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Natural stilbenoids isolated from grapevine exhibiting inhibitory effects against HIV-1 integrase and eukaryote MOS1 transposase in vitro activities.

Pflieger A, Waffo Teguo P, Papastamoulis Y, Chaignepain S, Subra F, Munir S, Delelis O, Lesbats P, Calmels C, Andreola ML, Merillon JM, Auge-Gouillou C, Parissi V - PLoS ONE (2013)

Bottom Line: Some of the molecules were found to be active against both proteins while others were specific for one of the two models.Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions.Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells.

View Article: PubMed Central - PubMed

Affiliation: Université François Rabelais de Tours, EA 6306, UFR Sciences Pharmaceutiques, Parc Grandmont, Tours, France.

ABSTRACT
Polynucleotidyl transferases are enzymes involved in several DNA mobility mechanisms in prokaryotes and eukaryotes. Some of them such as retroviral integrases are crucial for pathogenous processes and are therefore good candidates for therapeutic approaches. To identify new therapeutic compounds and new tools for investigating the common functional features of these proteins, we addressed the inhibition properties of natural stilbenoids deriving from resveratrol on two models: the HIV-1 integrase and the eukaryote MOS-1 transposase. Two resveratrol dimers, leachianol F and G, were isolated for the first time in Vitis along with fourteen known stilbenoids: E-resveratrol, E-piceid, E-pterostilbene, E-piceatannol, (+)-E-ε-viniferin, E-ε-viniferinglucoside, E-scirpusin A, quadragularin A, ampelopsin A, pallidol, E-miyabenol C, E-vitisin B, hopeaphenol, and isohopeaphenol and were purified from stalks of Vitis vinifera (Vitaceae), and moracin M from stem bark of Milliciaexelsa (Moraceae). These compounds were tested in in vitro and in vivo assays reproducing the activity of both enzymes. Several molecules presented significant inhibition on both systems. Some of the molecules were found to be active against both proteins while others were specific for one of the two models. Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions. Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells. Consequently, in addition to representing new original lead compounds for further modelling of new active agents against HIV-1 integrase, these molecules could be good tools for identifying such reaction intermediates in DNA mobility processes.

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Selection by in vitro transposition of natural stilbene compounds active against Mos1 transposase.(A)in vitro transposition test principle. pBC-3T3 was used both as the donor of pseudo-Mos1 and as the target for integration. It contained the pBR322 tetracycline resistance gene (without promoter) flanked by two Mos1 3′ITR. This plasmid was unable to confer resistance to Escherichia coli cells in tetracycline concentrations over 10 µg/ml. Upon transposition, MOS1 excises the pseudo-Mos1 from one pBC-3T3 molecule, and then triggers its reinsertion within the cat gene of another pBC-3T3 molecule [28]. Transposition events are revealed by promoter tagging, the tetracycline resistance being activated through the cat gene promoter. Transposition rates were quantified as reported in materials and methods section and the transposition inhibition is shown in (B) using the transposition rate obtained in presence of DMSO as reference. Values are the means from at least two independent experiments ± standard deviation (error bars). Only compounds giving a transposition rate inhibition over 50% at 25 µM were assayed at 10 µM.
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pone-0081184-g006: Selection by in vitro transposition of natural stilbene compounds active against Mos1 transposase.(A)in vitro transposition test principle. pBC-3T3 was used both as the donor of pseudo-Mos1 and as the target for integration. It contained the pBR322 tetracycline resistance gene (without promoter) flanked by two Mos1 3′ITR. This plasmid was unable to confer resistance to Escherichia coli cells in tetracycline concentrations over 10 µg/ml. Upon transposition, MOS1 excises the pseudo-Mos1 from one pBC-3T3 molecule, and then triggers its reinsertion within the cat gene of another pBC-3T3 molecule [28]. Transposition events are revealed by promoter tagging, the tetracycline resistance being activated through the cat gene promoter. Transposition rates were quantified as reported in materials and methods section and the transposition inhibition is shown in (B) using the transposition rate obtained in presence of DMSO as reference. Values are the means from at least two independent experiments ± standard deviation (error bars). Only compounds giving a transposition rate inhibition over 50% at 25 µM were assayed at 10 µM.

Mentions: To identify the natural stilbenes active on MOS1, the 17 molecules were tested using an in vitro genetic “hop” experiment, which is the most sensitive test available as yet. Purified MOS1 was incubated with the pBC-3T3 plasmid, which was used both as the transposon donor and the target plasmid. This plasmid contains the pBR322 tetracycline resistance gene (without promoter) framed by two Mos1 ITRs. This reconstitutes a pseudo-Mos1 element named 3T3. We took advantage of the fact that the cat gene (present in the pBC backbone) is a hotspot for Mos1 integration [27]. Consequently, transposition events are revealed by promoter tagging, the tetracycline resistance being activated through the cat gene promoter (figure 6A). Transposition events were recovered by bacterial transformation with selection for tetracycline resistance, as a gain-of-function landmark for transposition. A first round of selection was performed by testing the molecules at a final concentration of 25 µM. Controls correspond to assays performed without molecule but with DMSO, which is routinely used as the solvent. As shown in figure 6B, leachianol G, leachianol F and quadrangularin A, which were previously shown to be active against HIV-1 IN, also induced over 50% inhibition of the whole MOS1 transposition activity. In addition, three other compounds were shown to be active against MOS1: hopeaphenol, E-vitisin B and pallidol. To confirm the efficiency of the six molecules, they were similarly assayed at 10 µM (figure 6B). Since they appeared to be still active at 10 µM, they were further characterized in order to identify the targeted step of the transposition cycle.


Natural stilbenoids isolated from grapevine exhibiting inhibitory effects against HIV-1 integrase and eukaryote MOS1 transposase in vitro activities.

Pflieger A, Waffo Teguo P, Papastamoulis Y, Chaignepain S, Subra F, Munir S, Delelis O, Lesbats P, Calmels C, Andreola ML, Merillon JM, Auge-Gouillou C, Parissi V - PLoS ONE (2013)

Selection by in vitro transposition of natural stilbene compounds active against Mos1 transposase.(A)in vitro transposition test principle. pBC-3T3 was used both as the donor of pseudo-Mos1 and as the target for integration. It contained the pBR322 tetracycline resistance gene (without promoter) flanked by two Mos1 3′ITR. This plasmid was unable to confer resistance to Escherichia coli cells in tetracycline concentrations over 10 µg/ml. Upon transposition, MOS1 excises the pseudo-Mos1 from one pBC-3T3 molecule, and then triggers its reinsertion within the cat gene of another pBC-3T3 molecule [28]. Transposition events are revealed by promoter tagging, the tetracycline resistance being activated through the cat gene promoter. Transposition rates were quantified as reported in materials and methods section and the transposition inhibition is shown in (B) using the transposition rate obtained in presence of DMSO as reference. Values are the means from at least two independent experiments ± standard deviation (error bars). Only compounds giving a transposition rate inhibition over 50% at 25 µM were assayed at 10 µM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842960&req=5

pone-0081184-g006: Selection by in vitro transposition of natural stilbene compounds active against Mos1 transposase.(A)in vitro transposition test principle. pBC-3T3 was used both as the donor of pseudo-Mos1 and as the target for integration. It contained the pBR322 tetracycline resistance gene (without promoter) flanked by two Mos1 3′ITR. This plasmid was unable to confer resistance to Escherichia coli cells in tetracycline concentrations over 10 µg/ml. Upon transposition, MOS1 excises the pseudo-Mos1 from one pBC-3T3 molecule, and then triggers its reinsertion within the cat gene of another pBC-3T3 molecule [28]. Transposition events are revealed by promoter tagging, the tetracycline resistance being activated through the cat gene promoter. Transposition rates were quantified as reported in materials and methods section and the transposition inhibition is shown in (B) using the transposition rate obtained in presence of DMSO as reference. Values are the means from at least two independent experiments ± standard deviation (error bars). Only compounds giving a transposition rate inhibition over 50% at 25 µM were assayed at 10 µM.
Mentions: To identify the natural stilbenes active on MOS1, the 17 molecules were tested using an in vitro genetic “hop” experiment, which is the most sensitive test available as yet. Purified MOS1 was incubated with the pBC-3T3 plasmid, which was used both as the transposon donor and the target plasmid. This plasmid contains the pBR322 tetracycline resistance gene (without promoter) framed by two Mos1 ITRs. This reconstitutes a pseudo-Mos1 element named 3T3. We took advantage of the fact that the cat gene (present in the pBC backbone) is a hotspot for Mos1 integration [27]. Consequently, transposition events are revealed by promoter tagging, the tetracycline resistance being activated through the cat gene promoter (figure 6A). Transposition events were recovered by bacterial transformation with selection for tetracycline resistance, as a gain-of-function landmark for transposition. A first round of selection was performed by testing the molecules at a final concentration of 25 µM. Controls correspond to assays performed without molecule but with DMSO, which is routinely used as the solvent. As shown in figure 6B, leachianol G, leachianol F and quadrangularin A, which were previously shown to be active against HIV-1 IN, also induced over 50% inhibition of the whole MOS1 transposition activity. In addition, three other compounds were shown to be active against MOS1: hopeaphenol, E-vitisin B and pallidol. To confirm the efficiency of the six molecules, they were similarly assayed at 10 µM (figure 6B). Since they appeared to be still active at 10 µM, they were further characterized in order to identify the targeted step of the transposition cycle.

Bottom Line: Some of the molecules were found to be active against both proteins while others were specific for one of the two models.Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions.Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells.

View Article: PubMed Central - PubMed

Affiliation: Université François Rabelais de Tours, EA 6306, UFR Sciences Pharmaceutiques, Parc Grandmont, Tours, France.

ABSTRACT
Polynucleotidyl transferases are enzymes involved in several DNA mobility mechanisms in prokaryotes and eukaryotes. Some of them such as retroviral integrases are crucial for pathogenous processes and are therefore good candidates for therapeutic approaches. To identify new therapeutic compounds and new tools for investigating the common functional features of these proteins, we addressed the inhibition properties of natural stilbenoids deriving from resveratrol on two models: the HIV-1 integrase and the eukaryote MOS-1 transposase. Two resveratrol dimers, leachianol F and G, were isolated for the first time in Vitis along with fourteen known stilbenoids: E-resveratrol, E-piceid, E-pterostilbene, E-piceatannol, (+)-E-ε-viniferin, E-ε-viniferinglucoside, E-scirpusin A, quadragularin A, ampelopsin A, pallidol, E-miyabenol C, E-vitisin B, hopeaphenol, and isohopeaphenol and were purified from stalks of Vitis vinifera (Vitaceae), and moracin M from stem bark of Milliciaexelsa (Moraceae). These compounds were tested in in vitro and in vivo assays reproducing the activity of both enzymes. Several molecules presented significant inhibition on both systems. Some of the molecules were found to be active against both proteins while others were specific for one of the two models. Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions. Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells. Consequently, in addition to representing new original lead compounds for further modelling of new active agents against HIV-1 integrase, these molecules could be good tools for identifying such reaction intermediates in DNA mobility processes.

Show MeSH
Related in: MedlinePlus