KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing.

Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated

Therapeutic target-site variability in α1-antitrypsin characterized at high resolution

The intrinsic propensity of [alpha]1-antitrypsin to undergo conformational transitions from its metastable native state to hyperstable forms provides a motive force for its antiprotease function. However, aberrant conformational change can also occur via an intermolecular linkage that results in polymerization. This has both loss-of-function and gain-of-function effects that lead to deficiency of the protein in human circulation, emphysema and hepatic cirrhosis. One of the most promising therapeutic strategies being developed to treat this disease targets small molecules to an allosteric site in the [alpha]1-antitrypsin molecule. Partial filling of this site impedes polymerization without abolishing function. Drug development can be improved by optimizing data on the structure and dynamics of this site. A new 1.8 Å resolution structure of [alpha]1-antitrypsin demonstrates structural variability within this site, with associated fluctuations in its upper and lower entrance grooves and ligand-binding characteristics around the innermost stable enclosed hydrophobic recess. These data will allow a broader selection of chemotypes and derivatives to be tested in silico and in vitro when screening and developing compounds to modulate conformational change to block the pathological mechanism while preserving function

In silico assessment of potential druggable pockets on the surface of α1-Antitrypsin conformers

The search for druggable pockets on the surface of a protein is often performed on a single conformer, treated as a rigid body. Transient druggable pockets may be missed in this approach. Here, we describe a methodology for systematic in silico analysis of surface clefts across multiple conformers of the metastable protein α1-antitrypsin (A1AT). Pathological mutations disturb the conformational landscape of A1AT, triggering polymerisation that leads to emphysema and hepatic cirrhosis. Computational screens for small molecule inhibitors of polymerisation have generally focused on one major druggable site visible in all crystal structures of native A1AT. In an alternative approach, we scan all surface clefts observed in crystal structures of A1AT and in 100 computationally produced conformers, mimicking the native solution ensemble. We assess the persistence, variability and druggability of these pockets. Finally, we employ molecular docking using publicly available libraries of small molecules to explore scaffold preferences for each site. Our approach identifies a number of novel target sites for drug design. In particular one transient site shows favourable characteristics for druggability due to high enclosure and hydrophobicity. Hits against this and other druggable sites achieve docking scores corresponding to a Kd in the µM–nM range, comparing favourably with a recently identified promising lead. Preliminary ThermoFluor studies support the docking predictions. In conclusion, our strategy shows considerable promise compared with the conventional single pocket/single conformer approach to in silico screening. Our best-scoring ligands warrant further experimental investigation

Fragment docking to the A site targets the pharmacophore defined by Asn104, Thr114, and His139.

<p>Best poses of the top-scoring 20 fragments (coloured sticks) from the ZINC dataset docked in the A site of A1AT (cartoon, blue). The majority of these fragments fill the pocket defined by Thr114 and Asn104 at the top, and His139 at the bottom (thin sticks, cyan), identified in our previous study as a potential allosteric site for targeting A1AT polymerization. Some of the fragments take advantage of hydrogen bonding opportunities presented by His139 and Thr114.</p

The best-scoring and “best-efficient” small molecules from DrugBank docked against each of the sites A-I on A1AT.

<p>Diagrams, IUPAC names and PubChem CIDs for all DrugBank entries in this table can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036612#pone-0036612-g007" target="_blank">Figure 7</a>.</p

Properties of surface pockets in crystal structures and <i>in silico</i> conformers of α₁-antitrypsin.

<p>Persistence of clefts A–I among A1AT crystal structures (A) and computationally produced conformers (B). Where the sites C and E overlapped, the data are presented under the label “C_E”. The distribution of SiteMap calculated properties for the 100 <i>in silico</i> conformers are shown as boxplots: SiteScore (C), DScore (D), site volume (E) and hydrophobic vs. hydrophilic character balance (F). The corresponding data for crystal structures are shown as red symbols superimposed on the boxplots; 1qlp (circle), 2qug (plus sign), 3cwm (square), 1hp7 (diamond), 3drm (triangle point up), 1oph (triangle point down). Data are shown only for sites identified within PDB entries for native (stressed, ‘S’) forms of A1AT, as these are likely to be the appropriate target states for the design of polymerization inhibitors.</p

The structure of the wild type α₁-antitrypsin.

<p>Front (A) and back (B) views of the structure of A1AT in cartoon representation (PDB entry: 1qlp). The secondary elements are coloured as follows. ß-sheets: A (red), B (blue), and C (yellow); helices: A (cyan), B (apricot), C (blue), D (grey-green), E (purple), F (yellow), G (orange), H (pink), I (olive); loops: reactive centre loop (RCL, red), all other loops (green).</p

Results from docking the DrugBank collection against nine pockets on α₁-antitrypsin.

<p>(A) Boxplot distributions of docking scores for DrugBank molecules docked to each of the nine sites A to I. Only the top-ranking pose is included for each ligand and only ligands of molecular weight less than 500 Daltons are included in this plot. (B) The best-scoring ligand for each site is assigned a worse score when docked against each of the other sites. The red diamonds represent the best docking score for each ligand depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036612#pone-0036612-t002" target="_blank">Table 2</a>, when docked to the site where it is ranked top. The black diamonds correspond to the scores for each of these ligands when docked to all other sites. The x-axis labels correspond to the DrugBank ID of the ligand and, in brackets, the site against which it is selected as “best-scoring”, e.g. 07124(A) refers to DrugBank entry DB07124 which achieves its best score against site A.</p

Shifts in melting temperature of A1AT in the presence of selected small molecule ligands (ThermoFluor assay).

<p>The quoted <i>p</i>-values are the result of a Welch two-sample t-test (performed using the R statistical software) testing the null hypothesis that the difference in the mean values of the distribution of the thermal shift values for DMSO and the distribution of the thermal shift values observed for each ligand is zero. The null hypothesis was rejected for <i>p</i>-values <0.01.</p

The nine top-ranking surface pockets identified by SiteMap on α₁-antitrypsin.

<p>Coloured spheres represent the SiteMap predictions for eight top-ranking surface clefts on the wild type α₁-antitrypsin (PDB entry 1qlp, in grey cartoon representation): site A: green, B: cyan, C: blue, D: purple, E: fuchsia, F: orange, G: slate blue, H: brown. The yellow spheres correspond to the ninth site, I, a cleft identified on crystal structures of A1AT containing the Ala70Gly mutation.</p