Identification of L. infantum trypanothione synthetase inhibitors with leishmanicidal activity from a (non-biased) in-house chemical library

Redox homeostasis in trypanosomatids is based on the low-molecular-weight trypanothione, an essential dithiol molecule that is synthetized by trypanothione synthetase (TryS) and maintained in its reduced state by trypa- nothione disulfide reductase (TryR). The fact that both enzymes are indispensable for parasite survival and absent in the mammalian hosts makes them ideal drug targets against leishmaniasis. Although many efforts have been directed to developing TryR inhibitors, much less attention has been focused on TryS. The screening of an in-house library of 144 diverse molecules using two parallel biochemical assays allowed us to detect 13 inhibitors of L. infantum TryS. Compounds 1 and 3 were characterized as competitive inhibitors with Ki values in the low micromolar range and plausible binding modes for them were identified by automated ligand docking against refined protein structures obtained through computational simulation of an entire catalytic cycle. The proposed binding site for both inhibitors overlaps the polyamine site in the enzyme and, additionally, 1 also occupies part of the ATP site. Compound 4 behaves as a mixed hyperbolic inhibitor with a Ki of 0.8 μM. The activity of 5 is clearly dependent on the concentration of the polyamine substrate, but its kinetic behavior is clearly not compatible with a competitive mode of inhibition. Analysis of the activity of the six best inhibitors against intracellular amastigotes identified 5 as the most potent leishmanicidal candidate, with an EC50 value of 0.6 μM and a selectivity index of 35.This work has been supported by the Spanish MICINN (Projects PID2019-104070RB-C21 and PID2019-104070RBC22), the Spanish National Research Council (CSIC, Projects CSIC-PIE-201980E100 and CSIC-PIE-201980E028), and the Comunidad de Madrid (PLATESA2-CM ref S-2018/BAA-4370). The MCIN is also acknowledged for the pre- doctoral fellowship to M.A.C.Peer reviewe