Abstract
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.
Footnotes
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Our sincere appreciation goes to Dr. Verne Schirch, Virginia Commonwealth University, for valuable advice and assistance. We also acknowledge the support from the grants funded by the Program for Promoting Academic Excellence of Universities, National Cheng Kung University (NCKU): Grant D96-3500 to T.-F.F. and Grant D96-2200 to H.-L.W., Director of Cardiovascular Research Center, NCKU.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.019299.
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ABBREVIATIONS: DHFR, dihydrofolate reductase; PVDF, polyvinylidene difluoride; bp, base pair; GSE, grape seed extract; EGCG, epigallocatechin-3-gallate; RT-PCR, reverse transcription-polymerase chain reaction; IPTG, isopropyl β-d-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; hpf, hours postfertilization; dpf, days postfertilization.
- Received October 17, 2007.
- Accepted November 30, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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