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

Wheat leaf properties affecting the absorption and subsequent translocation of foliar-applied phosphoric acid fertiliser

Background and aims. Although foliar fertilisation using liquid forms of phosphorus (P) is not a new concept, its adoption has been hindered by a limited understanding of the variability in performance of fluid forms of foliar P applied to broadacre crops. There is a need to identify how the surface structure of leaves influences the absorption and subsequent translocation of foliar-applied P in above ground plant parts. Methods. This study examined the surface properties of wheat leaves using scanning electron microscopy and measured the recovery of foliar-applied fertiliser that was labelled with either 32P or 33P from both the adaxial (upper) and abaxial (lower) leaf sides into untreated plant parts. Results. We found that the adaxial leaf surface absorbed and translocated more foliar-applied P away from the treated leaf than the abaxial surface, likely related to the higher abundance of trichomes and stomata present on that side of the leaf. The recovery of the foliar-applied fertiliser varied with rate and timing of application; ranging from <30%to as much as 80% of the adaxial-applied fertiliser translocated from the treated leaf into the wheat ear. Conclusions. This study demonstrated that the differences in surface morphological features between leaf sides influenced the combined absorption and subsequent translocation of foliar-applied P in the above ground plant parts. This is due to a direct effect on the foliar pathway and/or due to differences in wettability affecting both the leaf coverage and drying time of fertilisers on the leaves. Although foliar fertilisation in this study contributed less than 10 % of the total P in the plant, it provided a more efficient pathway for P fertilisation than soil-applied P.C.A.E. Peirce, T.M. McBeath, V. Fernández, M.J. McLaughli