Virtual Screening Models of Cruzain Inhibitors
Transcrição
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 10 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. 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