Paulo Otávio Lourenço Moreira

Context: Neglected Tropical Diseases (NTDs) face growing challenges due to parasite resistance and the declining efficacy of available treatments. For diseases such as Chagas and malaria, limited private sector investment further hinders therapeutic innovation, reinforcing the urgent need for more effective interventions. Aims: To analyze the antiprotozoal potential of known medicinal species by providing novel insights into their biological activities. Methods: Five ethnomedicinal plant species were selected based on their limited documentation regarding antiprotozoal activity. Twenty crude extracts were obtained by maceration with solvents of increasing polarity from the branches of Anchietea salutaris A. St.-Hil. and Davilla rugosa Poir., and the aerial parts of Banisteriopsis argyrophylla A. Juss., Grindelia robusta Nutt. and Pyrostegia venusta (Ker Gawl.) Miers. The extracts were tested in vitro against Trypanosoma cruzi, Leishmania amazonensis, and Plasmodium falciparum and evaluated for cytotoxicity to determine selectivity indexes (SI). Results: In the preliminary screening at 25 µg/mL, all species exhibited some antiprotozoal activity. None of the extracts were considered active against T. cruzi. Hexane and dichloromethane extracts from A. salutaris, B. argyrophylla, and D. rugosa demonstrated potent antiplasmodial effects (IC50  9.2 g/mL; SI ≥ 22). Additionally, the dichloromethane extract of G. robusta showed strong activity against L. amazonensis (IC50 = 2.2 g/mL; SI = 78). Conclusions: These findings highlight the potential of underexplored medicinal plant species as sources of bioactive compounds for developing treatments for NTDs.

Leishmania presents a complex life cycle that involves both invertebrate and vertebrate hosts. By regulating gene expression, protein synthesis, and metabolism, the parasite can adapt to various environmental conditions. This regulation occurs mainly at the post‐transcriptional level and may involve epitranscriptomic modifications of RNAs. Recent studies have shown that mRNAs in humans undergo a modification known as N4‐acetylcytidine (ac4C) catalyzed by the enzyme N‐acetyltransferase (NAT10), impacting mRNAs stability and translation. Here, we characterized the NAT10 homologue of L. mexicana, finding that the enzyme exhibits all the conserved acetyltransferase domains although failed to functionally complement the Kre33 mutant in Saccharomyces cerevisiae. We also discovered that LmexNAT10 is nuclear, and seems essential, as evidenced by unsuccessful attempts to obtain null mutant parasites. Phenotypic characterization of single‐knockout parasites revealed that LmexNAT10 affects the multiplication of procyclic forms and the promastigote‐amastigote differentiation. Additionally, in vivo infection studies using the invertebrate vector Lutzomyia longipalpis showed a delay in the parasite differentiation into metacyclics. Finally, we observed changes in the cell cycle progression and protein synthesis in the mutant parasites. Together, these results suggest that LmexNAT10 might be important for parasite differentiation, potentially by regulating ac4C levels.

Leishmania presents a complex life cycle that involves both invertebrate and vertebrate hosts. By regulating gene expression, protein synthesis, and metabolism, the parasite can adapt to various environmental conditions. This regulation occurs mainly at the post-transcriptional level and may involve epitranscriptomic modifications of RNAs. Recent studies have shown that mRNAs in humans undergo a modification known as N4-acetylcytidine (ac4C) catalyzed by the enzyme N-acetyltransferase (NAT10), impacting mRNAs stability and translation. Here, we characterized the NAT10 homologue of L. mexicana , finding that the enzyme exhibits all the conserved acetyltransferase domains and although failed to functionally complement the Kre33 mutant in Saccharomyces cerevisia e, has in vitro acetyltransferase activity. We also discovered that LmexNAT10 is nuclear, and seems essential, as evidenced by unsuccessful attempts to obtain null mutant parasites. Phenotypic characterization of single-knockout parasites revealed that LmexNAT10 affects the multiplication of procyclic forms and the promastigote-amastigote differentiation. Additionally, in vivo infection studies using the invertebrate vector Lutzomyia longipalpis showed a delay in the parasite differentiation into metacyclics. Finally, we observed changes in the cell cycle progression and protein synthesis in the mutant parasites. Together, these results suggest that LmexNAT10 might be important for parasite differentiation, potentially by regulating ac4C levels.

Despite decades of research devoted to finding a vaccine against leishmaniasis, we are still lacking a safe and effective vaccine for humans. Given this scenario, the search for a new prophylaxis alternative for controlling leishmaniasis should be a global priority. Inspired by leishmanization—a first generation vaccine strategy where live L. major parasites are inoculated in the skin to protect against reinfection—live-attenuated Leishmania vaccine candidates are promising alternatives due to their robust elicited protective immune response. In addition, they do not cause disease and could provide long-term protection upon challenge with a virulent strain. The discovery of a precise and easy way to perform CRISPR/Cas-based gene editing allowed the selection of safer null mutant live-attenuated Leishmania parasites obtained by gene disruption. Here, we revisited molecular targets associated with the selection of live-attenuated vaccinal strains, discussing their function, their limiting factors and the ideal candidate for the next generation of genetically engineered live-attenuated Leishmania vaccines to control leishmaniasis.

Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which makes the development of new drugs urgent. To achieve this goal, the integration of kinetic and DSF assays against parasitic validated targets, along with phenotypic assays, can help the identification and optimization of bioactive compounds. Pteridine reductase 1 (PTR1), a validated target in Leishmania sp., is responsible for the reduction of folate and biopterin to tetrahydrofolate and tetrahydrobiopterin, respectively, both of which are essential for cell growth. In addition to the in vitro evaluation of 16 thiazolidine-2,4-dione derivatives against Leishmania major PTR1 (LmPTR1), using the differential scanning fluorimetry (ThermoFluor®), phenotypic assays were employed to evaluate the compound effect over Leishmania braziliensis (MHOM/BR/75/M2903) and Leishmania infantum (MHOM/BR/74/PP75) promastigotes viability. The ThermoFluor® results show that thiazolidine-2,4-dione derivatives have micromolar affinity to the target and equivalent activity on Leishmania cells. 2b is the most potent compound against L. infantum (EC₅₀ = 23.45 ± 4.54 μM), whereas 2a is the most potent against L. braziliensis (EC₅₀ = 44.16 ± 5.77 μM). This result suggests that lipophilic substituents on either—meta and/or—para positions of the benzylidene ring increase the potency against L. infantum. On the other hand, compound 2c (CE₅₀ = 49.22 ± 7.71 μM) presented the highest selectivity index.