Virtual Screening Models of Cruzain Inhibitors

Brazilian Chemical Society (SBQ). Division of Medicinal Chemistry. 4th Brazilian Symposium on Medicinal Chemistry
Virtual Screening Models of Cruzain Inhibitors: Development and
Experimental Validation
1
1
Malvezzi, A.* ; Rezende, L ; Amaral, A. T. –do
1
[email protected]
1 - Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
Keywords: virtual screening, cruzain, docking; pharmacophore model; drug-like
Introduction
1
Seeking for new cruzain inhibitor(s) – a cysteine
protease of Trypanosoma cruzi, the etiologic agent
2
of Chagas disease – two virtual screening schemes
(Models I and II) were proposed, validated and
3
applied to the ZINC
database (3.294.714
compounds).
The proposed virtual screening models, composed
4,5
of a sequence of different physical-chemical ,
6
pharmacophore (Catalyst program ), docking (Gold
7
program ) and visual inspection filters, were built
from information taken from 33 PDB complexes of
cruzain and other cysteine proteases.
A detailed recognition of the cruzain structural
features and characteristics was performed through
visual inspection of the enzyme environment;
8
followed by the analysis of GRID generated
molecular interaction fields. Also, the molecular
interaction properties on the enzyme cavity were
9
analyzed using the CAVBASE program; and the
flexibility and induced-fix of the protein was analyzed
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by molecular dynamics simulations (Gromacs ).
Results and Discussion
The virtual screening Model I, when applied to the
ZINC database selected 10 compounds. Six of these
compounds were acquired and tested as cruzain
11
inhibitors . Three of the tested compounds (1, 2 and
3, respectively) did not show any significant cruzain
inhibition, up to 7 mM, while the other three tested
compounds (4, 5 and 6, respectively) showed
unspecific cruzain inhibition, suggesting an enzyme
12
inhibition by the promiscuous mechanism . This
mechanism was verified by the addition of 0.1%
Triton X-100 on the enzymatic assay with a
concomitant loss of cruzain inhibition activity. For
these compounds, the corroboration of the
promiscuous mechanism was achieved observing
the loss of enzyme inhibition after a ten times
increase in the cruzain concentration on the
13
enzymatic assay .
The virtual screening Model II selected 55
compounds, when applied to the ZINC database.
Nineteen of these compounds were acquired and
tested as cruzain inhibitors. One compound, showed
a specific cruzain inhibition (Ki = 21 µM), while the
other eighteen showed no significant inhibition, up to
592µM concentration. The promiscuous mechanism
of enzymatic inhibition was not observed, since 0.1%
of Triton X-100 was added in all assays. Additionally,
Model II identified two other cruzain inhibitors,
however, these compounds were not acquired or
tested, since they are known cruzain inhibitors 14
already described in the literature
– and are
structurally similar to the inhibitors used in the
construction of the model.
Conclusions
For Model I three out of six tested compounds
were promiscuous inhibitors. This high proportion of
promiscuous acting compounds shows that this kind
of artifact can be prevalent in vitro assays and is a
real concern in both HTS and VS programs
Model II found one cruzain inhibitor out of 19
tested compounds. This represents a hit rate of
5,3% of the VS program. This value is in agreement
15
with those found in the literature , that ranges from
1 to 50%.
Acknowledgements
The present study was financially supported by
FAPESP, PRP-USP Procontes and CAPES
PROBRAL. The authors acknowledge the valuable
collaboration of Prof. Hugo Kubinyi with this project.
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4th Brazilian Symposium on Medicinal Chemistry – BrazMedChem2008
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