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Brazilian Chemical Society (SBQ). Division of Medicinal Chemistry. 4th Brazilian Symposium on Medicinal Chemistry Non-Selective Inhibition of Trypanosoma cruzi GAPDH and rabbit GAPDH determined by Isothermal Titration Calorimetry 1,2 1 1 1 Helton José Wiggers , Juliana Cheleski , Renato F. Freitas , Vinicius Sciuti , Adriano Defini 3 1* Andricopulo , Carlos Alberto Montanari 1 Grupo de Química Medicinal de Produtos Naturais, NEQUIMED-PN. Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador Sancarlense, 400, 13560-970, São 2 Carlos, SP, Brazil. Tel: +55 16 3373-9986; Fax: +55 16 3373-9985. Departamento de Química, Universidade Federal 3 de São Carlos Laboratório de Química Medicinal e Computacional, Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo. Email: [email protected]. Keywords: Enzyme kinetics, ITC, GAPDH, Introduction Isothermal titration calorimetry (ITC) has been employed in kinetics parameters determination of enzymes belonging to trypanosomatids. In this work, we report a calorimetric assay for glycosomal glyceraldehyde-3-phophate dehydrogenase enzyme (EC 1.2.1.12 - GAPDH) an important enzyme in the life cycle of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas´ disease. Michaelis-Menten kinetics parameters were determined for the conversion of glyceraldehyde 3-phosphate to 1-arseno-3phosphoglycerate by TcGAPDH. Novel TcGAPDH inhibitors were found from our in silico screening and a bisphosphonate was tested as inhibitor of the TcGAPDH enzyme. A plausible binding site was proposed using docking as a tool for describing the pose it should take when interacting with the enzyme. Data are in close agreement with previous X-ray crystallographic data. Assaying the bisphosphonate against rabbit GAPDH (rGAPDH) has yielded a similar mode of binding as the one found for TcGAPDH. Analysis of proton-linked binding during the rGAPDH catalysis was evaluated, and the intrinsic enthalpy of reaction was determined. Results and Discussion Figure 1 (A) shows the selection of compounds by drug-like filters and (B) docking in the TcGAPDH database-screening step. Trypanosoma cruzi, Chagas´ disease Titration experiments were carried out on an Isothermal Titration Calorimetry instrument (VP-ITC, MicroCal, Northampton, USA), at 25 oC. The conversion rates of glyceraldehyde-3-phosphate to 1arseno-3-phosphoglycerate catalyzed by TcGAPDH in solutions containing no cosolvent and 5% v/v DMSO in TEA buffer were monitored via ITC. Firstly, the steady state condition was verified via the varied concentration of the substrate in the cell chamber. We then measured the inhibitory activity of 4butylphenil-amine-methylene-phosphonic acid that has shown to be a TcGAPDH inhibitor. The inhibition constant (Ki) was determined by fitting graphically the rate against substrate concentration in the MichaelisMenten equation. A docking study followed by molecular dynamics simulations of the inhibitor posed it nearby the active site. Rabbit muscle GAPDH (rabGAPDH) exhibits a high degree of homology to the human GAPDH. Thus, commercial rabGAPDH (Sigma) was assayed using the same protocol as the one used for TcGAPDH. A set of four buffered solutions was employed to the analysis of proton-linked binding by ploting of ∆Hion vs ∆Happ. Conclusions We have successfully used virtual screening as a computational approach to assess potential novel noncovalent inhibitors for TcGAPDH and rabGAPDH. Its direct integration with Isothermal Titration Calorimetry allowed the identification of the bisphosphonate inhibitory activity against the two enzymes. Inhibition of rabGAPDH was comparable to that of TcGAPDH when the same TEA buffer was used, via the same mode of binding. Acknowledgements (CNPq, FAPESP) ____________________ 1 Figure 1. In silico selection of TcGAPDH inhibitors Télles-Valencia et al. J. Mol. Biol. 2004, 341, 1355–1365 Wiggers et al. Anal. Biochem. 2007, 370, 107-114 3 Ladame et al. Eur. J. Biochem. 2003, 270, 4574-4586 2 4th Brazilian Symposium on Medicinal Chemistry – BrazMedChem2008