The Binding Mode of Second-Generation Sulfonamide Inhibitors of MurD: Clues for Rational Design of Potent MurD Inhibitors

<div><p>A series of optimized sulfonamide derivatives was recently reported as novel inhibitors of UDP-<em>N</em>-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD). These are based on naphthalene-<em>N</em>-sulfonyl-D-glutamic acid and have the D-glutamic acid replaced with rigidified mimetics. Here we have defined the binding site of these novel ligands to MurD using ¹H/¹³C heteronuclear single quantum correlation. The MurD protein was selectively ¹³C-labeled on the methyl groups of Ile (δ1 only), Leu and Val, and was isolated and purified. Crucial Ile, Leu and Val methyl groups in the vicinity of the ligand binding site were identified by comparison of chemical shift perturbation patterns among the ligands with various structural elements and known binding modes. The conformational and dynamic properties of the bound ligands and their binding interactions were examined using the transferred nuclear Overhauser effect and saturation transfer difference. In addition, the binding mode of these novel inhibitors was thoroughly examined using unrestrained molecular dynamics simulations. Our results reveal the complex dynamic behavior of ligand–MurD complexes and its influence on ligand–enzyme contacts. We further present important findings for the rational design of potent Mur ligase inhibitors.</p> </div

Expected Ile (δ1), Val, and Leu methyl signals in the MurD protein.

<p>Expected Ile (δ1), Val, and Leu methyl signals in the MurD protein.</p

Nontrivial NOE connectivities and corresponding distances (Å) calculated from the transferred NOESY spectra for arylalkyloxy derivatives.

<p>For the sake of clarity, the atom labels do not strictly follow IUPAC rules for all compounds.</p>a<p>Distances from X-ray structure, PDB code 2VTD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Humljan1" target="_blank">[8]</a>.</p>b<p>Distances from X-ray structure, PDB code 2XPC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Sosi1" target="_blank">[11]</a>.</p>c<p>Overlapped.</p>d<p>Medium NOE cross-peak that can belong either to H1” or H5” due to signal overlap.</p

Nontrivial NOE connectivities and corresponding distances (Å) calculated from the transferred NOESY spectra for alkyloxy derivatives.

<p>For the sake of clarity, the atom labels do not strictly follow IUPAC rules for all compounds.</p>a<p>Distances from X-ray structure, PDB code 2JFF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Kotnik1" target="_blank">[7]</a>. ^b Observed in 1D trace from 2D spectrum.^c Medium NOE cross-peak that can belong either to H1” or H5” due to signal overlap.</p

MurD domain flexibility.

<p>Snapshot of the most “closed” (red) and the most “open” (blue) conformations during the simulation of the <b>4b</b>–MurD complex. The trajectory frames were aligned using the root mean square deviation trajectory tool extension in VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Humphrey1" target="_blank">[42]</a>.</p

Comparison of the binding mode of the first and second generation sulfonamide inhibitors.

<p>The snapshot from the MD trajectory shows the binding mode of the compound <b>6b</b> (in orange). The compound <b>1b</b> (in cyan) is superimposed (crystal structure PDB code 2VTD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Humljan1" target="_blank">[8]</a>). The <i>N</i>-terminal domain, central domain, and <i>C</i>-terminal domains are colored in red, green, and blue respectively. The MurD structures were aligned using Visual Molecular Dynamics (VMD) program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Humphrey1" target="_blank">[42]</a>.</p

Ile (δ1), Val, and Leu methyl groups in MurD protein.

<p>(<b>A</b>) MurD protein in complex with compound <b>1b</b> (PDB entry 2VTD) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Humljan1" target="_blank">[8]</a>. Ile (δ1), Val, and Leu methyl groups are represented as spheres. The <i>N</i>-terminal domain, central domain, and <i>C</i>-terminal domains are colored in red, green, and blue respectively. (<b>B</b>) Close-up view of MurD binding site (PDB entry 2VTD) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Humljan1" target="_blank">[8]</a> with bound compound <b>1b</b>. Only the methyl groups within 12 Å of the ligand are shown. Methyl groups in the range of 5 Å are marked as transparent Van der Waals spheres.</p

Overlay of ¹H/¹³C HSQC NMR spectra in absence (black) and presence (red) of compound 6b.

<p>The ligand/protein ratio is 10∶1. The proposed assignment of crucial methyl groups is presented. ^a The signals of Leu416 methyl groups disappear at a ligand/protein ratio of 0.5∶1 The new position of these signals cannot be identified because of the signal overlap. In such cases, the minimum possible CSPs are calculated. The Ile and Val/Leu regions are marked with orange and light blue boxes respectively. ^b One Ile signal was deliberately folded into the Leu region.</p

Selected expansions from transferred NOESY spectra.

<p>(<b>A</b>) The mutually exclusive H5–CH₂(1′) and H7–CH₂(1′) and the mutually exclusive H1–H^α and H3–H^α NOEs of compound <b>1a</b>. (<b>B</b>) The mutually exclusive H5–CH₂(1′) and H7–CH₂(1′) of compound <b>6a</b>. (<b>C</b>) The H1–H5′′ NOE of compound <b>6a</b>. Its H3–H5′′ NOE is detectable only at a lower contour level.</p

STD NMR spectrum of the compound 6b.

<p>(<b>A</b>) Reference spectrum. (<b>B</b>) STD spectrum. Note: The spectra intensities are not to scale.</p