Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

This work was supported by the Leverhulme Trust (Grant number RL2012-025). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1 , a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3 , a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1 , to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.Publisher PDFPeer reviewe

Simplifying nature:Towards the design of broad spectrum kinetoplastid inhibitors, inspired by acetogenins

The need for new treatments for the neglected tropical diseases African sleeping sickness, Chagas disease and Leishmaniasis remains urgent with the diseases widespread in tropical regions, affecting the world's very poorest. We have previously reported bis-tetrahydropyran 1,4-triazole analogues designed as mimics of the annonaceous acetogenin natural product chamuvarinin, which maintained trypanocidal activity. Building upon these studies, we here report related triazole compounds with pendant heterocycles, mimicking the original butenolide of the natural product. Analogues were active against T. brucei, with a nitrofuran compound displaying nanomolar trypanocidal activity. Several analogues also showed strong activity against T. cruzi and L. major. Importantly, select compounds gave excellent selectivity over mammalian cells with a furan-based analogue highly selective while remaining active against all three cell lines, thus representing a potential lead for a new broad spectrum kinetoplastid inhibitor.</p

Design and application of a low-temperature continuous flow chemistry platform

A flow reactor platform technology applicable to a broad range of low temperature chemistry is reported. The newly developed system captures the essence of running low temperature reactions in batch and represents this as a series of five flow coils, each with independently variable volume. The system was initially applied to the functionalization of alkynes, Grignard addition reactions, heterocycle functionalization, and heteroatom acetylation. This new platform has then been used in the preparation of a 20-compound library of polysubstituted, fluorine-containing aromatic substrates from a sequential metalation-quench procedure and can be readily adapted to provide gaseous electrophile inputs such as carbon dioxide using a tube-in-tube reactor

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

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

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