A comparative study of purine nucleobase and nucleoside transporters in protozoan species

Al-Salabi, Mohammed I. (2006) A comparative study of purine nucleobase and nucleoside transporters in protozoan species. PhD thesis, University of Glasgow.

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Abstract

Selective delivery of drugs to parasites via plasma membrane transporters offers an effective approach to specifically target pathogens. Using biochemical techniques, we have identified and characterised a number of purine nucleobase and nucleoside transporters in protozoan parasites such as Leishmania major, Leishmania mexicana, Trypanosoma brucei and Toxoplasma gondii. In one study, the uptake of [3H]adenine, [3H]hypoxanthine, and [3H]allopurinol, and antileishmanial hypoxanthine analogue, by Leishmania major promastigotes was investigated. The results showed that these compounds were all taken up by a single high-affinity transporter, LmajNBT1, with Km values of 4.6±0.9, 0.71±0.07, and 54±3 μM and Vmax­ values of 3.2±0.3, 2.8±0.6, and 0.24±0.06 pmol (107 cells)-1s-1, respectively. [3H]adenine transport was fully inhibited by the natural purines guanine and xanthine, with Ki values of 2.8±0.7 and 23±8 μM, respectively. Using purine analogues, an extensive inhibitor profile for LmajNBT1 was obtained, which allowed the construction of a quantitative model for the interactions between the transporter binding site and the permeant. The model predicts that hypoxanthine was bound through hydrogen bonds to N(1)H, N3, N7, and N(9)H of the purine ring, with a total Gibbs free energy of -39.5 kJ/mol. The interactions with adenine were similar, except for a weak hydrogen bond to N1 (unprotonated in adenine). The predicted model of substrate binding for LmajNBT1 was almost identical to that for the Trypanosoma brucei H2 (TbH2) transporter. It is proposed that the architecture of their respective binding sites is very similar and that LmajNBT1 can be named a functional homolog of TbH2. This thesis also reports the first identification and characterization of a purine nucleobase transporter in Leishmania amastigotes. Uptake of [3H]hypoxanthine by Leishmania mexicana amastigotes was mediated by a single high-affinity transporter, LmexNBT1, with Km and Vmax values of 1.6±0.4μM and 0.092 ± 0.057 pmol (107 cells)-1s-1 respectively, with high affinity for adenine, guanine, and xanthine, with Ki values of 4.2±0.8, 1.7±0.1, and 13±2 μM respectively, but low affinity for nucleosides and pyrimidine nucleobases. Allopurinol was apparently taken up by the same transporter (Km of 33.6±6.0 μM). All evidence was compatible with a model of a single purine nucleobase transporter being expressed in amastigotes. Using various purine nucleobase analogues, a model for the interactions between hypoxanthine and the transporter's permeant binding site was constructed and compared with previously obtained models for substrate recognition by nucleobase transporters, and found to be very similar to the models of the LmajNBT1 and TbH2 transporters, but markedly different from the human FNT1 transporter.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Funder's Name: UNSPECIFIED
Supervisor's Name: de Koning, Professor Harry
Date of Award: 2006
Depositing User: Ms Mary Anne Meyering
Unique ID: glathesis:2006-6224
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Mar 2015 14:59
Last Modified: 31 Mar 2015 15:36
URI: http://theses.gla.ac.uk/id/eprint/6224

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