Download Non-Selective Inhibition of Trypanosoma cruzi GAPDH and rabbit

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
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Helton José Wiggers , Juliana Cheleski , Renato F. Freitas , Vinicius Sciuti , Adriano Defini
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Andricopulo , Carlos Alberto Montanari
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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
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Carlos, SP, Brazil. Tel: +55 16 3373-9986; Fax: +55 16 3373-9985. Departamento de Química, Universidade Federal
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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)
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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
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Ladame et al. Eur. J. Biochem. 2003, 270, 4574-4586
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4th Brazilian Symposium on Medicinal Chemistry – BrazMedChem2008