Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate

The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP(+) and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6-α6 loop and α6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis

<i>Trypanosoma brucei</i> DHRF-TS revisited:characterisation of a bifunctional and highly unstable recombinant dihydrofolate reductase-thymidylate synthase

<div><p>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^- <i>Escherichia coli</i>, 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 (<i>K</i>_i 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (<i>K</i>_i 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.</p></div

Stabilisation of recombinant <i>T</i>. <i>brucei</i> TS activity by substrate dUMP.

<p>A. Determination of TS stability relative to dUMP concentration using Tsf-<i>Tb</i>DHFR-TS from <i>thyA</i>^- lysate. Tsf-<i>Tb</i>DHFR-TS (circles), <i>Lm</i>DHFR-TS (squares), human TS (triangles). Incubation mixtures omitted either 200 μM dUMP (open symbols) or 100 μM CH₂THF (closed symbols); post-incubation, addition of the missing reagent was used to initiate reactions. The <i>inset</i> shows the loss of TS activity Tsf-<i>Tb</i>DHFR-TS in absence of dUMP over a shorter time scale. All activities were determined in triplicate spectrophotometrically using 100 μM CH₂THF, as described in the methods. B. Optimum concentration of dUMP required to stabilise the TS activity. Activity was determined by incubating the diluted lysate for 10 min in assay buffer with varying concentrations of dUMP prior to initiation of the reaction with CH₂THF and excess (200 μM) dUMP.</p

Kinetic properties of recombinant Tsf-<i>Tb</i>DHFR-TS compared with those of related species and the human homologues.

<p>Kinetic properties of recombinant Tsf-<i>Tb</i>DHFR-TS compared with those of related species and the human homologues.</p

Chemical structures of DHFR and TS inhibitors.

<p>Chemical structures of DHFR and TS inhibitors.</p

Sensitivity of recombinant <i>Tb</i>DHFR-TS to DHFR and TS inhibitors compared with their human counterparts.

<p>Sensitivity of recombinant <i>Tb</i>DHFR-TS to DHFR and TS inhibitors compared with their human counterparts.</p

Purification of recombinant Tsf-<i>Tb</i>DHFR-TS from <i>E</i>. <i>coli</i>.

<p>A. SDS-PAGE gel stained with Coomassie blue. Lane 1. Clarified <i>thyA</i>^- <i>E</i>. <i>coli</i> lysate expressing Tsf-<i>Tb</i>DHFR-TS. Lane 2. Glycerol-diluted lysate treated with TEV protease. Lane 3. Methotrexate agarose elution. Proteins indicated by arrows identified by mass spectrometry to be Tsf-<i>Tb</i>DHFR-TS, cleaved <i>Tb</i>DHFR-TS, and the Tsf tag. B. Size exclusion chromatography of purified product. Closed circles: DHFR activity. Dashed line: absorbance at 280 nm.</p