title: Cloning, recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly, Lucilia cuprina.
abstract: While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non-specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC50 values at µg levels. Trimethoprim and pyrimethamine were 5-30-fold less active. Relative IC50 values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active-site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.
authors.affiliation: CSIRO Animal, Food and Health Sciences, Brisbane, Queensland, Australia.
authors.forename: A C
authors.initials: AC
authors.lastname: Kotze
authors.forename: N H
authors.initials: NH
authors.lastname: Bagnall
authors.forename: A P
authors.initials: AP
authors.lastname: Ruffell
authors.forename: R
authors.initials: R
authors.lastname: Pearson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004175:Diptera / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D020128:Inhibitory Concentration 50; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007306:Insecticides / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011679:Pupa / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment / Q000662:veterinary; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D007306:Insecticides; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase
keywords: DHFR; Lucilia cuprina; dihydrofolate reductase; inhibitor profile; insecticide