title: Cloning, expression, and characterization of Babesia gibsoni dihydrofolate reductase-thymidylate synthase: inhibitory effect of antifolates on its catalytic activity and parasite proliferation.
abstract: Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed K(m) values of 4.70 +/- 0.059 (mean +/- standard error of the mean) and 9.75 +/- 1.64 microM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC(50)] = 68.6 +/- 5.20 nM) than pyrimethamine (IC(50) = 55.0 +/- 2.08 microM) and trimethoprim (IC(50) = 50 +/- 12.5 microM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan.
authors.forename: Gabriel O
authors.initials: GO
authors.lastname: Aboge
authors.forename: Honglin
authors.initials: H
authors.lastname: Jia
authors.forename: Mohamad A
authors.initials: MA
authors.lastname: Terkawi
authors.forename: Youn-Kyoung
authors.initials: YK
authors.lastname: Goo
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Nishikawa
authors.forename: Fujiko
authors.initials: F
authors.lastname: Sunaga
authors.forename: Kuzuhiko
authors.initials: K
authors.lastname: Namikawa
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Tsuji
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
authors.forename: Kozo
authors.initials: K
authors.lastname: Fujisaki
authors.forename: Xuenan
authors.initials: X
authors.lastname: Xuan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D001403:Babesia / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005493:Folic Acid Antagonists / Q000494:pharmacology; D015870:Gene Expression; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics*
chemical_list: D000981:Antiprotozoal Agents; D017931:DNA Primers; D016054:DNA, Protozoan; D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase
references.citation: Vet Clin North Am Small Anim Pract. 2003 Jul;33(4):885-904, viii
references.pmid: 12910748
references.citation: J Med Chem. 2006 Feb 9;49(3):1055-65
references.pmid: 16451071
references.citation: Antimicrob Agents Chemother. 2000 Apr;44(4):1047-50
references.pmid: 10722510
references.citation: Vet Parasitol. 2007 Oct 21;149(1-2):85-94
references.pmid: 17706873
references.citation: Antimicrob Agents Chemother. 2005 Sep;49(9):3652-7
references.pmid: 16127035
references.citation: Mol Biochem Parasitol. 1997 Feb;84(2):203-14
references.pmid: 9084040
references.citation: Mol Biochem Parasitol. 2006 Aug;148(2):190-8
references.pmid: 16675041
references.citation: Antimicrob Agents Chemother. 1996 Jul;40(7):1628-32
references.pmid: 8807052
references.citation: Int J Parasitol. 1990 Jul;20(4):503-13
references.pmid: 2210944
references.citation: Int J Parasitol. 1993 May;23(3):399-402
references.pmid: 8359989
references.citation: Annu Rev Biochem. 1995;64:721-62
references.pmid: 7574499
references.citation: Antimicrob Agents Chemother. 2006 Oct;50(10):3435-43
references.pmid: 17005826
references.citation: J Biol Chem. 1993 Oct 15;268(29):21637-44
references.pmid: 8408015
references.citation: DNA Res. 2005 Feb 28;12(1):53-62
references.pmid: 16106752
references.citation: J Clin Microbiol. 2001 Jul;39(7):2603-9
references.pmid: 11427577
references.citation: Antimicrob Agents Chemother. 1991 Jul;35(7):1348-55
references.pmid: 1929292
references.citation: J Med Chem. 1998 Apr 23;41(9):1367-70
references.pmid: 9554869
references.citation: Int J Parasitol. 2007 Jun;37(7):787-93
references.pmid: 17320089
references.citation: Antimicrob Agents Chemother. 2003 Feb;47(2):800-3
references.pmid: 12543697
references.citation: Pharmacotherapy. 2004 May;24(5):673-6
references.pmid: 15162903
references.citation: Biochemistry. 1987 Dec 29;26(26):8606-13
references.pmid: 3126806
references.citation: Mol Biochem Parasitol. 1991 Apr;45(2):317-30
references.pmid: 2038362
references.citation: Gene. 1998 Apr 28;211(1):177-85
references.pmid: 9573357
references.citation: J Vet Med Sci. 2001 Sep;63(9):977-81
references.pmid: 11642286
references.citation: J Vet Med Sci. 2002 Jul;64(7):571-5
references.pmid: 12185309
references.citation: Exp Parasitol. 1990 Apr;70(3):367-71
references.pmid: 2178951
references.citation: Antimicrob Agents Chemother. 2005 Sep;49(9):3948-51
references.pmid: 16127079
references.citation: Antimicrob Agents Chemother. 2001 Sep;45(9):2517-23
references.pmid: 11502523
references.citation: Vet Parasitol. 2006 Oct 10;141(1-2):177-80
references.pmid: 16765518
references.citation: Vector Borne Zoonotic Dis. 2007 Summer;7(2):163-6
references.pmid: 17627433
references.citation: Protein Eng Des Sel. 2005 Oct;18(10):457-64
references.pmid: 16120637
references.citation: J Biochem Mol Biol. 2004 Jul 31;37(4):474-9
references.pmid: 15469736
references.citation: Mol Biochem Parasitol. 2004 Feb;133(2):209-19
references.pmid: 14698433
references.citation: Pharmacogenomics. 2005 Jan;6(1):17-26
references.pmid: 15723602
references.citation: Am J Trop Med Hyg. 1988 Oct;39(4):328-36
references.pmid: 3142285
references.citation: J Chemother. 2004 Feb;16(1):3-12
references.pmid: 15077993
references.citation: Mol Biochem Parasitol. 1990 Jun;41(2):259-68
references.pmid: 2204832
references.citation: Science. 2003 Aug 29;301(5637):1196-202
references.pmid: 12947189
references.citation: Parasitology. 2007 Aug;134(Pt 9):1185-94
references.pmid: 17381888
references.citation: Int J Parasitol. 2000 Dec;30(14):1501-5
references.pmid: 11428342
references.citation: Biochemistry. 1990 Dec 4;29(48):10779-85
references.pmid: 2176883
references.citation: Plant Mol Biol. 1999 Dec;41(6):733-9
references.pmid: 10737138
references.citation: Oncology. 1998 Nov-Dec;55(6):564-8
references.pmid: 9778624
references.citation: Parasitology. 1994 Feb;108 ( Pt 2):147-52
references.pmid: 8159459
references.citation: J Antimicrob Chemother. 2006 Jun;57(6):1043-54
references.pmid: 16617066
references.citation: Trans R Soc Trop Med Hyg. 1986;80(2):201-3
references.pmid: 3538522
references.citation: Parasitol Res. 1991;77(4):346-50
references.pmid: 1866423
references.citation: Biochem Pharmacol. 1999 Nov 15;58(10):1529-37
references.pmid: 10535743
references.citation: Anal Biochem. 1987 Apr;162(1):156-9
references.pmid: 2440339