Andre Lacerda Braga Teles, Roseane R. Silva, Michelle Ko, Glaucio Monteiro Ferreira, Samuel da Rocha Pita, Gustavo Henrique Goulart Trossini, Paulo Carvalho and Marcelo Santos Castilho* Pages 406 - 417 ( 12 )
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 macromolecular target.
Schistosoma mansoni, DHFR, 2, 4-diaminopyrimidine, Thermal shift assay, Kinetic assay, Molecular modelling.
Programa de Pos-graduacao em Biotecnologia – PPGBiotec, Universidade Estadual de Feira de Santana, Feira de Santana, Washington University School of Medicine, St. Louis, Dulaney High School, Timonium, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, Faculdade de Farmacia, Universidade Federal da Bahia, Salvador, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, Notre Dame of Maryland University, Baltimore, Programa de Pos-graduacao em Biotecnologia – PPGBiotec, Universidade Estadual de Feira de Santana, Feira de Santana