silica gel particles (QIAEX II gel extraction system, Qiagen), and cloned in pGEMT-Easy vector (Promega). Candidate plasmids containing the correct sized inserts were confirmed by digestion and dideoxy sequencing on an ABI Prism 377 semiadaptive sequencer Version 3.0. The insert present in pGEMT-Easy was reamplified using the abovementioned primers and subcloned in pET-28a(+) vector (Novagen) in-frame with the N terminus His6 tag. The constructs were transformed into E. coli BL21(DE3) cells (Novagen), and cultures were grown at 37 °C in Luria Broth (Hi-media) until A 600 of 0.6. These were induced with 1 mm isopropyl-β-d-thiogalactopyranoside and further incubated at 12 °C for 12 h. Cells were harvested at 8000 rpm for 10 min, and the resultant pellet was stored at -70 °C until further use. The pellet was resuspended in lysis buffer containing 20 mm Tris-Cl (pH 7.5), 0.5 m NaCl, and 5 mm imidazole. Cells were disrupted using a probe-type ultrasonicator (Vibra-Cell, Sonics and Materials). Cell debris were removed by centrifugation at 15,000 rpm for 30 min. The supernatant obtained was applied to a Ni-NTA metal affinity column (His-bind resin, Novagen) equilibrated with the lysis buffer. The column was initially washed with lysis buffer and subsequently washed with the same buffer containing 60 mm imidazole. The protein was eluted using a step gradient of 0.3–0.5 m imidazole, and fractions were tested for purity by SDS-PAGE. The protein was applied on a fast protein liquid chromatography desalting column to remove imidazole followed by concentration of the protein using Centriprep-10. Protein concentration was determined from A 280, assuming the molar extinction coefficient E280 = 9530 m-1 cm-1, which was calculated using the formula from Ref. 17Mulvey R.S. Gualtieri R.J. Beychok S. Biochemistry. 1974; 13: 782-787Crossref PubMed Scopus (28) Google Scholar. We further truncated the protein by removing 10 residues from the N terminus by using primers (forward, 5′-ACGTCCATGGTGCATCATCATCATCATCATATTGATATAGAAGATATTAAGAAAATTCTTCCACATAGATATCCTTTCC-3′, and reverse, 5′-CGGGATCCTTATTTCGATAAGGCAAACGTCATTTC-3′ with NcoI and BamHI sites underlined, respectively), and the PCR conditions used were as described above. The truncated gene was ligated into pET-28a(+) vector. The truncation led to an increase in the protein yield by 3-fold, i.e. from 5 to 15 mg/liter. Purified PfFabZ (2 mg/ml) was injected onto a Superdex™ 200 HR 10- × 300-mm column (Amersham Biosciences) equilibrated in 20 mm Tris, 500 mm NaCl (pH 7.5) connected to an ÄKTA™ design system. The column flow rate was maintained at 0.3 ml/min. The molecular weight of PfFabZ was determined by plotting V e/V 0 versus elution volume for standard proteins. V e corresponds to the peak elution volume of the standard protein, and V 0 represents the void volume of the column determined using blue dextran (M r < 2,000,000). Dynamic light scattering studies were performed on a Brookhaven Instruments Dynamic Light Scattering setup that can measure sizes from 2 to 4000 nm. The sample of PfFabZ (1 mg/ml) in 20 mm Tris, 500 mm NaCl (pH 7.5) was centrifuged at 14,000 rpm for 15 min and filtered through a 0.2-μ filter. The data acquisition time was 3 min. The routines used to fit the data points were cumulants, and non-negative least squares analysis was used to obtain the radius of gyration of PfFabZ. All experiments were carried out on a Jasco V-530 UV-visible spectrophotometer. FabZ was assayed at 25 °C by monitoring the forward as well as the reverse reaction, i.e. increase and decrease in A 260, respectively, due to the conversion of β-hydroxybutyryl-CoA to crotonoyl-CoA and vice versa. The standard reaction mixture in a total volume of 100 μl contained 20 mm Tris-HCl buffer, pH 7.5, 500 mm NaCl, 8 μg of FabZ, and 100 μm β-hydroxybutyryl-CoA or crotonoyl-CoA. The K m for each substrate analog was determined by varying its concentration. The kinetic parameters were obtained by fitting initial velocity data to the Michaelis-Menten equation by non-linear regression analysis using SigmaPlot 2000 software. The forward (k forward) and reverse (k reverse) rate constants were determined by using the following first order rate equation (Equation 1), log[S]=(-kt/2.3)+log[S]0(Eq. 1) where a plot of log[S] versus t is linear with a slope of -k/2.3 and an intercept of log[S]0 on the log[S] axis. [S] is the substrate concentration at time t, and k is the first order rate constant. The reaction was performed as described above and stopped by the addition of 3% chloroform, and the compounds were separated by reverse phase HPLC as described earlier (18DeBuysere M.S. Olson M.S. Anal. Biochem. 1983; 133: 373-379Crossref PubMed Scopus (49) Google Scholar). Briefly, the mixtures were loaded onto a Sephasil Peptide C18 column (4.6 mm × 25 cm; particle diameter, 5 μm; column volume, 4.155 ml; Amersham Biosciences), and the compounds were separated on a gradient of 220 mm phosphate buffer, pH 4.0 and methanol:chloroform (98:2). The retention times of β-hydroxybutyryl-CoA, crotonoyl-CoA, butyryl-CoA, and NADH were 14.8, 39.61, 48.47, and 4.31 min, respectively, at a flow rate of 0.5 ml/min. To determine the K m of PfFabZ for crotonoyl-CoA by HPLC, the peaks obtained while running different reaction mixtures were compared with the peaks of the standard run. For the FabZ-FabI coupled assay, PfFabI was purified according to published protocols (5Kapoor M. Dar M.J. Surolia A. Surolia N. Biochem. Biophys. Res. Commun. 2001; 289: 832-837Crossref PubMed Scopus (71) Google Scholar). The structure of FabZ is not available from any source, but P. falciparum FabZ shares 21% sequence identity with E. coli FabA. We modeled P. falciparum FabZ using E. coli FabA (Protein Data Bank code 1MKB) as the template. Modeling was done using MOE (Molecular Operating Environment) (19Inc Chemical Computing Group Molecular Operating Environment (MOE). Version 2001.07. Chemical Computing Group, Inc., Montreal, Quebec, Canada2001Google Scholar). Ten intermediate homology models were built as a result of the permutational selection of different loop candidates and side chain rotamers. The intermediate models were averaged to produce the final model by Cartesian average. We used Swiss-Pdb-Viewer to generate the dimeric structure with transformation matrix from the template structure (20Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9507) Google Scholar). Of the four compounds synthesized, NAS-21, NAS-75, and NAS-79 were synthesized using published procedures (21Penning T.D. Talley J.J. Bertenshaw S.R. Carter J.S. Collins P.W. Docter S. Graneto M.J. Lee L.F. Malecha J.W. Miyashiro J.M. Rogers R.S. Rogier D.J. Yu S.S. Anderson G.D. Burton E.G. Cogburn J.N. Gregory S.A. Koboldt C.M. Perkins W.E. Seibert K. Veenhuizen A.W. Zhang Y.Y. Isakson P.C. J. Med. Chem. 1997; 40: 1347-1365Crossref PubMed Scopus (1984) Google Scholar, 22Bebernitz G.R. Argentieri G. Battle B. Brennan C. Balkan B. Burkey B.F. Eckhardt M. Gao J. Kapa P. Strohschein R.J. Schuster H.F. Wilson M. Xu D.D. J. Med. Chem. 2001; 44: 2601-2611Crossref PubMed Scopus (135) Google Scholar). NAS-91 is a novel compound, therefore, its synthesis is described in detail below. Preparation of 4,4,4-Trifluoro-1-(4-nitrophenyl)butane-1,3-dione (NAS-21)—To a solution of ethyl trifluoroacetate (1.4 ml, 0.012 m) in methyl tert-butyl ether 25% NaOCH3 in methanol (4 ml, 1.5 eq) was added slowly. A solution of 4-nitroacetophenone (1.65 g, 0.01 m) in methyl tert-butyl ether was added to this mixture dropwise over 10 min. The reaction mixture was stirred at room temperature for 18 h. After the completion of the reaction the mixture was quenched with 3 n HCl. The organic layer was collected, washed with water and brine, and dried over Na2SO4, and the product (1.80 g) was crystallized from chloroform (70% yield). Preparation of 1-(4-Methoxyphenyl)ethanone [(4-Trifluoromethyl)pyrimidine-2-yl]hydrazone (NAS-75)—To 4-methoxyacetophenone (150 mg, 1 mm) in 5 ml of acetic acid and 1 ml of water 2-hydrazino-4-(trifluoromethyl)pyrimidine (178 mg, 1 mm) was added. Shortly after addition, a solid formed to which acetic acid was added to maintain stirring for 16 h. The reaction mixture was diluted with 4 ml of water, and subsequently the solid mass was filtered and dried in vacuo to afford 270 mg (90% yield) of the product. Preparation of 1-(4-Methylphenyl)ethanone [(4-Trifluoromethyl)pyrimidine-2-yl]hydrazone (NAS-79)—NAS-79 was prepared as in Ref. 22Bebernitz G.R. Argentieri G. Battle B. Brennan C. Balkan B. Burkey B.F. Eckhardt M. Gao J. Kapa P. Strohschein R.J. Schuster H.F. Wilson M. Xu D.D. J. Med. Chem. 2001; 44: 2601-2611Crossref PubMed Scopus (135) Google Scholar except that 4-methylacetophenone was taken instead of 4-methoxyacetophenone. Preparation of 4-Chloro-2-[(5-chloroquinolin-8-yl)oxy]phenol (NAS-91)—Cesium carbonate (2 mm, 651 mg) was added to 5-chloro-8-hydroxyquinoline (2 mm, 360 mg) in 5 ml of N-methylpyrrolidinone, and the slurry was heated with stirring under regular flow of N2 at 110 °C for 45 min. After cooling the reaction mixture to room temperature, 2-bromo-4-chlorophenol (1 mm, 207 mg) and copper (I) chloride (0.5 mm, 50 mg) was added. The flask was degassed and filled with N2 four to five times. The reaction mixture was heated at 140 °C for 70 h under the continuous flow of N2. The completion of the reaction was monitored by thin layer chromatography. After completion the reaction mixture was diluted 10 times with ethyl acetate and filtered through celite. The organic layer was washed with water and brine and dried over sodium sulfate. The product in the organic layer thus obtained was purified on a silica gel (100–200 mesh) column equilibrated in petroleum ether. Adsorbed material was eluted with a gradient of 1–12% ethyl acetate in petroleum ether. NAS-91 eluted at 10% ethyl acetate in petroleum ether. Yield 45% (136 mg); 1H NMR (CDCl3) δ 8.9 (d, 1H); 8.63 (d, J = 8.4 Hz, 1H); 7.64–7.56 (m, 3H); 7.23 (d, J = 8.4 Hz, 1H); 7.01 (d, J = 17.7 Hz, 1H); 6.80 (d, J = 8.4 Hz, 1H). 13C NMR (CDCl3) δ 153.09, 150.68, 149.66, 144.72, 141.17, 134.70, 132.32, 131.20, 127.28, 122.83, 122.50, 120.02, 119.91, 118.81, 115.93. Mass spectra found 306.1, calculated for C15H9O2NCl2 306.0. Analysis (C H N) found C, 58.38; H, 3.07; N, 4.56; calculated for C15H9O2NCl2; C, 58.85; H, 2.96; N, 4.58. The inhibition of PfFabZ activity was monitored by the spectrophotometric assay performed as described above except that a given inhibitor was added prior to the initiation of the reaction by addition of crotonoyl-CoA. The studies were performed in the presence of 1% Me2SO used for solubilizing the inhibitors. The inhibition constant K i was determined by the Dixon plot and the Lineweaver-Burk equation. In Dixon's method (23Dixon M. Biochem. J. 1953; 55: 170-171Crossref PubMed Scopus (3280) Google Scholar), the enzyme activity was measured at four different concentrations of substrate (50, 75, 100, and 125 μm) as a function of the inhibitor concentration (0.1–2.5 μm). For the determination of K i by the Lineweaver-Burk equation the enzyme assays were performed in the absence and presence (1 μm) of each inhibitor at a range of substrate concentrations (10–60 μm). Fluorescence measurements were performed on a Jobin-Yvon Horiba fluorimeter under computer control. The excitation and emission monochromator slit widths were 3 and 5 nm, respectively. Measurements were performed at 25 °C in a 3-ml quartz cuvette, and the solutions were mixed continuously on a magnetic stirrer. The solutions containing PfFabZ were excited at 280 nm, and the emission was recorded from 300 to 500 nm. For inhibitor binding studies, PfFabZ (22 μm) in 20 mm Tris, 500 mm NaCl, pH 7.5 was titrated with different concentrations of the inhibitors (0–8.3 μm). The magnitude of fluorescence decrease (F 0 - F) upon addition of each inhibitor concentration was fitted to Equation 2 to determine the value of K i, (F0-F)=ΔFmax/{1+(Ki/[I])}(Eq. 2) Corrections for the inner filter effect were performed according to Equation 3 (24Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 44-45Google Scholar), Fc=Fantilog[(Aex+Aem)/2](Eq. 3) where F c and F are the corrected and measured fluorescence intensities, respectively. A ex and A em are the solution absorbances at the excitation and emission wavelengths, respectively. K i represents the dissociation constant of the inhibitor for PfFabZ. ΔG, the change in free energy upon binding of inhibitor to the protein, was calculated from Equation 4, ΔG=-RTlnKb(Eq. 4) where R is the gas constant, T is the temperature in Kelvin, and K b is the binding constant. The experiments were performed using P. falciparum FCK2 strain (chloroquine-sensitive, IC50, 18 nm), an isolate from Karnataka, India. P. falciparum was cultured using standard techniques (25Trager W. Jenson J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6121) Google Scholar) and routinely synchronized using 5% sorbitol (26Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2813) Google Scholar). Growth inhibition by the compounds of interest was assessed by [3H]hypoxanthine uptake. Typically uninfected or infected (1–2% parasitemia) red blood cells (2% hematocrit) were added to the culture medium in the wells of a 96-well plate (Nunc), and different concentrations of inhibitor in Me2SO were added such that the final concentration of Me2SO did not exceed 0.05%. The experiment was started with the synchronized parasite culture in the early trophozoite stage, and [3H]hypoxanthine was added to the culture after incubation with the inhibitors for 48 h. The culture from the 96-well plate was harvested after 31 h (27Arnot D.E. Gull K. Ann. Trop. Med. Parasitol. 1998; 92: 361-365Crossref PubMed Scopus (80) Google Scholar, 28Suarez J.E. Urquiza M. Curtidor H. Rodriguez L.E. Ocampo M. Torres E. Guzman F. Patarroyo M.E. Mem. Inst. Oswaldo Cruz. 2000; 95: 495-501Crossref PubMed Scopus (13) Google Scholar) using a Nunc cell harvester, and P. falciparum growth was assessed by measuring the incorporation of [3H]hypoxanthine (29Ancelin M.L. Calas M. Bompart J. Cordina G. Martin D. Ben Bari M. Jei T. Druilhe P. Vial H.J. Blood. 1998; 91: 1426-1437Crossref PubMed Google Scholar) using a liquid scintillation counter (Wallac). The IC50 was calculated from a plot of relative percent parasitemia versus log concentration of the inhibitor by fitting it to non-linear regression analysis using SigmaPlot 2000 software. The in vitro fatty acid synthesis was performed as described earlier (4Surolia N. Surolia A. Nat. Med. 2001; 7: 167-173Crossref PubMed Scopus (402) Google Scholar, 30Bergler H. Fuchsbichler S. Hogenauer G. Turnowsky F. Eur. J. Biochem. 1996; 242: 689-694Crossref PubMed Scopus (131) Google Scholar). Trophozoites isolated from 100-ml cultures with 8–10% parasitemia were resuspended and sonicated in 0.2 ml of 70 mm potassium phosphate buffer (pH 7.0) for 5 s and centrifuged at 48,000 × g for 1 h at 4 °C. The supernatant fraction was used as crude extract for determining the in vitro fatty acid synthesis. The assay mixture in 70 mm potassium phosphate (pH 7.0) contained 1.4 mm dithiothreitol, 20 μm acetyl-CoA, 3.6 mm glucose 6-phosphate, 0.14 mm EDTA, 200 μm NADH, 200 μm NADPH, 1 unit of glucose-6-phosphate dehydrogenase, 250 μg of parasite protein, and 80 μm [2-14C]malonyl-CoA (4Surolia N. Surolia A. Nat. Med. 2001; 7: 167-173Crossref PubMed Scopus (402) Google Scholar). The reaction was initiated by addition of solution containing 120 μg of freshly reduced ACP. The reaction mixture was incubated for 35 min with or without 10 μm NAS-91 or NAS-21, which was added just before the addition of ACP, at 37 °C. The reaction mixture was then treated with 4 m HCl at 100 °C for 2 h. The fatty acids were extracted in chloroform and methylated at 4 °C with diazomethane in ether, and the incorporation of [2-14C]malonyl-CoA into fatty acids was monitored by thin layer chromatography on silanized silica thin layer plates (Merck) developed with a solvent system of acetone:water:methanol:acetic acid (70:50:35:1) as described previously (4Surolia N. Surolia A. Nat. Med. 2001; 7: 167-173Crossref PubMed Scopus (402) Google Scholar, 30Bergler H. Fuchsbichler S. Hogenauer G. Turnowsky F. Eur. J. Biochem. 1996; 242: 689-694Crossref PubMed Scopus (131) Google Scholar). Inhibitors (10 and 100 μm) were added to P. falciparum cultures (100 ml, 9–10% parasitemia) for 70 h after which cultures were resuspended in 7 ml of the complete medium while retaining the same concentrations of the inhibitors. To this [1,2-14C]acetic acid was added (50 μCi/ml) (4Surolia N. Surolia A. Nat. Med. 2001; 7: 167-173Crossref PubMed Scopus (402) Google Scholar). After 2 h, parasites were isolated, washed thoroughly with phosphate-buffered saline, lysed, sonicated, spotted onto a Whatman No. 3MM paper disc, and counted using the scintillation fluid (4Surolia N. Surolia A. Nat. Med. 2001; 7: 167-173Crossref PubMed Scopus (402) Google Scholar). The data reported are from an average of two independent duplicate experiments. Preparation of the Ligand and Receptor Molecules—The protein target and the ligands were prepared for docking using AutoDock Version 3.05 (31Goodsell D.S. Morris G.M. Olson A.J. J. Mol. Recognit. 1996; 9: 1-5Crossref PubMed Scopus (1267) Google Scholar, 32Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9050) Google Scholar). Charges were assigned using the Kollman algorithm (33Chiche L. Gregoret L.M. Cohen F.E. Kollman P.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3240-3243Crossref PubMed Scopus (102) Google Scholar). Atomic solvation parameters and fragmental volumes were determined using the Addsol program. AutoTors was used to define torsion angles in the ligand. Polar hydrogen charges of the Gasteiger type (34Gasteiger J. Marsili M. Tetrahedron. 1980; 36: 3219-3228Crossref Scopus (3563) Google Scholar) were assigned, and the non-polar hydrogens were merged with the c" @default.
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