Introduction: Schistosoma mansoni is responsible for virtually all reported cases of schistosomiasis
in Latin America and the emergence of praziquantel- and oxaminiquine-resistant strains
makes it urgent to develop new schistosomicide agents. Dihydrofolate reductases (DHFR) from bacteria
and protozoan parasites are considered validated macromolecular targets for this goal, but S. mansoni
DHFR (SmDHFR) has been largely overlooked. To fill this gap in knowledge, the present work describes
optimized conditions to carry out thermal shift assays with SmDHFR, as well as a balanced kinetic
assay that supports 2,4-diaminopyrimidine derivatives as SmDHFR inhibitors. The most potent
inhibitor (2a) shows a large shift of the melting temperature (ΔTm = + 8 ± 0,21 ºC) and a low micromolar
IC50 value (12 ± 2,3 μM). Both thermal shift and classical kinectic assay suggest that 2a binds to the
substrate binding site (competitive inhibition mechanism). This information guided docking and molecular
dynamics studies that probed 2a interaction profile towards SmDHFR.
Conclusion: In conclusion, this work not only provides standardized assay conditions to identify
SmDHFR inhibitors, but also describes the binding profile of the first low micromolar inhibitor of this
Keywords: Schistosoma mansoni, DHFR, 2, 4-diaminopyrimidine, Thermal shift assay, Kinetic assay, Molecular modelling.
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