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Discovery of novel triple helical DNA intercalators by an integrated virtual and actual screening platform.

Holt PA, Ragazzon P, Strekowski L, Chaires JB, Trent JO - Nucleic Acids Res. (2009)

Bottom Line: Further molecular docking studies using compounds with high structural similarity resulted in two compounds that were then demonstrated by competition dialysis to have a superior affinity and selectivity for the triplex nucleic acid than MHQ-12.Biophysical characterization of these compounds by circular dichroism and thermal denaturation studies confirmed their binding mode and selectivity.These studies provide a proof-of-principle for our integrated screening strategy, and suggest that this platform may be extended to discover new compounds that target therapeutically relevant nucleic acid morphologies.

View Article: PubMed Central - PubMed

Affiliation: James Graham Brown Cancer Center, Department of Biochemistry and Molecular Biology, University of Louisville, 529 S. Jackson Street, Louisville, KY 40202, USA.

ABSTRACT
Virtual Screening is an increasingly attractive way to discover new small molecules with potential medicinal value. We introduce a novel strategy that integrates use of the molecular docking software Surflex with experimental validation by the method of competition dialysis. This integrated approach was used to identify ligands that selectively bind to the triplex DNA poly(dA)-[poly(dT)](2). A library containing approximately 2 million ligands was virtually screened to identify compounds with chemical and structural similarity to a known triplex intercalator, the napthylquinoline MHQ-12. Further molecular docking studies using compounds with high structural similarity resulted in two compounds that were then demonstrated by competition dialysis to have a superior affinity and selectivity for the triplex nucleic acid than MHQ-12. One of the compounds has a different chemical backbone than MHQ-12, which demonstrates the ability of this strategy to 'scaffold hop' and to identify small molecules with novel binding properties. Biophysical characterization of these compounds by circular dichroism and thermal denaturation studies confirmed their binding mode and selectivity. These studies provide a proof-of-principle for our integrated screening strategy, and suggest that this platform may be extended to discover new compounds that target therapeutically relevant nucleic acid morphologies.

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Chemical structures of the ligands used in virtual screening and competition dialysis experiments. (A) MHQ-12, (B) LS-08, (C) MHQ-15, (D) OZ-85H, (E) compound 1 and (F) compound 2.
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Figure 1: Chemical structures of the ligands used in virtual screening and competition dialysis experiments. (A) MHQ-12, (B) LS-08, (C) MHQ-15, (D) OZ-85H, (E) compound 1 and (F) compound 2.

Mentions: The initial step in virtual screening was performing Surflex-Sim to determine which of the ligands in the library were most structurally similar to the known, triplex selective intercalator MHQ-12. Of the approximately 2 million ligands screened for similarity against MHQ-12, 350 ligands had a Surflex-Sim score of greater than 0.70 (range 0.875–0.704) and were selected for Surflex-Docking studies. A cutoff Surflex-Sim score of 0.70 was selected based on previous studies which suggested that this is the lowest score where the ligand structure–function relationship is typically maintained (25). The next step in the virtual screening process involved performing Surflex-Dock studies with the top 350 ranked Surflex-Sim ligands using the intercalation site and the three grooves (major–major, major–minor and minor) of the triplex as individual docking targets. Interestingly, MHQ-12 has the top Surflex Raw Score out of all 350 ligands that were docked to the intercalation site, which directly supports the ability of Surflex-Dock to successfully dock and rank a known selective triplex intercalator. We propose a new metric to evaluate the Surflex-Dock results, the ‘Normalized Surflex Raw Score (NSRS)’. The rationale behind the normalization of the Surflex Raw Score is that the score for a ligand binding to a single site on a target measures only the interaction with that one site. However, a ligand may have multiple interaction sites on a particular target. Therefore, for selectivity for a particular mode of binding, it is crucial to determine the binding of the ligand to the site of interest relative to the binding to other potential sites on the target. Since ligands interact with nucleic acids typically through either the groove-binding or intercalation, protomols were constructed at the three grooves and the intercalation site (48). Binding of the ligand to the intercalation site relative to binding in the three grooves embodies the ‘normalized’ affinity and specificity of the ligand for triplex intercalation. The following metric determines the NSRS for the intercalation site for each of the top 350 Surflex-Dock results:2Ranking of the 350 intercalation site Surflex-Dock results by NSRS shows that only three ligands have a higher NSRS score than MHQ-12 (NSRS of 6.8) (Figure 1A). The ligands are LS-08 (49) (Figure 1B), compounds 1 (Figure 1E) and 2 (Figure 1F) and have NSRS values of 7.03, 7.34 and 7.39, respectively (Figure 1). Interestingly, LS-08 (Figure 1B) which was identified by our virtual screening methodology, was previously tested by Chaires (46) and shown to be highly triplex selective, which adds validity to our virtual screening approach used to identify triplex selective ligands. Based on the NSRS values, compounds 1 (Figure 1E) and 2 (Figure 1F) were hypothesized to have superior affinity and selectivity for binding to the triplex nucleic acid, and were tested by competition dialysis. Two known triplex selective compounds, MHQ-15 (Figure 1C) and OZ-85H (Figure 1D) served as positive controls, as these compounds have been extensively studied and characterized (18). Biophysical characterization was performed by circular dichroism and thermal denaturation to assess the ability of the compounds to intercalate into the DNA triplex.Figure 1.


Discovery of novel triple helical DNA intercalators by an integrated virtual and actual screening platform.

Holt PA, Ragazzon P, Strekowski L, Chaires JB, Trent JO - Nucleic Acids Res. (2009)

Chemical structures of the ligands used in virtual screening and competition dialysis experiments. (A) MHQ-12, (B) LS-08, (C) MHQ-15, (D) OZ-85H, (E) compound 1 and (F) compound 2.
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Figure 1: Chemical structures of the ligands used in virtual screening and competition dialysis experiments. (A) MHQ-12, (B) LS-08, (C) MHQ-15, (D) OZ-85H, (E) compound 1 and (F) compound 2.
Mentions: The initial step in virtual screening was performing Surflex-Sim to determine which of the ligands in the library were most structurally similar to the known, triplex selective intercalator MHQ-12. Of the approximately 2 million ligands screened for similarity against MHQ-12, 350 ligands had a Surflex-Sim score of greater than 0.70 (range 0.875–0.704) and were selected for Surflex-Docking studies. A cutoff Surflex-Sim score of 0.70 was selected based on previous studies which suggested that this is the lowest score where the ligand structure–function relationship is typically maintained (25). The next step in the virtual screening process involved performing Surflex-Dock studies with the top 350 ranked Surflex-Sim ligands using the intercalation site and the three grooves (major–major, major–minor and minor) of the triplex as individual docking targets. Interestingly, MHQ-12 has the top Surflex Raw Score out of all 350 ligands that were docked to the intercalation site, which directly supports the ability of Surflex-Dock to successfully dock and rank a known selective triplex intercalator. We propose a new metric to evaluate the Surflex-Dock results, the ‘Normalized Surflex Raw Score (NSRS)’. The rationale behind the normalization of the Surflex Raw Score is that the score for a ligand binding to a single site on a target measures only the interaction with that one site. However, a ligand may have multiple interaction sites on a particular target. Therefore, for selectivity for a particular mode of binding, it is crucial to determine the binding of the ligand to the site of interest relative to the binding to other potential sites on the target. Since ligands interact with nucleic acids typically through either the groove-binding or intercalation, protomols were constructed at the three grooves and the intercalation site (48). Binding of the ligand to the intercalation site relative to binding in the three grooves embodies the ‘normalized’ affinity and specificity of the ligand for triplex intercalation. The following metric determines the NSRS for the intercalation site for each of the top 350 Surflex-Dock results:2Ranking of the 350 intercalation site Surflex-Dock results by NSRS shows that only three ligands have a higher NSRS score than MHQ-12 (NSRS of 6.8) (Figure 1A). The ligands are LS-08 (49) (Figure 1B), compounds 1 (Figure 1E) and 2 (Figure 1F) and have NSRS values of 7.03, 7.34 and 7.39, respectively (Figure 1). Interestingly, LS-08 (Figure 1B) which was identified by our virtual screening methodology, was previously tested by Chaires (46) and shown to be highly triplex selective, which adds validity to our virtual screening approach used to identify triplex selective ligands. Based on the NSRS values, compounds 1 (Figure 1E) and 2 (Figure 1F) were hypothesized to have superior affinity and selectivity for binding to the triplex nucleic acid, and were tested by competition dialysis. Two known triplex selective compounds, MHQ-15 (Figure 1C) and OZ-85H (Figure 1D) served as positive controls, as these compounds have been extensively studied and characterized (18). Biophysical characterization was performed by circular dichroism and thermal denaturation to assess the ability of the compounds to intercalate into the DNA triplex.Figure 1.

Bottom Line: Further molecular docking studies using compounds with high structural similarity resulted in two compounds that were then demonstrated by competition dialysis to have a superior affinity and selectivity for the triplex nucleic acid than MHQ-12.Biophysical characterization of these compounds by circular dichroism and thermal denaturation studies confirmed their binding mode and selectivity.These studies provide a proof-of-principle for our integrated screening strategy, and suggest that this platform may be extended to discover new compounds that target therapeutically relevant nucleic acid morphologies.

View Article: PubMed Central - PubMed

Affiliation: James Graham Brown Cancer Center, Department of Biochemistry and Molecular Biology, University of Louisville, 529 S. Jackson Street, Louisville, KY 40202, USA.

ABSTRACT
Virtual Screening is an increasingly attractive way to discover new small molecules with potential medicinal value. We introduce a novel strategy that integrates use of the molecular docking software Surflex with experimental validation by the method of competition dialysis. This integrated approach was used to identify ligands that selectively bind to the triplex DNA poly(dA)-[poly(dT)](2). A library containing approximately 2 million ligands was virtually screened to identify compounds with chemical and structural similarity to a known triplex intercalator, the napthylquinoline MHQ-12. Further molecular docking studies using compounds with high structural similarity resulted in two compounds that were then demonstrated by competition dialysis to have a superior affinity and selectivity for the triplex nucleic acid than MHQ-12. One of the compounds has a different chemical backbone than MHQ-12, which demonstrates the ability of this strategy to 'scaffold hop' and to identify small molecules with novel binding properties. Biophysical characterization of these compounds by circular dichroism and thermal denaturation studies confirmed their binding mode and selectivity. These studies provide a proof-of-principle for our integrated screening strategy, and suggest that this platform may be extended to discover new compounds that target therapeutically relevant nucleic acid morphologies.

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