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. 2008 Oct 17;3(10):635-44.
doi: 10.1021/cb8001039. Epub 2008 Oct 3.

Vinylogous Ureas as a Novel Class of Inhibitors of Reverse Transcriptase-Associated Ribonuclease H Activity

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Vinylogous Ureas as a Novel Class of Inhibitors of Reverse Transcriptase-Associated Ribonuclease H Activity

Michaela Wendeler et al. ACS Chem Biol. .
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Abstract

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4 H-cyclohepta[ b]thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT). A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor beta-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Site-directed mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or non-natural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, divalent metal-chelating inhibitors that have been reported.

Figures

Figure 1
Figure 1
Inhibition of retroviral, bacterial, and human RNases H by vinylogous ureas NSC727447 and NSC727448. IC50 values are the average of triplicate analyses and were determined under initial-rate conditions from the slope of the time-dependent increase in fluorescence.
Figure 2
Figure 2
Yonetani–Theorell plot for inhibition of HIV-1 RNase H activity in the presence of β-thujaplicinol (NSC18806) and vinylogous urea NSC727447. The inverse of the rate of RNase H cleavage (expressed as the change in relative fluorescence per minute) is plotted as a function of NSC727447 concentration at β-thujaplicinol concentrations of 0, 0.1, 0.2, and 0.4 μM. Concentrations of the titrated inhibitor NSC727447 were 0, 0.25, 0.5, 1, 2, 4, 8, and 16 μM. The assay was performed at fixed concentrations of substrate and enzyme (250 nM and 4 nM, respectively). The parallel set of lines indicates that the two compounds bind in a mutually exclusive fashion.
Figure 3
Figure 3
RNA-dependent DNA polymerase activity of HIV-1 RT is not inhibited by NSC727447. a) Substrate was an end-labeled 21 nt DNA oligomer, annealed to a 45 nt RNA template (45R/21D RNA/DNA). In the first step, the DNA oligomer was extended to a 33 nt AZT-monophosphate-terminated primer. The resultant RNA/DNA hybrid (45R/33D) was purified, after which DNA synthesis was examined in the presence of a pyrophosphate donor and NSC727447. b) Lane 1, 21nt/33nt marker mix; lane 2, 45R/33D –RT, +PPi; lane 3, 45R/33D +RT, +PPI, –dNTPs; lane 4, 45R/33D –PPi, +RT, +dNTPs; lane 5, 45R/33D +RT, +PPI, +dNTPs, lanes 6–9, 45R/33D +RT, +PPI, +dNTPs, +NSC727447 at final concentrations of 50.0, 25.0, 12.5, and 6.25 μM, respectively.
Figure 4
Figure 4
Mass spectrometric protein footprinting strategy. Biotinylation reactions of free heterodimeric HIV-1 RT and of RT in complex with NSC727447 are performed in parallel, allowing accessible lysine residues to be covalently modified by NHS-biotin. RT subunits p66 and p51 are then separated by SDS-PAGE and subsequently subjected to proteolysis by trypsin. Comparative analysis of the generated peptide fragments by MALDI-TOF MS identifies lysine residues shielded from biotinylation in the RT/inhibitor complex.
Figure 5
Figure 5
MALDI-TOF analysis of RT tryptic peptides. a–c) Representative segments of the mass spectra. a) Free RT was treated with NHS-biotin. b) The RT–inhibitor complex was treated with NHS-biotin. c) Unmodified RT. The indicated peaks correspond to the following peptides: p1 = aa 278–284 containing the modified residues Cys280 and Lys281; p2 = aa 66–72 containing modified lysines Lys66 and Lys70; p3 = aa 264–275; p4 = aa 144–154. Comparison of the modification patterns of free RT (a) with the RT–inhibitor complex (b) reveals that p1 was specifically shielded in the preformed complex. Unmodified peptide fragments p3 and p4 (a, b, and c) serve as internal controls. d) Postsource decay data for p1. The fragmentation pattern of the parent ion (1326.5 Da) confirms the following sequence for the modified peptide: QL(C+biot.)(K+biot.)LLR. The “b” and “y” ions are derived from fragmentation of the peptide bonds and provide amino acid sequence information read from the peptide N-terminus toward C-terminus and the peptide C-terminus toward its N-terminus, respectively. During fragmentation of tryptic peptides the loss of ammonia from “b” and “y” ions is frequently observed. The respective peaks in our spectrum are accordingly labeled. We also detected the “a4 – NH3” ion, which resulted from the combined loss of the CO and NH3 groups from the “b4” ion. In addition to the conventional fragmentation pattern, PSD analyses commonly yield other internal fragmentation reactions. We did not attempt to identify these additional ion peaks because the assigned “b” and “y” ions provided unequivocal identification of the peptide sequence and modified residues. The spectrum in the range of 0–1300 m/z has been magnified to better visualize the fragmented ions.
Figure 6
Figure 6
Structure of HIV-1 RT depicting, in magenta, residues of the p66 and p51 thumb subdomains whose biotinylation is affected by NSC727447 binding. p66 subdomains have been color-coded blue (fingers), red (palm), green (thumb), yellow (connection), and gold (RNase H), For simplicity, only the p51 thumb is color-coded in green. Catalytic carboxylates of the RNase H domain are in cyan and Gln500 and Tyr501 of the RNase H primer grip in white. This figure was generated based on PDB entry 1RTD using Discovery Studio 2.0 software (Accelrys).
Figure 7
Figure 7
Sensitivity of HIV-1 RT Tyr501 variants to NSC727447 inhibition. Each IC50 value reported is the average of three independent experiments.

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