The online version of this article (doi:10.1186/1475-2875-11-194) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
KS performed the study and drafted the manuscript. CT and TV provided anti-folates. YY discussed and commented on the manuscript. PC and UL conceived of the study and drafted the manuscript. All authors read and approved the final manuscript.
There is an urgent need for the discovery of new anti-malarial drugs. Thus, it is essential to explore different potential new targets that are unique to the parasite or that are required for its viability in order to develop new interventions for treating the disease. Plasmodium serine hydroxymethyltransferase (SHMT), an enzyme in the dTMP synthesis cycle, is a potential target for such new drugs, but convenient methods for producing and assaying the enzyme are still lacking, hampering the ability to screen inhibitors.
Production of recombinant Plasmodium falciparum SHMT (PfSHMT) and Plasmodium vivax SHMT (PvSHMT), using auto-induction media, were compared to those using the conventional Luria Bertani medium with isopropyl thio-β-D-galactoside (LB-IPTG) induction media. Plasmodium SHMT activity, kinetic parameters, and response to inhibitors were measured spectrophotometrically by coupling the reaction to that of 5,10-methylenetetrahydrofolate dehydrogenase (MTHFD). The identity of the intermediate formed upon inactivation of Plasmodium SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). The active site environment of Plasmodium SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate.
Auto-induction media resulted in a two to three-fold higher yield of Pf- and PvSHMT (7.38 and 29.29 mg/L) compared to that produced in cells induced in LB-IPTG media. A convenient spectrophotometric activity assay coupling Plasmodium SHMT and MTHFD gave similar kinetic parameters to those previously obtained from the anaerobic assay coupling SHMT and 5,10-methylenetetrahydrofolate reductase (MTHFR); thus demonstrating the validity of the new assay procedure. The improved method was adopted to screen for Plasmodium SHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase. Plasmodium SHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLP-thiosemicarbazone.
Auto-induction media offers a cost-effective method for the production of Plasmodium SHMTs and should be applicable for other Plasmodium enzymes. The SHMT-MTHFD coupled assay is equivalent to the SHMT-MTHFR coupled assay, but is more convenient for inhibitor screening and other studies of the enzyme. In addition to inhibitors of malarial SHMT, the development of species-specific, anti-SHMT inhibitors is plausible due to the presence of differential active sites on the Plasmodium enzymes.
Additional file 1: Chemical structures of anti-folates (concentration indicated) and amino acid analogues (1 mM) and their inhibition activities. NA; no inhibition activity. (A) 2,4-diaminopyrimidine anti-folates. (B) amino acid analogues. (DOC 172 KB)12936_2012_2487_MOESM1_ESM.doc
Authors’ original file for figure 112936_2012_2487_MOESM2_ESM.pdf
Authors’ original file for figure 212936_2012_2487_MOESM3_ESM.pdf
Authors’ original file for figure 312936_2012_2487_MOESM4_ESM.pdf
Authors’ original file for figure 412936_2012_2487_MOESM5_ESM.pdf
Authors’ original file for figure 512936_2012_2487_MOESM6_ESM.pdf
Lucumi E, Darling C, Jo H, Napper AD, Chandramohanadas R, Fisher N, Shone AE, Jing H, Ward SA, Biagini GA, DeGrado WF, Diamond SL, Greenbaum DC: Discovery of potent small-molecule inhibitors of multidrug-resistantPlasmodium falciparumusing a novel miniaturized high-throughput luciferase-based assay. Antimicrob Agents Chemother. 2010, 54: 3597-3604. 10.1128/AAC.00431-10. PubMedCentralCrossRefPubMed
Coteron JM, Marco M, Esquivias J, Deng X, White KL, White J, Koltun M, El Mazouni F, Kokkonda S, Katneni K, Bhamidipati R, Shackleford DM, Angulo- Barturen I, Ferrer SB, Jiménez-Díaz MB, Gamo FJ, Goldsmith EJ, Charman WN, Bathurst I, Floyd D, Matthews D, Burrows JN, Rathod PK, Charman SA, Phillips MA: Structure-guided lead optimization of triazolopyrimidine-ring substituents identifies potent Plasmodium falciparum dihydroorotate dehydrogenase inhibitors with clinical candidate potential. J Med Chem. 2011, 54: 5540-5561. 10.1021/jm200592f. PubMedCentralCrossRefPubMed
Olliaro P, Yuthavong V: An overview of chemotherapeutic targets for antimalarial drug discovery. Pharmacol Ther. 1999, 89: 91-110. CrossRef
Schirch V: Folates in serine and glycine metabolism. Folate and pterins. Edited by: Blankley RL, Benkovic SJ. 1984, New York: Wiley, 399-431.
Manohar R, Ramesh KS, Rao NA: Purification, physicochemical and regulatory properties of serine hydroxymethyltransferase from sheep liver. J Biosci. 1982, 4: 31-50. 10.1007/BF02702579. CrossRef
Daidone F, Florio R, Rinaldo S, Contestabile R, di Salvo M, Cutruzzolà F, Bossa F, Paiardini A: In silico andin vitrovalidation of serine hydroxymethyltransferase as a chemotherapeutic target of the antifolate drug pemetrexed. Eur J Med Chem. 2011, 46: 1616-1621. 10.1016/j.ejmech.2011.02.009. CrossRefPubMed
Franca TC, Pascutti PG, Ramalho TC, Figueroa-Villar JD: A three-dimensional structure ofPlasmodium falciparumserine hydroxymethyltransferase in complex with glycine and 5-formyl-tetrahydrofolate. Homology modeling and molecular dynamics. Biophys Chem. 2005, 115: 1-10. 10.1016/j.bpc.2004.12.002. CrossRefPubMed
Sirichaiwat C: Target guided synthesis of 5-benzyl-2,4-diaminopyrimidine antimalarials.PhD thesis. 2003, Mahidol University: Chemistry Department
Sirichaiwat C, Intaraudom C, Kamchonwongpaisan S, Vanichtanankul J, Thebtaranonth Y, Yuthavong Y: Target guided synthesis of 5-benzyl-2,4-diaminopyrimidines: their antimalarial activities and binding affinities to wild type and mutant dihydrofolate reductases fromPlasmodium falciparum. J Med Chem. 2004, 47: 345-354. 10.1021/jm0303352. CrossRefPubMed
Yuthavong Y, Vilaian T, Kamchonwongpaisan S, Tarnchompoo B, Thongpanchang C, Chitnumsub P, Yuvaniyama J, Matthews D, Charman W, Charman S, Vivas L, Katiyar SB: Antimalarial compounds with flexible side-chains. US patent application no. 20090099220. Medicines for Malaria Venture. 2009
Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976, 72: 245-248. CrossRef
SIB Bioinformatics Resource Portal. [ http://web.expasy.org/protparam/]
Petrovskaya LE, Shulga AA, Bocharova OV, Ermolyuk YS, Kryukova EA, Chupin VV, Blommers MJ, Arseniev AS, Kirpichnikov MP: Expression of G-protein coupled receptors inEscherichia colifor structural studies. Biochemistry (Mosc). 2010, 75: 881-891. 10.1134/S0006297910070102. CrossRef
Wang P, Wang Q, Yang Y, Coward JK, Nzila A, Sims PF, Hyde JE: Characterisation of the bifunctional dihydrofolate synthase-folylpolyglutamate synthase fromPlasmodium falciparum; a potential novel target for antimalarial antifolate inhibition. Mol Biochem Parasitol. 2010, 172: 41-51. 10.1016/j.molbiopara.2010.03.012. PubMedCentralCrossRefPubMed
Taylor RT, Weissbach H: Radioactive assay for serine transhydroxymethylase. Anal Biochem. 1965, 13: 80-84. 10.1016/0003-2697(65)90120-X. CrossRef
Acharya JK, Rao NA: A novel intermediate in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase. J Biol Chem. 1992, 267: 19066-19071. PubMed
Khomutov RM, Dixon HB, Vdovina LV, Kirpichnikov MP, Morozov YV, Severin ES, Khurs EN: N-(5'-phosphopyridoxyl)glutamic acid and N-(5'-phosphopyridoxyl)-2-oxopyrrolidine-5-carboxylic acid and their action on the apoenzyme of aspartate aminotransferase. Biochem J. 1971, 124: 99-106. PubMedCentralCrossRefPubMed
- Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay
- BioMed Central
Neu im Fachgebiet Innere Medizin
Meistgelesene Bücher aus der Inneren Medizin
Mail Icon II