Enzymes of Purine Salvage Pathway in \kur{Trypanosoma brucei} and the Trypanocidal Action of Acyclic Nucleoside Phosphonates

This study aims to functionally characterize two enzymes, HGPRT and XPRT, of an essential purine salvage pathway in the infection stage of Trypanosoma brucei. Localization, in vivo function and in vitro activity of these enzymes were characterized. Effect of acyclic nucleoside phosphonates, putative inhibitors of HGXPRT, on the viability of bloodstream form of T. brucei was evaluated

Validation of acyclic nucleoside phosphanates as inhibitors of 6-oxo purine phosphorybosyltransferases in \kur{Trypanosoma Brucei}

The aim of this study was to create RNAi double-knockdown of two enzymes HGPRT and XPRT and to determine if these enzymes are essential for growth of the bloodstream stage of Trypanosoma brucei. Further, we aim to investigate the intracellular localization of these two enzymes using digitonin fractionation and immunofluorescent assay. In addition, cytotoxicity screen of three selected acyclic nucleoside phosphanates was permormed using T. brucei cell line overexpressing HGPRT and XPRT enzymes to validate their putative targets

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites' viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery

General structures of ANPs 1–7 and their phosphoramidate prodrugs 1p-7p.

<p>General structures of ANPs 1–7 and their phosphoramidate prodrugs 1p-7p.</p

K_i values of the selected ANPs for HGPRT-I, HGPRT-II and HGXPRT and a comparison of the <i>in vitro</i> antitrypanosomal activity of their prodrugs against <i>T</i>. <i>brucei</i> BF427 cell line with their cytotoxicity in human cell lines.

<p>*Estimated selectivity index (SI) = average CC₅₀ for A549 (Human lung carcinoma cells) divided by the average EC₅₀ for <i>T</i>. <i>brucei</i> BF427 cell line. Synthesis of ANP inhibitors and their prodrugs is described in [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006301#pntd.0006301.ref020" target="_blank">20</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006301#pntd.0006301.ref022" target="_blank">22</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006301#pntd.0006301.ref028" target="_blank">28</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006301#pntd.0006301.ref029" target="_blank">29</a>].^aData from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006301#pntd.0006301.ref029" target="_blank">29</a>]; ^bData from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006301#pntd.0006301.ref028" target="_blank">28</a>]; ^cData from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006301#pntd.0006301.ref020" target="_blank">20</a>]; ^dData from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006301#pntd.0006301.ref022" target="_blank">22</a>].</p

Subcellular localization of HGPRT-I, HGPRT-II and HGXPRT in the bloodstream form of <i>T</i>. <i>brucei</i>.

<p>(A) Immunoblot analysis of BF cells over-expressing v5-tagged HGPRT-I, HGPRT-II and HGXPRT was performed to reveal the subcellullar localization of these proteins. Cytosolic (CYT) and organellar (ORG) fractions were obtained by digitonin fractionation. Purified fractions were analyzed by immunoblot with the following antibodies: anti-V5, anti-enolase (cytosol), anti-hexokinase (organellar fraction, glycosomes), anti-mt hsp70 (organellar fraction, mitochondrion). The relevant sizes of the proteins are indicated on the left. (B) Immunofluorescence microscopy of the same cell lines as in (A) was used to determine subcellular localization of the v5-tagged HGPRT-I, HGPRT-II and HGXPRT proteins within the cell. HGPRT-I, HGPRT-II and HGXPRT were visualized by immunostaining using a monoclonal anti-v5 antibody and anti-mouse secondary antibody conjugated with fluorescein isothiocyanate (FITC). Antibodies against enolase and hexokinase served to mark cytosolic and glycosomal localization, respectively. The DNA content (nucleus and kinetoplast) was visualized using DAPI (4,6-diamidino-2-phenylindole).</p

The active site of HGPRT-I and HGXPRT.

<p>Each enzyme is represented by its Connolly surface and the inhibitor is shown as a stick model. The F_o-F_c electron density for each inhibitor is overlaid. (A) HGPRT-I.6 complex, (B) HGPRT-I.1 complex, (C) HGPRT-I.7 complex, (D) HGPRT-I.2 complex, (E) HGXPRT.7 complex and (F) HGXPRT.2 complex.</p

Effects of RNAi silencing of HGPRT-I, HGXPRT and simultaneous RNAi silencing of HGPRT-I/HGXPRT on <i>T</i>. <i>brucei</i> BF cell growth.

<p>Growth curves of the noninduced (NON) and RNAi induced (IND) cells in which the expression of HGPRT-I (A) HGXPRT (B) and HGPRT-I/HGXPRT (C) was RNAi silenced. Cells were cultured in HMI-9^full media and the cumulative cell number was calculated from cell densities adjusted by the dilution factor needed to see the cultures at 10⁵ cells/ml each day. The figure is representative of at least three independent RNAi inductions. The steady-state abundance of HGPRT-I and HGXPRT in noninduced (NON) RNAi cells and in cells induced with tetracycline for 2, 4 and 6 days (A, B and C, bottom panels) was determined by immunoblotting using specific anti-HGPRT-I and anti-HGXPRT serums. Densitometric analysis was performed using the Image Lab 4.1 software and the determined values were normalized to an enolase loading control signal. The protein marker sizes are indicated on the left.</p

Evaluation of the <i>Trypanosoma brucei</i> 6-oxopurine salvage pathway as a potential target for drug discovery

<div><p>Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for <i>de novo</i> synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of <i>Trypanosoma brucei</i> demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including <i>Plasmodium falciparum</i>. The most potent of these compounds have K_i values as low as 60 nM, and IC₅₀ values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.</p></div

Active sites of HGPRT-I, HGXPRT and human HGPRT after superimposition.

<p>(A) compound 6 complexes. HGPRT-I. 6 in cyan, human HGPRT.6 in gold. (B) compound 1 complexes. HGPRT-I.1 in orange and human HGPRT.1 in purple. (C) compound 2 complexes. HGPRT-I.2 in green, human HGPRT.2 in grey (D) compound 2 complexes. HGXPRT.2 in pink, human HGPRT.2 in grey. Black labels are for HGPRT-I, green labels are for the HGXPRT and red labels are for human HGPRT.</p