The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure-based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small-molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so-called "mepacrine binding site". The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi.

No abstract

Trypanothione reductase (TR) is an essential enzyme of trypanosomatids and therefore a promising target for the development of new drugs against African sleeping sickness and Chagas' disease. Diaryl sulfides with a central anilino moiety, decorated with a flexible N-alkyl side chain bearing a terminal ammonium ion, are a known class of inhibitors. Using computer modelling, we revised the binding model for this class of TR inhibitors predicting simultaneous interactions of the ammonium ion-terminated N-alkyl chain with Glu18 as well as Glu465'/Glu466' of the second subunit of the homodimer, whereas the hydrophobic substituent of the aniline ring occupies the "mepacrine binding site" near Trp21 and Met113. Systematic alteration of the carboxylate-binding fragments and the diaryl sulfide core of the inhibitor scaffold provided evidence for the proposed binding mode. In vitro studies showed IC(50) values in the low micromolar to submicromolar range against Trypanosoma brucei rhodesiense as well as the malaria parasite Plasmodium falciparum.

This is the first study examining functionality of subjects with anterior cruciate ligament (ACL) tears and a subsequent reconstruction comprehensively by multiple test sessions from pre- to six months post-reconstruction. The purpose was to evaluate if a generally applied rehabilitation program restores functionality to levels of healthy controls. Subjects with unilateral tears of the ACL were compared to matched healthy controls throughout the rehabilitation. 20 recreational athletes were tested: T1 (preoperative), 6 weeks after tear; T2, 6 weeks, T3, 3 months, T4, 6 months post-reconstruction. At all test sessions, subjects self-evaluated their activity level with the Tegner activity score and their knee state with the Knee Injury and Osteoarthritis Outcome Score. Passive range of motion during knee flexion and extension and leg circumference were measured as functional clinical tests. Bilateral countermovement jumps, one-leg jumps for distance and isometric force tests in knee flexion and extension with 90° and 110° knee angle were conducted as functional performance tests. For determination of functionality, leg symmetry indices (LSIs) were calculated by dividing values of the injured by the uninjured leg. In the ACL group most LSIs decreased from T1 to T2, and increased from T2 and T3 to T4. LSIs of ACL subjects remained lower than LSIs of healthy controls at 6 months post-reconstruction in nearly all parameters. Self-evaluation of ACL subjects showed, additionally, that activity level was lower than the pre-injury level at 6 months post-reconstruction. Low LSIs and low self-evaluation indicate that knee joint functionality is not completely restored at 6 months post-reconstruction. The study shows that multiple comprehensive testing throughout the rehabilitation gives detailed images of the functional state. Therefore, the functional state of ACL reconstructed individuals should be evaluated comprehensively and continuously throughout the rehabilitation to detect persisting deficiencies detailed and adapt rehabilitation programs individually depending on the functionality.

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.