Mechanistic and biological characterisation of novel N 5 -substituted paullones targeting the biosynthesis of trypanothione in Leishmania .

Trypanothione synthetase (TryS) producesN(1),N-8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimisedN(5)-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC(50)against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC(50)0.5-10 mu M) and selectivity (20-35) against the clinically relevant stage ofLeishmania braziliensis(mucocutaneous leishmaniasis) andL. infantum(visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against differentLeishmaniaspecies.