title: Investigation of the anti-toxoplasma activity of arprinocid and the application of proteomics to the analysis of drug-resistance
Creators: name: Cohen, Adrian Mark
Publisher: name: University of Glasgow
Year: 2001
Subjects: subject: Pharmacology.

description: The in vitro efficacy and mechanism of action of two purine analogues, arprinocid and its in vivo metabolite arprinocid-1-N-oxide, were investigated against T. gondii tachyzoites using two contrasting approaches. Firstly, a global proteomics approach was undertaken for the analysis of proteins expressed in the tachyzoite stage of T. gondii, as a preliminary to analysing differences between arprinocid-1-N-oxide-resistant and - sensitive parasites. Secondly, a biochemical approach to investigate purine transporters of T. gondii as possible conduits or targets for arprinocid and arprinocid-1-N-oxide. Initial work using 3H-uracil growth uptake to measure the growth of tachyzoites enabled the measurement of the basic anti-parasitic properties of the compounds. The IC50 values for arprinocid and arprinocid-1-N-oxide were 22.4 +/- 5.0 muM and 0.061 +/- 0.028 muM, respectively. Both compounds were specific to T. gondii at therapeutic concentrations and acted irreversibly within a short period of time. High-resolution two-dimensional electrophoresis (2-DE) using the pH ranges 4-7 and 6- 11 reproducibly separated over 1,000 polypeptides, whilst further separations using narrow range gels suggested that at least 3,000-4,000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Peptide mass fingerprint (PMF) data acquired by MALDI-time-of flight mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of the T. gondii EST database using PMF data was less reliable. In contrast, peptide fragmentation data, acquired by MALDI-post-source decay mass spectrometry, proved a successful strategy for the putative identification of proteins using the T. gondii EST database. Moreover, peptide fragmentation data permitted the identification of T. gondii proteins based on peptide homology to known proteins from other organisms. The data demonstrated that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Work presented in this thesis demonstrated the usefulness of proteomics for the investigation of strain variation, protein changes as a consequence of genetic manipulation, and protein expression differences between arprinocid-l-N-oxide-sensitive and -resistant T. gondii lines. Detailed analysis of these drug-sensitive and -resistant mutants indicated that they reproducibly differed in only one protein, although it is suspected that further analysis using narrow range IPG strips may yield more differences. Unfortunately, this differentially expressed protein remains unidentified probably because of the incompatibility of silver-staining with mass spectrometry analysis. Although proteomics is a powerful collection of tools for the investigation of biological questions, currently it is in an early development phase. In contrast, classical biochemical approaches, such as the oil-stop technique for measurements of purine transport, are more standardised. Characterisation of purine transport in T. gondii resulted in the identification of a high affinity transporter for hypoxanthine (TgHTl; Km = 0.91 +/- 0.19 muM) and low affinity transporters for inosine (TglTl; 656 +/- 259 muM) and adenosine (TgATl; Km = 105 +/- 22 muM). No saturable transport of [3H]-adenine was observed. The discovery of a hypoxanthine transporter with a 100-fold higher affinity for substrate than the purine nucleoside transporters indicates that TgATl may not be the main carrier responsible for purine salvage in this organism. Both arprinocid and arprinocid-l-N-oxide inhibited TgATl with high affinity (K = 3.3 +/- 1.1 muM and Ki = 10.4 +/- 3.4 muM, respectively), suggesting that these drugs may be substrates for, or inhibitors of, this transporter. Although their exact mechanisms of action remain to be elucidated, neither drug acts on T. gondii by interfering with the hypoxanthine-xanthine- guanine-phosphoribosyltransferase (HXGPRT)-mediated purine salvage.