Synthesis, Inhibition Potency, Binding Mode, and Antiprotozoal Activities of Fluorescent Inhibitors of Trypanothione Reductase Based on Mepacrine‐Conjugated Diaryl Sulfide Scaffolds

Abstract

Trypanothione reductase (TR) is a flavoenzyme unique to trypanosomatid parasites and a target for lead discovery programs. Various inhibitor scaffolds have emerged in the past, exhibiting moderate affinity for the parasite enzyme. Herein we show that the combination of two structural motifs of known TR inhibitors - diaryl sulfides and mepacrine - enables the simultaneous addressing of two hydrophobic patches in the active site. The binding efficacy of these conjugates is enhanced over that of the respective parent inhibitors. They show K(ic) values for the parasite enzyme down to 0.9+/-0.1 microm and exhibit high selectivity for TR over human glutathione reductase (GR). Despite their considerable molecular mass and in some cases permanent positive charges, in vitro studies revealed IC(50) values in the low micromolar to sub-micromolar range against Trypanosoma brucei rhodesiense and Trypanosoma cruzi, as well as the malaria parasite Plasmodium falciparum, which lack trypanothione metabolism. The inhibitors exhibit strong fluorescence due to their aminoacridine moiety. This feature allows visualization of the drugs in the parasite where high accumulation was observed by fluorescence microscopy even after short exposure times.

Improved Inhibitors of Trypanothione Reductase by Combination of Motifs: Synthesis, Inhibitory Potency, Binding Mode, and Antiprotozoal Activities

“…The significantly lower inhibition of bromo derivative 21, which does not have a second aryl substituent, supports our hypothesis that additional binding energy is gained by simultaneous occupation of the mepacrine binding site and the hydrophobic groove near Cys 52. [17] Quaternary benzyldimethylammonium ligands 26 and 27 displayed significantly higher inhibition potency compared to the corresponding piperazines or dimethylamines. Kinetic analysis showed competitive inhibition of T. cruzi TR with K ic values of 5 AE 1 mm (for 26) and 2 AE 1 mm (for 27).…”

“…[17] Replacement of the benzylamine moiety with a central imidazole platform is proposed to enable binding to Glu 18 (as seen for 1 in Figure 1). Two aryl sulfides appended to a phenyl substituent occupy the mepacrine binding site and the hydrophobic patch roughly framed by Cys 52, Val 53, and Ile 106.…”

“…mimics the positively charged trypanothione. Tricyclic scaffolds such as acridines, [17] related compounds, such as phenothiazine-based derivatives, [18,19] and ring-opened 2-aminodiphenyl sulfide-based structures are known to interact with TR. [20] In general, clear structure-activity relationships (SAR) are difficult to determine for TR inhibitors.…”

Synthesis, biological profiling and mechanistic studies of 4-aminoquinoline-based heterodimeric compounds with dual trypanocidal–antiplasmodial activity

“…Because it is unique and essential to these parasites, TryR constitutes a valuable target for trypanocidal drug discovery programs. 46,47 A number of quinoline (ref 20) or aminoacridine derivatives have been reported to be inhibitors of TryR. 20,46,48,49 In these compounds the presence of a protonatable nitrogen atom seems to be crucial for the TryR inhibitory activity because it mimics the positively charged substrate, trypanothione disulfide.…”

“…46,47 A number of quinoline (ref 20) or aminoacridine derivatives have been reported to be inhibitors of TryR. 20,46,48,49 In these compounds the presence of a protonatable nitrogen atom seems to be crucial for the TryR inhibitory activity because it mimics the positively charged substrate, trypanothione disulfide. Interestingly, it has been reported that dimerization of several classes of compounds with known TryR inhibitory activity leads to increased potency, [50][51][52] which might be related to the presence of two interacting binding sites in the large active site of TryR.…”