title: Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase.
abstract: Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Marc W
authors.initials: MW
authors.lastname: Gibson
authors.affiliation: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Simon
authors.initials: S
authors.lastname: Dewar
authors.affiliation: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Han B
authors.initials: HB
authors.lastname: Ong
authors.affiliation: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Natasha
authors.initials: N
authors.lastname: Sienkiewicz
authors.affiliation: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Alan H
authors.identifier: 0000-0001-5134-0329
authors.initials: AH
authors.lastname: Fairlamb
mesh_terms: D000818:Animals; D004795:Enzyme Stability; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development
chemical_list: D005493:Folic Acid Antagonists; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase