Optimization of Potent and Specific Trypanothione Reductase Inhibitors: A Structure-Based Drug Discovery Approach

Abstract

Leishmania spp. are responsible for up to 1 million new cases each year. The current therapeutic arsenal against Leishmania is largely inadequate, and there is an urgent need for better drugs. Trypanothione reductase (TR) represents a druggable target since it is essential for the parasite and not shared by the human host. Here, we report the optimization of a novel class of potent and selective LiTR inhibitors realized through a concerted effort involving X-ray crystallography, synthesis, structure–activity relationship (SAR) investigation, molecular modeling, and in vitro phenotypic assays. 5-Nitrothiophene-2-carboxamides 3, 6e, and 8 were among the most potent and selective TR inhibitors identified in this study. 6e and 8 displayed leishmanicidal activity in the low micromolar range coupled to SI > 50. Our studies could pave the way for the use of TR inhibitors not only against leishmaniasis but also against other trypanosomatidae due to the structural similarity of TR enzymes.

Fragment Merging, Growing, and Linking Identify New Trypanothione Reductase Inhibitors for Leishmaniasis

“…The Dixon plots showed a linear competitive inhibition with the K i values of 5.5 ± 0.2, 0.2 ± 0.1, and 0.8 ± 0.2 μM, respectively (Figure S4). Particularly, 10 and 14 are among the most active Li TR competitive inhibitorsin terms of K i valuesyet identified. , …”

“…Among all, 10 is the most potent fragment-derived TR inhibitor with a K i value of 0.2 μM. To the best of our knowledge, it is among the strongest competitive inhibitors of Li TR enzyme yet identified. , Unfortunately, compound 10 turned out to be also the most potent GR inhibitor (IC 50 = 2.3 μM) of the set, which may account for its cytotoxicity on murine macrophages (CC 50 = 12.5 μM). Nevertheless, a correlation between TR inhibition and phenotypic effects on axenic amastigotes (EC 50 = 11.0 μM) might be observed for 10 .…”

“…Particularly, 10 and 14 are among the most active LiTR competitive inhibitors�in terms of K i values�yet identified. 14,19 Modifications Are Allowed on the Solvent-Exposed Region. Modifications at the solvent-exposed position (region C, Figure 4B) had little impact on enzyme activity.…”

“… 17 This “three-point attachment” was realized upon the introduction of a N -3,4-dichlorobenzyl substituent on chlorpromazine, leading to a 2-order-of-magnitude-increase in TR inhibitory potency. 17 Moreover, in a recent work, Ilari and co-workers 19 reported a class of inhibitors targeting the Z-site and endowed with high activity and selectivity for TR.…”

Experimental structure based drug design (SBDD) applications for anti‐leishmanial drugs: A paradigm shift?

“…187 Structural studies of Leishmania TryR in complex with inhibitors show three binding locations (Figure 10): (1) There are a few inhibitors binding at the NADPH site and its entrance, 188 (2) cysteines C52 and C57 behind the isoalloxazine system of NAD that drive the electron flux towards the final product provide a binding site for metals and their ligands, 60,189,190 and (3) inhibitors binding and partially occupying the wide TS2 pocket and even extending to its opening, 191,192 in what has been referred to as the mepacrine binding site (MBS) or Z-site in the Trypanosoma brucei enzyme. [193][194][195] NADPH pocket. A target-oriented HTS on 120,000 compounds for inhibitory activity against L. infantum TryR located 290 hits, and 64 of them were later confirmed in secondary assays, but only 15 were selected based on biochemical and physicochemical parameters.…”