Cloning, expression, and characterization of Babesia gibsoni dihydrofolate reductase-thymidylate synthase: inhibitory effect of antifolates on its catalytic activity and parasite proliferation.
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.
Aboge
Gabriel O
GO
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan.
Jia
Honglin
H
Terkawi
Mohamad A
MA
Goo
Youn-Kyoung
YK
Nishikawa
Yoshifumi
Y
Sunaga
Fujiko
F
Namikawa
Kuzuhiko
K
Tsuji
Naotoshi
N
Igarashi
Ikuo
I
Suzuki
Hiroshi
H
Fujisaki
Kozo
K
Xuan
Xuenan
X
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*
D000981:Antiprotozoal Agents; D017931:DNA Primers; D016054:DNA, Protozoan; D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase
Vet Clin North Am Small Anim Pract. 2003 Jul;33(4):885-904, viii
12910748
J Med Chem. 2006 Feb 9;49(3):1055-65
16451071
Antimicrob Agents Chemother. 2000 Apr;44(4):1047-50
10722510
Vet Parasitol. 2007 Oct 21;149(1-2):85-94
17706873
Antimicrob Agents Chemother. 2005 Sep;49(9):3652-7
16127035
Mol Biochem Parasitol. 1997 Feb;84(2):203-14
9084040
Mol Biochem Parasitol. 2006 Aug;148(2):190-8
16675041
Antimicrob Agents Chemother. 1996 Jul;40(7):1628-32
8807052
Int J Parasitol. 1990 Jul;20(4):503-13
2210944
Int J Parasitol. 1993 May;23(3):399-402
8359989
Annu Rev Biochem. 1995;64:721-62
7574499
Antimicrob Agents Chemother. 2006 Oct;50(10):3435-43
17005826
J Biol Chem. 1993 Oct 15;268(29):21637-44
8408015
DNA Res. 2005 Feb 28;12(1):53-62
16106752
J Clin Microbiol. 2001 Jul;39(7):2603-9
11427577
Antimicrob Agents Chemother. 1991 Jul;35(7):1348-55
1929292
J Med Chem. 1998 Apr 23;41(9):1367-70
9554869
Int J Parasitol. 2007 Jun;37(7):787-93
17320089
Antimicrob Agents Chemother. 2003 Feb;47(2):800-3
12543697
Pharmacotherapy. 2004 May;24(5):673-6
15162903
Biochemistry. 1987 Dec 29;26(26):8606-13
3126806
Mol Biochem Parasitol. 1991 Apr;45(2):317-30
2038362
Gene. 1998 Apr 28;211(1):177-85
9573357
J Vet Med Sci. 2001 Sep;63(9):977-81
11642286
J Vet Med Sci. 2002 Jul;64(7):571-5
12185309
Exp Parasitol. 1990 Apr;70(3):367-71
2178951
Antimicrob Agents Chemother. 2005 Sep;49(9):3948-51
16127079
Antimicrob Agents Chemother. 2001 Sep;45(9):2517-23
11502523
Vet Parasitol. 2006 Oct 10;141(1-2):177-80
16765518
Vector Borne Zoonotic Dis. 2007 Summer;7(2):163-6
17627433
Protein Eng Des Sel. 2005 Oct;18(10):457-64
16120637
J Biochem Mol Biol. 2004 Jul 31;37(4):474-9
15469736
Mol Biochem Parasitol. 2004 Feb;133(2):209-19
14698433
Pharmacogenomics. 2005 Jan;6(1):17-26
15723602
Am J Trop Med Hyg. 1988 Oct;39(4):328-36
3142285
J Chemother. 2004 Feb;16(1):3-12
15077993
Mol Biochem Parasitol. 1990 Jun;41(2):259-68
2204832
Science. 2003 Aug 29;301(5637):1196-202
12947189
Parasitology. 2007 Aug;134(Pt 9):1185-94
17381888
Int J Parasitol. 2000 Dec;30(14):1501-5
11428342
Biochemistry. 1990 Dec 4;29(48):10779-85
2176883
Plant Mol Biol. 1999 Dec;41(6):733-9
10737138
Oncology. 1998 Nov-Dec;55(6):564-8
9778624
Parasitology. 1994 Feb;108 ( Pt 2):147-52
8159459
J Antimicrob Chemother. 2006 Jun;57(6):1043-54
16617066
Trans R Soc Trop Med Hyg. 1986;80(2):201-3
3538522
Parasitol Res. 1991;77(4):346-50
1866423
Biochem Pharmacol. 1999 Nov 15;58(10):1529-37
10535743
Anal Biochem. 1987 Apr;162(1):156-9
2440339