Metacyclogenesis is a critical process during Trypanosoma cruzi life cycle. This transition naturally occurs in the insect vector’s gut, influenced by temperature, nutrient availability and pH fluctuations, among others. Triatomine Artificial Urine (TAU) medium is the widely adopted approach to metacyclogenesis in vitro. However, this becomes less effective when epimastigotes are cultured for long periods, limiting the study of the metacyclic trypomastigote stage. We aimed to establish a practical and efficient method for generating high concentrations and purity of metacyclic epimastigotes in vitro. Epimastigotes of the Dm28c strain were exposed to pH shifts in nutrient-rich (MT-LIT) and nutrient-poor (M16) media, under static or agitated conditions. Results showed that both media promoted higher metacyclogenesis rates than TAU, with epimastigotes adherence to the substrate being crucial factor. Metacyclogenesis efficiency varies depending on the strains and culture conditions. LIT and M16 at pH 6 produced metacyclic trypomastigotes with infective capacity on Vero cells. Under these conditions, a variety of intermediate forms were observed compared to those induced by TAU metacyclogenesis. Our findings further emphasize the strain-dependent nature of optimal in vitro metacyclogenesis conditions and offer new opportunities for studying the intermediate forms involved in this essential process.

Background: In recent years, compounds known as Proteolysis Targeted Chimeras (PROTACs) have revitalized the field of bioactive molecule design. These compounds promote proteolysis of therapeutic targets by recruiting them to ubiquitin ligases. One of the most commonly used classes of compounds in the synthesis of PROTACs are immunomodulatory imides (IMiDs), such as thalidomide (TLD), which interact with the E3 ligase CRL4^CRBN via the CULT domain of the cereblon protein (CRBN). This domain has been identified in proteins across various phylogenetic groups, including trypanosomatids, leading to the hypothesis that IMiD-derived PROTACs should be active in these organisms. Methods: The trypanocidal activity of the PROTAC dBET1 and its separated components (JQ1 and TLD) were assayed using a T. cruzi strain expressing β-glalactosidase. Potential CRL4-E3L complexes from humans and trypanosomatids were assembled in silico with MultimerMapper. The IMiD-binding site of HsCRBN and its trypanosomatid homologs were analyzed using molecular dynamics and docking simulations. Results: We demonstrate that the compound dBET1 does not function as a PROTAC in Trypanosoma cruzi. In silico structural analysis of CRL4-E3L complex orthologs revealed that the trypanosomal CULT-containing protein is not part of such a complex. Molecular dynamics simulations showed that the pocket of this CULT domain is smaller than that of mammalian CRBN and cannot accommodate IMiDs within. Conclusions: We underscore the importance of functional and structural validation in drug discovery, particularly when extrapolating mechanisms between evolutionarily distant species. While PROTACs hold promise in human therapeutics, our work advocates for re-evaluating the rationale behind thalidomide-based PROTACs in trypanosomatid research.

Metacyclogenesis is a critical process in the Trypanosoma cruzi life cycle. This transition naturally occurs in an insect vector’s gut. Although Triatomine Artificial Urine (TAU) medium is the widely adopted approach to metacyclogenesis in vitro, its efficiency decreases with prolonged epimastigote culture, limiting the availability of metacyclic trypomastigotes for study. We aimed to establish a practical and efficient method for generating high concentrations and purities of metacyclic trypomastigotes in vitro. Epimastigotes of the Dm28c strain were exposed to pH shifts in nutrient-rich (MT-LIT) and nutrient-poor (M16) media, under static or agitated conditions. Both media promoted higher metacyclogenesis rates than TAU, with epimastigotes’ adherence to the substrate being a crucial factor. Metacyclogenesis efficiency varies depending on the strains and culture conditions. Notably, both LIT and M16 at pH 6 produced metacyclic trypomastigotes with infective capacity on Vero cells. Under these conditions, a variety of intermediate forms were observed compared to those induced by TAU metacyclogenesis. Our findings further emphasize the strain-dependent nature of optimal in vitro metacyclogenesis conditions and offer new opportunities for studying the intermediate forms involved in this essential process.