Discussion

Improved drug therapy against trypanosomatid infections is urgently needed^1,2.The mitochondrial F_oF₁-ATP synthase represents an attractive drug target for the following reasons: i) The structure and composition of this large multisubunit complex in trypanosomatids significantly differs from its mammalian counterpart as it includes 13 unique proteins with no homology outside the Euglenozoa group (the functions of these components remain to be elucidated)^3,4. The catalytic core F₁-ATPase itself involves an additional subunit, p18, again with unknown function. Moreover, the essential α subunit is proteolytically cleaved into two separate polypeptides that remain associated with the complex, a feature that appears to have no parallel in any other group of organisms^5–7. Thus, despite the ubiquitous presence of this enzyme in all domains of life, it should be feasible to develop inhibitors that are specific for the trypanosomatid enzyme. ii) The absence of the traditional respiratory chain inthe infective bloodstream stage of Trypanosoma brucei (and potentially in other trypanosome species as well) requires this enzyme to continuously operate as an ATP-hydrolysis driven proton pump to generate the essential mitochondrial potential (Δψ_m)^8–10. The mechanism of maintaining theΔψm is even more unique in non-tsetse transmitted, dyskinetoplastic subspecies T. b. evansi and T. b. equiperdum, which lack functional mitochondrial DNA and maintain the Δψ_m using the hydrolytic activity of the F₁-ATPase coupled to the electrogenic exchange of ATP^4-/ADP^3- by the ATP/ADP carrier¹⁰. The aim of this project was to perform a pilot medium throughput screening campaign to identify small molecule inhibitors of T. brucei F₁-ATPase activity. Active, tagged F_oF₁-ATP synthase was purified from insect stage T.brucei by affinity chromatography and used to develop a robust ATPase enzymatic screening assay. The ADP Hunter Plus kit (DiscoverX) was optimized to meet screening requirements set by the European Lead Factory¹¹. The fluorescence based endpoint assay had excellent performance during the robustness testing, consistently generating a Z’ value ≥ 0.6, and a signal to background ratio of ≥ 2.6. DMSO tolerance was ≥ 2.5%. A pilot screen of 7623 compounds from four drug libraries (NIH Clinical Collection, Selleckchem FDA approved drug library, BioAscent and GSK Kinase Inhibitor Compounds) identified 54 primary hits with inhibition of pIC₅₀ > 4.3 and no off-target interference against the ADP hunter assay components. Twenty compounds from the BioAscent library that achieved the highest medicinal chemistry scores (1 and 2 - excellent or good candidates) were chosen for follow up studies. As a first step we retested the compounds using a modified Pullman ATPase assay that couples the production of ADP to the oxidation of NADH via the pyruvate kinase and lactate dehydrogenase reactions¹². Out of the twenty selected hits, two compounds showed inhibition of ATPase activity in low micromolar range (pIC₅₀ values of 5.4 and 5.6, respectively). These hits are currently being tested in cell viability assays to elucidate their trypanocidal potential. 1) Cullen, D. R. et al.  (2017) 2) Giordani, F. et al.  (2016) 3) Zikova, A. et al. (2009) 4) Perez, E. et al. (2014) 5) Speijer, D. et al. (1997) 6) Nelson, R. E. et al.  (2004) 7) Gahura, O. et al. (2018) 8) Nolan, D. P. et al. (1992) 9) Vercesi, A. E. et al.(1992) 10) Schnaufer, A. et al. (2005) 11) McElroy, S. P. et al. (2017) 12) Pullman, M. E. et (1960)