Effects of compounds on mitochondrial membrane potential.

<p>PF <i>T</i>. <i>brucei</i> were incubated with inhibitors and MitoTracker Red CMXRos, which is an indicator of the mitochondrial membrane potential (Δψ_m). <b>(A)</b> MitoTracker-loaded cells were imaged to qualitatively detect differences in the Δψ_m. Complex III inhibitor, antimycin A (AA at 2 μM) noticeably reduced MitoTracker Red CMXRos uptake, while the F_oF₁-ATP synthase (complex V) inhibitor oligomycin A (OA at 2 μM) and <b>compound 1</b> at 40 μM clearly enhanced MitoTracker Red CMXRos uptake as compared with the uninhibited control. <b>(B)</b> Fluorescence of MitoTracker-loaded cells were quantified using a microplate reader and normalised to MitoTracker green fluorescence. Data were consistent with microscope observations in which inhibitors of the electron transport chain such as Antimycin A (AA at 2 μM), or protonophores such as 2,4-dinitrophenol (DNP at 1 mM) decrease the mitochondrial membrane potential (Δψ_m), while inhibitors of the F_oF₁-ATP synthase such as Oligomycin A (OA at 2 μM) elevate the Δψ_m. Compounds 1 and 3 at 40 μM elevated the Δψ_m indicating that they, like OA, target the F_oF₁-ATP synthase.</p

B-THP-Ts decrease cellular ATP levels.

<p>PF <i>T</i>. <i>brucei</i> cells were incubated in buffered PBS with/without proline (as sole carbon source) and various inhibitors (concentrations indicated) incubated for 2 h, after which cells were harvested and cellular ATP levels were monitored by bioluminescence assay. Key: NS, no substrate; NI, no inhibitor control; Malonate (complex II inhibitor) AA, antimycin A (AA, complex III inhibitor). Background relative light units (RLU) were subtracted from measurements and % ATP production normalised to the no inhibitor control. Data were averaged from two independent experiments, each performed in duplicate (n = 4). Error bars show standard deviations. T-tests were performed against no inhibitor controls (black asterisks) and <b>compound 1</b> used at 10 μM (grey open circles) and significance values presented: <i>p</i> < 0.0005 (*** / °°°), <i>p</i> < 0.005 (** / °°), <i>p</i> < 0.05 (* / °). The B-THP-T <b>compound 1</b> significantly reduces ATP levels in a dose-response manner to levels indistinguishable from known inhibitors of oxidative phosphorylation.</p

B-THP-Ts bind to C-terminal GFP-PTP tagged F₁ α- and β- subunits.

<p><b>(A)</b> Stably transfected PF <i>T</i>. <i>brucei</i> cells endogenously-expressing F₁ α-GFP-PTP or F₁ β-GFP-PTP were loaded with MitoTracker Red CMXros (red) and DAPI (blue) and imaged microscopically to determine localisation of GFP-PTP-tagged protein. F₁ α-GFP-PTP and F₁ β-GFP-PTP were detected through natural fluorescence of their green fluorescent protein (GFP) tag, and both were found to co-localise with MitoTracker, confirming their mitochondrial association. <b>(B)</b> Following <i>in vivo</i> photo-affinity labelling with 50 μM <b>compound 3</b> or <b>compound 1</b> (negative control), GFP-PTP-tagged F₁ α- and β-subunits were affinity purified and Cy5.5 clicked on. Proteins were separated by SDS-PAGE and a western blot was performed with mouse anti-GFP / anti-mouse-DyLight 800. GFP-PTP-tagged proteins were revealed at 800 nm, Cy5.5-bound target proteins at 700 nm, and a merged image shows both together. Lanes 1 and 2 used <b>compounds 1 and 3</b> respectively with F₁ α-GFP-PTP-expressing cells and captured F₁ α-GFP-PTP migrating at 89 KDa was observed with anti-GFP at 800 nm in both lanes. F₁ α-GFP-PTP only fluoresced at 700 nm when bi-functional <b>compound 3</b> was used, indicating that Cy5.5 had bound specifically to the F₁ α-subunit via our bi-functional photo-affinity probe. Lanes 3 and 4 used <b>compounds 1 and 3</b> respectively with F₁ β-GFP-PTP-expressing cells, and captured F₁ β-GFP-PTP migrating at ~101 KDa was observed in both lanes with anti-GFP. As with the α-subunit, β-GFP-PTP only conjugated Cy5.5 when the bi-functional <b>compound 3</b> was used, confirming that both the α- and β-subunits are targets of our B-THP-T compounds.</p

Inhibition of ATP production in digitonin-permeabilsed cells.

<p><b>(A-C)</b> Digitonin-permeabilised PF <i>T</i>. <i>brucei</i> were probed with substrates in the presence of inhibitors and resulting ATP production quantified by bioluminescence assay. See experimental section for details. Inhibitors were used at saturating concentrations to ensure that relevant enzyme activities were ablated. Key: NS, no substrate; NI, no inhibitor; SHAM, salicylhydroxamic acid (TAO inhibitor at 100 μM); Rotenone (complex I inhibitor at 100 μM); Malonate (complex II inhibitor at 5 mM); AA, antimycin A (complex III inhibitor at 200 μM); OA, oligomycin A (complex V inhibitor at 200 μM); DNP, 2,4-dinitrophenol (protonophore at 1 mM); Comp.1, <b>compound 1</b> at 200 μM. Background relative light units (RLU) were subtracted from measurements and % ATP production normalised to the no inhibitor control. Data were averaged from three independent experiments, each performed in quadruplicate. Error bars show standard deviations. T-tests were performed against no inhibitor controls (black asterisks) and <b>compound 1</b> (grey open circles) and significance values presented: <i>p</i> < 0.00005 (*** / °°°), <i>p</i> < 0.0005 (** / °°), <i>p</i> < 0.005 (* / °). <b>(A)</b> Succinate was used as a substrate to generate ATP solely through oxidative phosphorylation (OxPhos). Oxidative phosphorylation inhibitors malonate, AA, OA and DNP virtually eliminated all ATP production. <b>Compound 1</b> almost eliminated ATP production and was indistinguishable from inhibitors of oxidative phosphorylation, suggesting that it too inhibits oxidative phosphorylation. <b>(B)</b> α-ketoglutarate yields 40% ATP through substrate-level phosphorylation (SubPhos) and 60% through oxidative phosphorylation. Malonate, AA, OA and DNP inhibit oxidative phosphorylation, but not substrate-level phosphorylation. <b>Compound 1</b> acted similarly, albeit with a minor additional effect (possibly on DLST, which was also a pull-down hit), suggesting oxidative phosphorylation is its major target. <b>(C)</b> Gly-3P acts as an alternative entry site to the electron transport chain yielding ATP through oxidative phosphorylation and the glycosome (GlyPhos). Malonate has no effect, as it acts at a different entry point. AA, OA and DNP inhibit oxidative phosphorylation, but not glycosomal phosphorylation. There is no significant difference between <b>compound 1</b> and oxidative phosphorylation inhibitors, suggesting <b>compound 1</b> acts downstream of complex II. <b>(D)</b> IC₅₀ determination of <b>compound 1</b> in digitonin-permeabilised cells when succinate (57.3 ± 14.5 μM) or Gly-3P (39.2 ± 4.6 μM) is used as substrate. The efficacy of <b>compound 1</b> was similar for both substrates indicating that blockade is downstream of them both.</p

Binding of nucleotide and compound 1 to yeast F₁ α- and β-subunits.

<p>Yeast F₁ subunits are oriented around the Walker A motif for comparisons. <b>(A and B)</b> The crystal structure yeast F₁ with bound nucleotide (PDB entry 2WPD [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005886#pntd.0005886.ref068" target="_blank">68</a>]). ATP occupies that regulatory site of the α-subunit <b>(A)</b>, while ADP occupies the catalytic site of the β-subunit <b>(B)</b>. The positioning of nucleotide is similar for both: the adenine anchors within a hydrophobic pocket while the phosphates interact with the Walker A nest. Hydrogen bonds are shown as dotted lines. <b>(C-D)</b> Docking of <b>compound 1</b> into the yeast ATP binding sites. In the regulatory α-subunit <b>(C)</b> the triazole moiety of <b>compound 1</b> interacts with the Walker A nest, THP2 occupies the position of the nucleotide ribose, the hydrophobic tail buries into the hydrophobic adenine pocket, and the terminal hydroxyl forms extensive H-bonds. The position is <b>compound 1</b> is different in β-subunit <b>(D)</b>, whereby THP2 is sandwiched between Tyr345 and Phe424, the hydrophobic tail buries into the adenine site, and THP1 and terminal hydroxyl form potential H-bonds. <b>(E) Compound 1</b>.</p

Localisation of B-THP-T target(s).

<p>PF <i>T</i>. <i>brucei</i> cells were <i>in vivo</i> pulse-chase photo-affinity labelled with lead B-THP-T <b>compound 1</b> or bi-functional <b>compound 3.</b> Cells were imaged following in-cell Cy5.5 cycloaddition. Cy5.5 labelling (green) was absent when bi-functional tags were absent (<b>compound 1</b>) or when <b>compound 3</b> was not UV-activated, indicating that the Cy5.5 labelling observed with UV-activated bi-functional <b>compound 3</b> is specific. Cy5.5-labelled proteins co-localise with MitoTracker (red), indicating that B-THP-T compounds target mitochondrial proteins.</p

Proline metabolism in PF <i>T</i>. <i>brucei</i> and reactions catalysed by potential B-THP-T targets.

<p>PF <i>T</i>. <i>brucei</i> grown in glucose-free medium are reliant on proline as a carbon source and enzymes of an incomplete TCA-cycle are used to metabolise it to alanine and actetate (shown in blue): 1, proline dehydrogenase; 2, δpyrroline-5-carboxylate dehydrogenase (δPCDH); 3, L-alanine aminotransferase; 4, a-ketoglutarate dehydrogenase complex (of which dihydrolipoamide succinyltransferase, DLST, is a component); 5, succinyl-CoA synthetase; 6, mitochondrial complex II; 7, fumarase; 8, malate dehydrogenasemalic enzyme; 9, pyruvate dehydrogenase complex; 10, acetate:succinate CoA-transferase. Substrate-level phosphorylation occurs with the conversion of succinyl-CoA to succinate at succinyl-CoA synthetase (step 5). Mitochondrial complex II reduces succinate to fumarate at step 6 and passes electrons into the electron transport chain (represented in green) via ubiquinol (Q). Complex III passes the electrons to cytochrome c (C), and complex IV passes them from cytochrome c to molecular oxygen forming water. Complexes III and IV together export six protons (green dashed line) for each molecule of succinate reduced, and the F_oF₁-ATP synthase (complex V) generates ATP by importing four protons, making 1.5 molecules of ATP for every molecule of succinate reduced. The sites of action of oxidative phosphorylation inhibitors are shown. Figure adapted from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005886#pntd.0005886.ref019" target="_blank">19</a>]. Roles of four of the six-mitochondrion pull-down hits are shown in red italic numbers.</p

Mitochondrial proteins identified as potential targets of B-THP-T compounds by biotin pull-down / LC-MSMS.

<p>Mitochondrial proteins identified as potential targets of B-THP-T compounds by biotin pull-down / LC-MSMS.</p