4E Interacting Protein as a Potential Novel Drug Target for Nucleoside Analogues in Trypanosoma brucei

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.

apparatus and are uncorrected. Purity was assessed by means of analytical LC-MS employing a Waters Alliance 2695 XE separation Module using a Phenomenex Luna® reversed-phase C18 (2) column (3 μm, 100x2.00 mm) and a gradient system of HCOOH in H2O (0.1 %, v/v)/HCOOH in MeCN (0.1 %,v/v) at a flow rate of 0.4 mL / min, 10:90 to 0:100 in 9 minutes. High-resolution MS spectra were recorded on a Waters LCT Premier XE Mass spectrometer. The obtained final compound had a purity of >95%, as assayed by analytical HPLC (UV detection).

d]pyrimidine (FH6357)
In a flame-dried culture tube, equipped with a stir bar, was added under argon: FH3120 (0.72 g, 1.0 mmol, 1 eq.), CuI (0.038 g, 0.20 mmol, 0.2 eq.), 1,10-phenanthroline (0.036 g, 0.20 mmol, 0.2 eq.) and KF (0.17 g, 3.0 mmol, 3 eq.). Then, the culture tube was capped with a septum and evacuated. Next, the flask was refilled with argon. This procedure was repeated three times in total. Then, anhydrous DMSO (2.0 mL, 2.0 mL / mmol SM) was added, followed by B(OMe)3 (0.33 mL, 3.0 mmol, 3 eq.) and TMSCF3 (0.45 mL, 3.0 mmol, 3 eq.). The mixture was stirred at ambient temperature for 1-2 min to ensure adequate homogenization, and then transferred to a pre-heated oil bath at 60 °C. After 18H, the reaction mixture was cooled to ambient temperature, and water added. Next, EA was added, and the layers were separated. The water layer was extracted twice more with EA. The organic layers were combined, dried over Na2SO4, filtered and evaporated till dryness. The residue was purified by column chromatography 0 → 20 % EA / hexanes to give FH6357 (0.29 g). The product still contained (~10 %) of unreacted iodide SM; and was therefore directly used in the next steps (azidation and Staudinger reduction).

d]pyrimidine (FH6366)
The mixture containing FH6357 (0.29 g) was dissolved in anhydrous DMF (10 mL / mmol SM). Next, NaN3 (2.05 eq.) was added. The resulting mixture was heated in a pre-heated oil bath at 65 °C for 30 min. Next, the mixture was cooled to ambient temperature.
Then, the mixture was poured into half-saturated NaHCO3 solution and EA (equal volumes). The layers were separated, and the water layer extracted two more times with EA. The organic layers were combined, dried over Na2SO4, filtered and evaporated till dryness. The residue was purified by column chromatography (generally a gradient of 10 → 35 % EA / Hexanes or PET) to yield the protected azido-nucleoside. The azido-nucleoside (1 eq.) was dissolved in THF (10 mL / mmol).
Then, PMe3 solution (1M in THF; 2.7 eq.) was added and the mixture stirred at ambient temperature until TLC analysis showed full conversion of starting material (generally 1 h). Next, the solution was evaporated till dryness, and subsequently re-dissolved in MeCN (10 mL / mmol). To this solution was added a 1M aq. HOAc solution (3.3 eq.), and the mixture heated in a pre-heated oil bath at 65 °C for 1H. Next, the mixture was cooled to ambient temperature and poured into sat. aq. NaHCO3 solution. DCM was added, layers were separated, and the water layer extracted two more times with DCM. The organic layers were combined, dried over Na2SO4, filtered and evaporated till dryness. Purification by column chromatography (a gradient of 40 → 75 % EA / hexanes). As