Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme

Abstract

Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS 2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.

Innovative Approach for a Classic Target: Fragment Screening on Trypanothione Reductase Reveals New Opportunities for Drug Design

“…TR from L. infantum was produced as previously reported. TR from T. brucei (TbTR) was produced as previously reported ( Turcano et al, 2020 ), with minor modifications. Briefly, the gene corresponding to TbTR (Uniprot ID: Q389T8) was cloned in the pET15b vector using the restriction site NdeI and XhoI to add an N-terminal His-tag.…”

“…Considerable effort has been put together to identify new hits targeting TR, rationalize the interaction through structural characterization, and improve known scaffolds through SAR studies or structure-based design ( Bernardes et al, 2013 ; Field et al, 2017 ; Battista et al, 2020 ), but despite some remarkable results ( Patterson et al, 2011 ; De Gasparo et al, 2018 ; Turcano et al, 2018 ; De Gasparo et al, 2019 ; Turcano et al, 2020 ), none of the leads proposed has been yet promoted to preclinical trials, due to sub-optimal activity or toxicity issue. Most characterized inhibitors were found to target the TSH cavity, mainly the so-called mepacrine binding site (MBS) located at the entrance of the TSH cavity.…”

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

“…TR from L. infantum and from T. brucei were expressed and purified as previously reported. 17,29,33 Briefly, the genes, coding for the enzymes (aa 1−492) and codon optimized for the expression in E. coli, were obtained from GenScript. The coding sequences were then cloned in the pET15b vector to have an N-terminal fused 6xHisTag for purification purposes.…”

“…Recent years have witnessed a significant research effort toward the identification of effective modulators of the enzymes involved in T­(SH) 2 metabolism, which are both necessary for parasite survival and absent in the human host. Since several years, we have focused on the design, synthesis, and evaluation of novel molecular scaffolds as anti- Leishmania agents that can also interfere with the trypanothione metabolism. − Among the drug targets explored in this field, it has been demonstrated that pentavalent antimonials affect the TR activity, which is essential for survival of Leishmania inside the macrophages. , Moreover, due to its structural and sequence similarity to TR from other trypanosomatids such as T. brucei and T.…”

Tackling Sleeping Sickness: Current and Promising Therapeutics and Treatment Strategies

“…Also, to achieve an adequate trypanocidal effect, TyrR activity must be inhibited by up to 90% due to the enzyme's elevated efficacy in terms of turnover [207,211]. A spiro derivative molecule has been determined to differentially inhibit trypanothione reductase, having no modulatory effect on human glutathione reductase [212]. Spiro molecules serve as a scaffold for BBB-penetrant molecules [212].…”