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ChemMedChem
2011 Oct 04;610:1797-805. doi: 10.1002/cmdc.201100281.
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Identification of small-molecule inhibitors of the XendoU endoribonucleases family.
Ragno R
,
Gioia U
,
Laneve P
,
Bozzoni I
,
Mai A
,
Caffarelli E
.
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The XendoU family of enzymes includes several proteins displaying high sequence homology. The members characterized so far are endoribonucleases sharing similar biochemical properties and a common architecture in their active sites. Despite their similarities, these proteins are involved in distinct RNA-processing pathways in different organisms. The amphibian XendoU participates in the biosynthesis of small nucleolar RNAs, the human PP11 is supposed to play specialized roles in placental tissue, and NendoU has critical function in coronavirus replication. Notably, XendoU family members have been implicated in human pathologies such as cancer and respiratory diseases: PP11 is aberrantly expressed in various tumors, while NendoU activity has been associated with respiratory infections by pathogenic coronaviruses. The present study is aimed at identifying small molecules that may selectively interfere with these enzymatic activities. Combining structure-based virtual screening and experimental approaches, we identified four molecules that specifically inhibited the catalytic activity of XendoU and PP11 in the low micromolar range. Moreover, docking experiments strongly suggested that these compounds might also bind to the active site of NendoU, thus impairing the catalytic activity essential for the coronavirus life cycle. The identified compounds, while allowing deep investigation of the molecular functions of this enzyme family, may also represent leads for the development of new therapeutic tools.
Figure 1. AutoDock proposed conformations of various XendoU substrates/ligands. 3′â€UMP (light grey), 2′,3′â€cyclicâ€UMP (magenta), UU (yellow), UUU (green) and CUUG (cyan). The van der Waals surface of XendoU is shown in white. The phosphate is shown in dark grey.
Figure 2. Functional validation of XendoU inhibitors 1–7. a) XendoU processing activity was analyzed in the presence of the best performing individual compounds (indicated above each lane). 32Pâ€labeled 003 RNA was incubated with Hisâ€XendoU alone (lanes –) or in the presence of decreasing amounts of inhibitors, at the specific concentrations. Untreated sample (input RNA) was fractionated in lane C. b) Schematic representation of U16 snoRNA processing. U16 primary transcript (003 RNA) as well as the complementary cleavage products (Iâ€1 and Iâ€2; Iâ€3 and Iâ€4) released by XendoU endonucleolytic clevages upstream (a, b, c and d sites) and downstream (e and f sites) of the U16 snoRNA are depicted. The double cleavage upstream and downstream of the U16â€coding region produces preâ€U16 molecules that, in the cell system, are converted by exonuclease trimming to the mature snoRNA. c) XendoU binding activity was tested in the presence of inhibitors. Hisâ€XendoU was incubated with a fixed amount of 32Pâ€labeled mini 003 RNA alone (lane –) or in the presence of each compound at a concentration of 200 μm. RNA incubated in buffer alone was loaded in lane C. Arrowheads point to free RNA (RNA) and protein/RNA complex (Complex).
Figure 3. Docked conformations of the XendoU inhibitors. A portion of the XendoU enzyme is displayed as a yellow ribbon. Surflex, AutoDock, Dock and FRED overlapped poses of a) NSC 13728 (1), b) NSC 26699 (2), c) NSC 45576 (3), d) NSC 106505 (4), e) NSC 109483 (5), f) NSC 117199 (6) and g) NSC 130796 (7) are shown, and their IC50 values are given.
Figure 4. Pharmacophore model derived from XendoU inhibitors. The surfaces derived from the most active compounds (4, 6 and 7) are colored in red, blue and grey. The peculiar areas from the less active agents (1–3 and 5) are depicted in purple. The hydrophobic features (HYâ€1, −2, −3 and −4) and the hydrogen acceptors (HAâ€1 and −2) are also indicated. Interaction (—) distances are given in angstroms (Ã…).
Figure 5. Analysis of inhibition of human PP11. a) Cleavage assay: in vitro processing reaction was performed by incubating 5′â€endâ€labeled P1 oligoribonucleotide (run as untreated molecule in lane C) with Hisâ€PP11 alone (lane –) or in the presence of each inhibitor at a concentration of 200 μm. The sequence of P1 oligoribonucleotide, as well as the PP11cleavage sites (indicated by arrows), are reported below. b) Doseâ€dependent inhibition assay of the four PP11 inhibitors. c) Binding assay: the four compound (1, 3, 4 and 7) that inhibit PP11cleavage activity were further tested for their ability to affect PP11 binding properties. A mobility shift assay was performed as already described for XendoU. Hisâ€PP11 was incubated with a fixed amount of 32Pâ€labeled RNA alone (lanes 3 and 4) or in the presence of each compound at a concentration of 200 μm (lanes 5–8). Untreated RNA was fractionated in lanes 1 and 2. The arrows point to the RNA–protein complex (Complex) or to the free RNA (RNA). d) Docked conformations of the PP11 inhibitors: NSC 13728 (1; cyan), NSC 45576 (3; purple), NSC 106505 (4; yellow), NSC 130796 (7; green). The van der Waals surface of PP11 is shown in white.
Figure 6. Binding mode comparisons of the XendoU/PP11 common, active compounds docked in the three RNAses. a) NendoU (pink), XendoU (cyan) and PP11 (orange) cumulative binding surfaces of the XendoU/PP11 common inhibitors (NCS 130796, NCS 106505, NCS 45576 and NSC 13728). b) AutoDock proposed binding conformations of the XendoU/PP11 active compounds into the NendoU opened form (PDB: 2H85[21]): NSC 13728 (1; purple), NCS 45576 (3; blue), NCS 106505 (4; white), NCS 130796 (7; yellow). c) Binding mode comparison of NCS 130796 (7) docked in NendoU (pink), PP11 (orange) and XendoU (cyan).
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