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Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

Wiggers HJ, Rocha JR, Fernandes WB, Sesti-Costa R, Carneiro ZA, Cheleski J, da Silva AB, Juliano L, Cezari MH, Silva JS, McKerrow JH, Montanari CA - PLoS Negl Trop Dis (2013)

Bottom Line: Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation.Two compounds were found to have trypanocidal activity.The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil.

ABSTRACT
A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3-60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.

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Crystal structure of Neq176 co-crystallized with cruzain showing the mode of binding (MOB) of the inhibitor at the catalytic site of chain B with the unbiased mFo-DFc electron density map shown in cyan.Figure prepared using CCP4mg software [43].
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pntd-0002370-g007: Crystal structure of Neq176 co-crystallized with cruzain showing the mode of binding (MOB) of the inhibitor at the catalytic site of chain B with the unbiased mFo-DFc electron density map shown in cyan.Figure prepared using CCP4mg software [43].

Mentions: The 2-acetamidothiophene-3-carboxamide is probably the one responsible for the activity presented by compound Neq42. As can be observed by the comparison between the active and inactive series shown in Figure 6, molecules lacking this moiety completely lose their activity against the cruzain enzyme. Nevertheless, potencies of compounds Neq165, Neq176, Neq177 are still in a similar order of magnitude as compound Neq42, notwithstanding a significant MW lowering that results in an increased LE for the analogs (0.33 kcal mol−1 atom−1 for compound Neq176). This observation signals that the piperidine and benzyl groups give rise only to a minor contribution to the potency, since removal of both groups (compound Neq176) resulted only in a slight decrease in potency, but with a significant LE improvement to 0.33 kcal mol−1 atom−1. The compound Neq172, which is a combination derived from compounds Neq38 and Neq42, resulted in smaller LE and potency, once again evincing the importance of 2-acetamidothiophene-3-carboxamide for the activity. Not only tailored LEs were substantially increased but also a new non-peptidic scaffold, which is an excellent starting point for optimization, was identified in agreement with the proposed binding mode shown in Figure 7. [43]


Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

Wiggers HJ, Rocha JR, Fernandes WB, Sesti-Costa R, Carneiro ZA, Cheleski J, da Silva AB, Juliano L, Cezari MH, Silva JS, McKerrow JH, Montanari CA - PLoS Negl Trop Dis (2013)

Crystal structure of Neq176 co-crystallized with cruzain showing the mode of binding (MOB) of the inhibitor at the catalytic site of chain B with the unbiased mFo-DFc electron density map shown in cyan.Figure prepared using CCP4mg software [43].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750009&req=5

pntd-0002370-g007: Crystal structure of Neq176 co-crystallized with cruzain showing the mode of binding (MOB) of the inhibitor at the catalytic site of chain B with the unbiased mFo-DFc electron density map shown in cyan.Figure prepared using CCP4mg software [43].
Mentions: The 2-acetamidothiophene-3-carboxamide is probably the one responsible for the activity presented by compound Neq42. As can be observed by the comparison between the active and inactive series shown in Figure 6, molecules lacking this moiety completely lose their activity against the cruzain enzyme. Nevertheless, potencies of compounds Neq165, Neq176, Neq177 are still in a similar order of magnitude as compound Neq42, notwithstanding a significant MW lowering that results in an increased LE for the analogs (0.33 kcal mol−1 atom−1 for compound Neq176). This observation signals that the piperidine and benzyl groups give rise only to a minor contribution to the potency, since removal of both groups (compound Neq176) resulted only in a slight decrease in potency, but with a significant LE improvement to 0.33 kcal mol−1 atom−1. The compound Neq172, which is a combination derived from compounds Neq38 and Neq42, resulted in smaller LE and potency, once again evincing the importance of 2-acetamidothiophene-3-carboxamide for the activity. Not only tailored LEs were substantially increased but also a new non-peptidic scaffold, which is an excellent starting point for optimization, was identified in agreement with the proposed binding mode shown in Figure 7. [43]

Bottom Line: Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation.Two compounds were found to have trypanocidal activity.The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil.

ABSTRACT
A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3-60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.

Show MeSH
Related in: MedlinePlus