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Comparative Study
. 2011 Aug 22;51(8):1986-98.
doi: 10.1021/ci200194w. Epub 2011 Jul 26.

Identification of Alternative Binding Sites for Inhibitors of HIV-1 Ribonuclease H Through Comparative Analysis of Virtual Enrichment Studies

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Free PMC article
Comparative Study

Identification of Alternative Binding Sites for Inhibitors of HIV-1 Ribonuclease H Through Comparative Analysis of Virtual Enrichment Studies

Anthony K Felts et al. J Chem Inf Model. .
Free PMC article

Abstract

The ribonuclease H (RNase H) domain on the p66 monomer of HIV-1 reverse transcriptase enzyme has become a target for inhibition. The active site is one potential binding site, but other RNase H sites can accommodate inhibitors. Using a combination of experimental and computational studies, potential new binding sites and binding modes have been identified. Libraries of compounds were screened with an experimental assay to identify actives without knowledge of the binding site. The compounds were computationally docked at putative binding sites. Based on positive enrichment of natural-product actives relative to the database of compounds, we propose that many inhibitors bind to an alternative, potentially allosteric, site centered on Q507 of p66. For a series of hydrazone compounds, a small amount of positive enrichment was obtained when active compounds were bound by induced-fit docking at the interface between the DNA:RNA substrate and the RNase H domain near residue Q500.

Figures

Figure 1
Figure 1
Representative AnalytiCon compounds are shown with carbon in black and oxygen in red. Clockwise from top left, the compounds are NP-003686, NP-004204, NP-011987, and NP-005114. The abundance of hydroxyl groups is the reason why these compounds are very water soluble and not prone to aggregation.
Figure 2
Figure 2
Representative Life Chemicals hydrazone and hydrazine compounds are shown with carbon in black, oxygen in red, sulfur in yellow, iodine in magenta, and bromine in brown. Clockwise from top left, the compounds are F0745-0032, F1092-0760, F1345-0373, and F1345-0193.
Figure 3
Figure 3
Images are shown to point out where the binding sites explored in this study are located on RT. On the left, the complex of RT and the DNA:RNA substrate is shown with p66 in blue (“fingers”), red (“palm”), green (“thumb”), yellow (“connection”), and orange for the RNase H domain with the active site residues in red spheres. The p51 domain is in brown, and the DNA:RNA substrate (stick representation) in transparent gray. Site 500S is highlighted with the bound hydrazone inhibitor F1345-0193 shown in cyan spheres behind the transparent gray substrate and located at the intersection of the yellow, orange and brown domains. On the right, the opposite side is shown after a 180° rotation of the molecule on the left, showing the AnalytiCon compound NP-003686 (in cyan) bound to site 507 (with residue 431 represented in sticks for a better perspective on NP-003686) located at the intersection of the orange and yellow domains of p66 and the p51 brown domain. These representations were generated with VMD.
Figure 4
Figure 4
The distribution of the GlideScore XP5.0 binding energies for the 84 AnalytiCon active compounds are plotted relative to each other for the binding sites at 498, 500S, and 507. The solid red curve is the distribution for site 507; the dotted and dashed green curve, for site 498; and the dashed blue curve, for site 500S. Figure was generated with R.
Figure 5
Figure 5
The histogram of the difference in GlideScore XP5.0 binding energies at site 507 (“BE(507)”) and at site 498 (“BE(498)”). Bars with negative energy differences show actives that favor site 507; bars with positive energy differences show actives that favor site 498. Figure was generated with R.
Figure 6
Figure 6
The receiver-operating characteristic (ROC) curve in red is plotted showing what percentage of AnalytiCon active compounds is found in the ranked database of all compounds bound to site 498. The selectivity (Se) of finding active compounds within some threshold is plotted against one minus the specificity (1-Sp) which indicates the number of inactive compounds found within that threshold. The blue line indicates the ROC if the actives were randomly distributed in the database. The enrichment is 37% of the actives found in the top 10% of the screened database. The area under the ROC curve is 0.780. Figure was generated with R.
Figure 7
Figure 7
The ROC curve in red is plotted showing what percentage of AnalytiCon active compounds is found in the ranked database of all inactive compounds bound to site 507. The blue line indicates the ROC if the actives were randomly distributed in the database. Figure details can be found in the caption of Fig. 6. The enrichment is 76% of the actives found in the top 10% of the screened database. The area under the ROC curve is 0.824. Figure was generated with R.
Figure 8
Figure 8
The binding orientations of AnalytiCon compound NP-003686 (in blue) and compound EN37 (in red) are shown superimposed in site 507. The RNase H domain of p66 is in orange, the connection region of p66 is in yellow, and p51 is in brown. In green are residues that interact hydrophobically with the compounds. The residue labels end in “B” if the residue belongs to p51. This representation was generated with VMD.
Figure 9
Figure 9
The ROC curve in red is plotted showing what percentage of AnalytiCon active compounds is found in the ranked database of all compounds bound to site 500S with the DNA:RNA substrate present. The blue line indicates the ROC if the actives were randomly distributed in the database. Figure details can be found in the caption of Fig. 6. The enrichment is 58% of the actives found in the top 10% of the screened database. The area under the ROC curve is 0.815. Figure was generated with R.
Figure 10
Figure 10
The ROC curve in red is plotted showing what percentage of hydrazone/hydrazine active compounds is found in the ranked database of all compounds bound to site 500S (between RNase H and the DNA:RNA substrate). The blue line indicates the ROC if the actives were randomly distributed in the database. Figure details can be found in the caption of Fig. 6. The enrichment is 15% of the actives found in the top 10% of the screened database. The area under the ROC curve is 0.512. Figure was generated with R.
Figure 11
Figure 11
The distribution of GlideScore XP5.0 binding energies of the hydrazone/hydrazine active compounds (red solid curve) is shown relative to the distribution of database energies (blue dotted curve) when docking to site 500S. Notice the shoulder peak comprises of the actives with binding scores between −9 to −6 kcal/mol. Figure was generated with R.
Figure 12
Figure 12
The ROC curve in red is plotted showing what percentage of hydrazone/hydrazine active compounds is found in the ranked database of all compounds bound to site 500S of the minimized RT-DNA:RNA complex. The blue line indicates the ROC if the actives were randomly distributed in the database. Figure details can be found in the caption of Fig. 6. The enrichment is 24% of the actives found in the top 10% of the screened database. The area under the ROC curve is 0.574. Figure was generated with R.
Figure 13
Figure 13
The distribution of GlideScore XP5.0 binding energies of the hydrazone/hydrazine active compounds (red solid curve) is shown relative to the distribution of database energies (blue dotted curve) when docking to site 500S of the minimized RT-DNA:RNA complex. Notice the shoulder peak comprises of the actives with binding scores between −9.5 to −6 kcal/mol. Figure was generated with R.

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