Characterization and comparative studies of zebrafish and human recombinant dihydrofolate reductases--inhibition by folic acid and polyphenols.
Dihydrofolate reductase (DHFR) catalyzes folic acid reduction and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is the main target of methotrexate, the most widely used agent for antifolate therapy. Nevertheless, the emergence of methotrexate-resistance has greatly impeded the curative potential of this drug. Therefore, drugs with improved efficacy are still in demand, as well as an efficient in vitro assay system and animal model for antifolate drug discovery. The aim of this study is to evaluate the suitability of using zebrafish DHFR as an alternative assay system for antifolate drug discovery. The cDNAs encoding zebrafish and human DHFR were cloned, overexpressed, and purified. Similar structural and kinetic properties were revealed between zebrafish and human recombinant DHFRs. The susceptibilities of both enzymes to known DHFR inhibitors, including methotrexate and trimethoprim, and compounds with antifolate potential, such as polyphenols, are also comparable. In addition, the DHFR-mediated dihydrofolate reduction was significantly inhibited by its own substrate folic acid. An unexpected tissue-specific distribution of DHFR was observed with the highest level present in ova and brains of zebrafish. DHFR is also abundant in zebrafish embryos of early stages and decreased abruptly after 3 days postfertilization. The substantial resemblance between zebrafish and human DHFRs, as demonstrated in this study, provides compelling evidence supporting the use of zebrafish DHFR as an in vitro assay system for folate-related studies and drug discovery.
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan.
Tseng-Ting
TT
Kao
Kuan-Chieh
KC
Wang
Wen-Ni
WN
Chang
Chia-Ying
CY
Lin
Bing-Hung
BH
Chen
Hua-Lin
HL
Wu
Guey-Yueh
GY
Shi
Jen-Ning
JN
Tsai
Tzu-Fun
TF
Fu
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology*; D005492:Folic Acid / Q000737:chemistry / Q000494:pharmacology*; D005493:Folic Acid Antagonists / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D010636:Phenols / Q000737:chemistry / Q000494:pharmacology*; D059808:Polyphenols; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish
D018076:DNA, Complementary; D005419:Flavonoids; D005493:Folic Acid Antagonists; D010636:Phenols; D059808:Polyphenols; D011994:Recombinant Proteins; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase