Mechanisms of drug-target complex formation.

<p><b>(a)</b> Equilibria for rapid reversible inhibition via a one-step mechanism and slow-binding inhibition via a two-step induced-fit mechanism along with a schematic free-energy profile for this reaction. E denotes the enzyme, I the inhibitor, EI the initial enzyme-inhibitor complex, and EI* the final enzyme-inhibitor complex. In the case of InhA, the diphenyl ether inhibitors bind to the enzyme with bound oxidized cofactor NAD⁺, forming a ternary complex. The double-headed arrows in the energy profile highlight the importance of the barrier height for the kinetics of the reaction. <b>(b)</b> Schematic free-energy profiles for a slow-binding inhibitor (left) and a destabilized EI* state as a consequence of ligand removal or the presence of a rapid-reversible inhibitor (right). Each macrostate (EI, EI*) is obviously associated with many microstates.</p

2D density plot for the ether dihedral angles <i>α</i> and <i>β</i> of the unbound ligands PT70 (left) and 6PP (right) based on a 150 ns MD simulation in aqueous solution.

<p>The dihedral angles <i>α</i> (C_<i>OH</i>-C-O-C) and <i>β</i> (C-O-C-C_{<i>Me</i>/<i>H</i>}) are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127009#pone.0127009.g002" target="_blank">Fig 2</a>.</p

Collective backbone RMSD values (C, N, and C_<i>α</i> atoms) of InhA monomers.

<p>Each monomer of the simulated homotetrameric systems (150 ns) was fitted individually onto chain A of the 2X23 crystal structure as reference for the RMSD measurements and the data of the four monomers were combined to one box plot per system. Boxes indicate the interquartile range (first to third quartile), black lines in the boxes show the median of each distribution. The whiskers extend to values 1.5 times the interquartile range from the box. Significant differences in the medians are indicated by non-overlapping notches. Average values are marked by white triangles.</p

Collective backbone RMSD values of the substrate binding loop in the InhA monomers.

<p>Each monomer of the simulated homotetrameric systems (150 ns) was fitted individually onto chain A of the 2X23 crystal structure as reference for the RMSD measurements and the data of the four monomers were combined to one box plot per system. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127009#pone.0127009.g004" target="_blank">Fig 4</a> for further explanations.</p

Open and closed conformations of InhA observed in the MD simulations.

<p>Figure (a) shows the closed state represented by the medoid of conformational Family 1, figure (b) illustrates the open state represented by the medoid of cluster 4 (belonging to conformational Family 3). The same view of the binding pocket as in Fig 3 of Li et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127009#pone.0127009.ref017" target="_blank">17</a>] is used for better comparison. In this view, the portal-forming elements are located left (helix <i>α</i>6) and right (strand-4) of the binding site. The distances highlighted as yellow dashed lines were measured between Ala198/Ile202 on helix <i>α</i>6 and Phe97 on strand-4. For comparison, in the crystal structure of the <b>PT70</b> complex (PDB 2X23) respresenting the closed state, a distance of 4 Å is found between Ile202 and Phe97, whereas the open state is characterized by a distance of about 10 Å between Ala198 and Phe97 in chain B of the <b>PT155</b>-complex crystal structure (PDB 4OXN) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127009#pone.0127009.ref017" target="_blank">17</a>].</p

Occurrence frequency (in % of the trajectory snapshots) of <i>α</i>-helix and 3₁₀-helix motifs in the substrate binding loop.

<p>Each monomer of the simulated homotetrameric systems (150 ns) was analyzed, and data of the four monomers were combined to one box plot per system.</p

Overview of the experimentally characterized diphenyl ethers analyzed in the MD simulations.

<p>The phenyl rings are referred to as A- and B-ring, respectively. The corresponding ether torsions are symbolized by curly arrows and labeled <i>α</i> and <i>β</i>. Experimental data were taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127009#pone.0127009.ref007" target="_blank">7</a>] and references provided therein. <b>PT70</b> is a slow-onset inhibitor, with measured dissociation rate constant <i>k</i>_<i>off</i> and residence time <i>t</i>_<i>r</i>. In contrast, <b>6PP</b> and <b>TCL</b> show rapid-reversible binding kinetics; <i>k</i>_<i>off</i> and <i>t</i>_<i>r</i> values were estimated assuming a value of 10⁹ M⁻¹s⁻¹ for <i>k</i>_<i>on</i>, as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127009#pone.0127009.ref007" target="_blank">7</a>].</p

Occurrence frequencies (in %) of the conformational families of the InhA binding pocket in the three analyzed simulations of the PT70-, 6PP- and TCL-complexes, based on the hierarchical clustering analysis.

<p>Occurrence frequencies (in %) of the conformational families of the InhA binding pocket in the three analyzed simulations of the PT70-, 6PP- and TCL-complexes, based on the hierarchical clustering analysis.</p

Distances between the NAD⁺ nicotinamide oxygen and the phenolic oxygen of the ligand or the Ile194 backbone nitrogen.

<p>Distances are shown as a function of time in a moving-average plot with a window of 20 frames. Monomers 1 and 4 are illustrated for the <b>TCL</b> complex. Continuous lines indicate distances to the ligand, whereas dotted lines are used for distances to Ile194. For each illustrated ligand a stable interaction with a distance below 3 Å can be observed after the binding-mode change, while the interaction of NAD⁺ with Ile194 (present in the starting structure) is only slightly affected.</p

Violin plots of distances between the phenolic oxygen of Tyr158 and the respective ligands.

<p>White dots depict the medians. Thick vertical lines indicate the interquartile ranges (IQR), thin lines extend to 1.5 ⋅ IQR from the third and first quartile, respectively. The shape of the violins illustrates the kernel density estimation of the respective distribution.</p