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. 2014 Jul 15;426(14):2617-31.
doi: 10.1016/j.jmb.2014.05.006. Epub 2014 May 17.

Structure of a Dihydroxycoumarin Active-Site Inhibitor in Complex With the RNase H Domain of HIV-1 Reverse Transcriptase and Structure-Activity Analysis of Inhibitor Analogs

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Free PMC article

Structure of a Dihydroxycoumarin Active-Site Inhibitor in Complex With the RNase H Domain of HIV-1 Reverse Transcriptase and Structure-Activity Analysis of Inhibitor Analogs

Daniel M Himmel et al. J Mol Biol. .
Free PMC article

Abstract

Human immunodeficiency virus (HIV) encodes four essential enzymes: protease, integrase, reverse transcriptase (RT)-associated DNA polymerase, and RT-associated ribonuclease H (RNase H). Current clinically approved anti-AIDS drugs target all HIV enzymatic activities except RNase H, which has proven to be a very difficult target for HIV drug discovery. Our high-throughput screening activities identified the dihydroxycoumarin compound F3284-8495 as a specific inhibitor of RT RNase H, with low micromolar potency in vitro. Optimization of inhibitory potency can be facilitated by structural information about inhibitor-target binding. Here, we report the crystal structure of F3284-8495 bound to the active site of an isolated RNase H domain of HIV-1 RT at a resolution limit of 1.71Å. From predictions based on this structure, compounds were obtained that showed improved inhibitory activity. Computational analysis suggested structural alterations that could provide additional interactions with RT and thus improve inhibitory potency. These studies established proof of concept that F3284-8495 could be used as a favorable chemical scaffold for development of HIV RNase H inhibitors.

Keywords: HIV ribonuclease H; RNase H inhibitors; dihydroxybenzopyrone derivatives; protein–inhibitor complex; structure-based drug design.

Figures

Figure 1
Figure 1. Ribbon diagram of the structure of full-length RT
RT is a heterodimer that consists of p66 (color) and p51 (gray) subunits. The two enzymatic active sites and the subdomains of the p66 subunit are labelled. Right inset: A closer view of the isolated RNase H domain of the current structure, with F3284-8495 bound at the active site. Selected alpha-helices and beta-sheets are labelled based on ref. 52. The chemical structure of F3284-8495 is shown at the bottom center of the figure.
Figure 2
Figure 2. Inhibitor electron density at the RNase H active site
Two Fo-Fc simulated annealing omit maps are shown at the RNase H active site. In one (light blue, contoured at 2.8σ), the inhibitor has been excluded from the phase calculation, and in the other (violet, contoured at 11σ), both the Mn2+ cations and the inhibitor have been omitted from phasing. F3284-8495 (yellow carbon atoms) and two Mn2+ cations (black orbs) are shown modeled into the electron density. The cations are designated A and B. Two vantage points are shown rotated 90° from each other about the vertical axis.
Figure 3
Figure 3. Interactions between the protein, cations, and inhibitor
Coordination about the cations (light blue orbs) is shown as solid red lines. Two water molecules participate in this coordination (red orbs). The separation distance between the cations is shown in blue. Black dashed lines designate protein-inhibitor contacts. Distances are in Å. Wherever the distances differ for each of the two protein-inhibitor complexes in the asymmetric unit, both distances are given. Two vantage points are given, rotated about 70° about the vertical axis.
Figure 4
Figure 4. Effect of F3284-8495 and cations on RNase H conformation
Shown is the superposition of the current structure (orange, inhibitor not shown) with the RNase H domain of several RT structures without inhibitors bound at the RNase H active site (referenced here by Protein Data Bank accession numbers): 3DLK (cyan), with no ligands bound; 4G1Q (violet), with Mg2+ bound at the Cation A position; and 2BE2 (gray), with Mn2+ bound at the Cation B position. Superposition is based on main-chain atoms for residues 441 to 448 of the RNase H domain. a) A view of the entire RNase H domain. The overall conformation of the RNase H domain is highly similar whether or not ligands are bound. b) A closer view of the active site, rotated toward the viewer by about 60 degrees. Glu478 tends to point away from the active site if there is no Cation B to coordinate (3DLK and 4G1Q). His539 may adjust its position and side-chain conformation to interact with a ligand such as F3284-8495 at the active site.
Figure 5
Figure 5. Docking result for F3385-2581
The docking experiment resulted in one pose. Predicted electrostatic interactions (defined as hydrogen bonds, ionic-ionic, ionic-dipole, and dipole-dipole) are depicted by red dashes, and hydrophobic interactions by black dashes. Two additional hydrophobic contacts between residue Pro537 and the analog are not shown. An inset of the chemical structure is shown on the upper left side of the figure. Predicted protein-analog contacts were dominated by electrostatic interactions. An induced fit Schrödinger Glide score of −10.25 was computed for this docking result.
Figure 6
Figure 6. Docking results for F3385-2588
An electrostatic potential surface diagram (calculated in PyMol) is shown of RT with analog F3385-2588 docked to the RNase H active site. An inset of the chemical structure is shown on the upper right side of the figure. The induced-fit docking experiments resulted in multiple poses that could be categorized into several pose groups based on contacts formed with the protein. The most energetically favorable pose obtained for each of these groups is shown. Moving clockwise about the potential surface diagram, these pose groups interacted primarily with either His539 and Ala538 (pink pose), Trp535 and Pro537 (violet "Pose Group 1"), Gln500 and Trp535 (yellow pose), Gln475 and Tyr501 (gold "Pose Group 3"), or Arg448 and Asn474 (cyan, "Pose Group 2"). For the top three docking results (Pose Groups 1 −3), the Schrödinger Glide score is given (more negative = more energetically favorable), and insets are shown giving representative protein-inhibitor interactions predicted by the docking experiment (interactions color scheme as in Figure 5). Residues of the RNase H primer grip are indicated by an asterisk (*), and residues that interact with the scissile phosphate of the substrate's RNA strand (or with adjacent nucleotides) are indicated by a caret (^).
Figure 7
Figure 7. Docking results for F3385-2590
An electrostatic potential surface diagram (calculated in PyMol) is shown of RT with analog F3385-2590 docked to the RNase H active site. An inset of the chemical structure is shown on the upper right side of the figure. The resulting poses are similar to those obtained for F3385-2588 and are grouped as described in Figure 6. The Schrödinger Glide scores are given for the top three scoring pose groups, and insets are shown giving representative protein-inhibitor interactions predicted by the docking experiment (interactions color scheme as in Figure 5). Residues of the RNase H primer grip are indicated by an asterisk (*), and residues that interact with the scissile phosphate of the substrate's RNA strand (or with adjacent nucleotides) are indicated by a caret (^)

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