Structural comparison of human GUSB with bacterial GUS.

<p>(<b>A</b>). Cartoon model of superimposed human (light red) and bacterial GUS (green). Residues involved in catalysis are shown in ball and stick model for both the human (light red) and bacterial GUS (light green). (<b>B</b>). Superposition of the longer loop of bacterial GUS (light green) involved in binding to the inhibitor (yellow stick) and superimposed side chains of active site residues of human and bacterial GUS. (<b>C</b>). Comparison of lysosomal target loop of human GUS with bacterial GUS. Atomic coordinates of bacterial GUS were taken pdb code 3LPG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079687#pone.0079687-Wallace1" target="_blank">[51]</a>.</p

Structure of GUS monomer shown in cartoon model.

<p>Jelly roll domain, immunoglobulin constant region domain and TIM barrel domains are shown in yellow, green and red respectively. Residues involved in catalysis are shown in ball and stick model (cyan). Potential glycosylation sites and N-linked oligosaccharide chains are shown in light grey and orange respectively (ball and stick model). The hairpin loop proposed to be involved in lysosomal targeting is shown in pink.</p

List of proteins showing structural similarities with human GUS.

a<p>Number of residues aligned.</p>b<p>Total number of residues.</p>c<p>Root mean square deviations for C^α atoms.</p

Crystallographic data and refinement statistics of human GUS.

a<p>– numbers in parenthesis correspond to the highest resolution shell of (1.67–1.73 Å for data collection, 1.7–1.74 Å for refinement).</p

Overall structure of human GUS illustrated in cartoon model.

<p>Subunits A, B, D, and E are colored in green, light red, grey and sky blue, respectively. Residues involved in the catalysis are shown in ball and stick model (cyan) on each monomer. N-linked oligosaccharide chains are shown in ball and stick model (orange). The hairpin loop of each monomer is shown in magenta. All structures are drawn using the molecular visualization tool, PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC).</p

Cartoon model of superimposed human GUS and bacterial β-galactosidas.

<p>(<b>A</b>). Roll jelly like domain and (<b>B</b>). TIM barrel domains. Human GUS is shown in light red and bacterial β-galactosidase in sky blue. The side chains of active site residues of human and bacterial GUS are shown in ball and stick. Atomic coordinates of bacterial protein were taken from pdb code 1DP0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079687#pone.0079687-Juers1" target="_blank">[52]</a>.</p

Multiple sequence alignment of human GUS with mouse and bacterial GUS.

<p>The percent sequence identities are given in parentheses. Completely conserved residues and homologous residues are shaded in dark and light grey, respectively. The secondary structure elements are given on the top of sequences, where α-helices are represented by blue rectangles, β-strands by green arrows. Domains 1, 2 and 3 are indicated by yellow, green and red line respectively, below the sequence. Conserved active site residues are highlighted in green boxes. Potential glycosylation sites are in pink. Glycosylation sites are in magenta boxes. Amino acid sequences of GUS were taken from the Uniprot database with their primary accession number as: human, P08236; mouse P12265; and <i>E. coli</i>, P05804.</p

Representation of N-linked oligosaccharide chain on GUSB.

<p>(<b>A</b>). N-linked oligosaccharide chain at Asn173 and the lysosome targeting loop. Superimposed structure of reported earlier (1BHG, yellow) and the current structure (orange) of human GUS showing different orientation of lysosomal targeting motif. The side chain of Lys197, which is believed to participate in phosphotransferase recognition, is coordinated by the glycan chain at Asn173. <b>B</b>. N-linked oligosaccharide chain at Asn272. Contour electron density map (at the electron level 1.00 for a 2Fo-F) is shown with the modeled glycan chain. <b>C</b>. Stereo view of cartoon diagram showing a comparison of lysosomal target motif of human GUS (light red) with cathepsin D (cyan). The structure was drawn from the atomic coordinates of cathepsin D with pdb code, 1LYA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079687#pone.0079687-Flores1" target="_blank">[40]</a>.</p

Potent Inhibitors of Hepatitis C Virus NS3 Protease: Employment of a Difluoromethyl Group as a Hydrogen-Bond Donor

The design and synthesis of potent, tripeptidic acylsulfonamide inhibitors of HCV NS3 protease that contain a difluoromethyl cyclopropyl amino acid at P1 are described. A cocrystal structure of <b>18</b> with a NS3/4A protease complex suggests the presence of a H-bond between the polarized C–H of the CHF₂ moiety and the backbone carbonyl of Leu135 of the enzyme. Structure–activity relationship studies indicate that this H-bond enhances enzyme inhibitory potency by 13- and 17-fold compared to the CH₃ and CF₃ analogues, respectively, providing insight into the deployment of this unique amino acid

Discovery and Early Clinical Evaluation of BMS-605339, a Potent and Orally Efficacious Tripeptidic Acylsulfonamide NS3 Protease Inhibitor for the Treatment of Hepatitis C Virus Infection

The discovery of BMS-605339 (<b>35</b>), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropyl­acylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1′ site of the protease. The identification of <b>35</b> was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound <b>35</b> which demonstrated antiviral activity in HCV-infected patients