Limits...
Slow-Onset Inhibition of Mycobacterium tuberculosis InhA: Revealing Molecular Determinants of Residence Time by MD Simulations.

Merget B, Sotriffer CA - PLoS ONE (2015)

Bottom Line: Whereas the diphenyl ether inhibitors 6PP and triclosan (TCL) do not show loop ordering and thus, no slow-binding inhibition and high koff values, the slightly modified PT70 leads to an ordered loop and a residence time of 24 minutes.The individual simulations show comparable conformational features with respect to both the binding pocket and the SBL, allowing to define five recurring conformational families.The most abundant conformation besides the stable EI* state is characterized by a shift of Ile202 and Val203 toward the hydrophobic pocket of InhA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074, Würzburg, Germany.

ABSTRACT
An important kinetic parameter for drug efficacy is the residence time of a compound at a drug target, which is related to the dissociation rate constant koff. For the essential antimycobacterial target InhA, this parameter is most likely governed by the ordering of the flexible substrate binding loop (SBL). Whereas the diphenyl ether inhibitors 6PP and triclosan (TCL) do not show loop ordering and thus, no slow-binding inhibition and high koff values, the slightly modified PT70 leads to an ordered loop and a residence time of 24 minutes. To assess the structural differences of the complexes from a dynamic point of view, molecular dynamics (MD) simulations with a total sampling time of 3.0 µs were performed for three ligand-bound and two ligand-free (perturbed) InhA systems. The individual simulations show comparable conformational features with respect to both the binding pocket and the SBL, allowing to define five recurring conformational families. Based on their different occurrence frequencies in the simulated systems, the conformational preferences could be linked to structural differences of the respective ligands to reveal important determinants of residence time. The most abundant conformation besides the stable EI* state is characterized by a shift of Ile202 and Val203 toward the hydrophobic pocket of InhA. The analyses revealed potential directions for avoiding this conformational change and, thus, hindering rapid dissociation: (1) an anchor group in 2'-position of the B-ring for scaffold stabilization, (2) proper occupation of the hydrophobic pocket, and (3) the introduction of a barricade substituent in 5'-position of the diphenyl ether B-ring.

No MeSH data available.


Related in: MedlinePlus

Illustration of conformational families of InhA.After summarizing the eight clusters of the hierarchical cluster analysis to five conformational families, a Partitioning Around Medoids (PAM) clustering was performed with R for each conformational family. The resulting medoids are illustrated as cluster representatives. The top left Fig Shows an entire monomer (A) of InhA from the crystal structure of the complex with PT70 (PDB 2X23). The substrate binding loop (SBL) is highlighted in yellow. The arrow represents the direction of the view for the subsequent images. (a) Family 1: crystal structure conformation; PT70 monomer 4 after 34 ns of MD simulation. SBL and pocket residues are labeled. The ligand carbon atoms are depicted in slate blue, the cofactor carbon atoms in magenta. (b) Family 2: Helical twist of ascending SBL branch with Ile202 shifted toward the ligand; PT70 monomer 3 after 141 ns of MD simulation. (c) Family 3: Enhanced movement of Ile202 far into the hydrophobic cavity; 6PP monomer 1 after 102 ns of MD simulation. (d) Family 4: Flip of Tyr158 toward the hydrophobic pocket; TCL monomer 3 after 119 ns of MD simulation. (e) Family 5: Ile202 movement toward the outside of the protein into the solvent; 6PP monomer 3 after 27 ns of MD simulation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4440617&req=5

pone.0127009.g003: Illustration of conformational families of InhA.After summarizing the eight clusters of the hierarchical cluster analysis to five conformational families, a Partitioning Around Medoids (PAM) clustering was performed with R for each conformational family. The resulting medoids are illustrated as cluster representatives. The top left Fig Shows an entire monomer (A) of InhA from the crystal structure of the complex with PT70 (PDB 2X23). The substrate binding loop (SBL) is highlighted in yellow. The arrow represents the direction of the view for the subsequent images. (a) Family 1: crystal structure conformation; PT70 monomer 4 after 34 ns of MD simulation. SBL and pocket residues are labeled. The ligand carbon atoms are depicted in slate blue, the cofactor carbon atoms in magenta. (b) Family 2: Helical twist of ascending SBL branch with Ile202 shifted toward the ligand; PT70 monomer 3 after 141 ns of MD simulation. (c) Family 3: Enhanced movement of Ile202 far into the hydrophobic cavity; 6PP monomer 1 after 102 ns of MD simulation. (d) Family 4: Flip of Tyr158 toward the hydrophobic pocket; TCL monomer 3 after 119 ns of MD simulation. (e) Family 5: Ile202 movement toward the outside of the protein into the solvent; 6PP monomer 3 after 27 ns of MD simulation.

Mentions: The hierarchical cluster analysis was carried out with R [20] using the complete linkage method. This method was preferred over others not only because it tends to produce clusters with similar diameter, but primarily because it provides readily interpretable results in terms of a maximum RMSD value between members of a cluster. Here, eight clusters of recurring conformations of the InhA binding pocket were identified at an RMSD cutoff of 3.5 Å (cf. Supporting Information S2 Fig for further details). On the basis of the cluster dendrogram and the corresponding structural similarities, the clusters were further summarized to five “monophyletic” conformational families. Subsuming the clusters to monophyletic families was achieved by visual inspection instead of raising the RMSD cutoff, since mere RMSD values might overestimate the importance of minor backbone movements while concealing important side chain flips. These families are hereinafter referred to as Families 1 to 5 (cf. Fig 3):


Slow-Onset Inhibition of Mycobacterium tuberculosis InhA: Revealing Molecular Determinants of Residence Time by MD Simulations.

Merget B, Sotriffer CA - PLoS ONE (2015)

Illustration of conformational families of InhA.After summarizing the eight clusters of the hierarchical cluster analysis to five conformational families, a Partitioning Around Medoids (PAM) clustering was performed with R for each conformational family. The resulting medoids are illustrated as cluster representatives. The top left Fig Shows an entire monomer (A) of InhA from the crystal structure of the complex with PT70 (PDB 2X23). The substrate binding loop (SBL) is highlighted in yellow. The arrow represents the direction of the view for the subsequent images. (a) Family 1: crystal structure conformation; PT70 monomer 4 after 34 ns of MD simulation. SBL and pocket residues are labeled. The ligand carbon atoms are depicted in slate blue, the cofactor carbon atoms in magenta. (b) Family 2: Helical twist of ascending SBL branch with Ile202 shifted toward the ligand; PT70 monomer 3 after 141 ns of MD simulation. (c) Family 3: Enhanced movement of Ile202 far into the hydrophobic cavity; 6PP monomer 1 after 102 ns of MD simulation. (d) Family 4: Flip of Tyr158 toward the hydrophobic pocket; TCL monomer 3 after 119 ns of MD simulation. (e) Family 5: Ile202 movement toward the outside of the protein into the solvent; 6PP monomer 3 after 27 ns of MD simulation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440617&req=5

pone.0127009.g003: Illustration of conformational families of InhA.After summarizing the eight clusters of the hierarchical cluster analysis to five conformational families, a Partitioning Around Medoids (PAM) clustering was performed with R for each conformational family. The resulting medoids are illustrated as cluster representatives. The top left Fig Shows an entire monomer (A) of InhA from the crystal structure of the complex with PT70 (PDB 2X23). The substrate binding loop (SBL) is highlighted in yellow. The arrow represents the direction of the view for the subsequent images. (a) Family 1: crystal structure conformation; PT70 monomer 4 after 34 ns of MD simulation. SBL and pocket residues are labeled. The ligand carbon atoms are depicted in slate blue, the cofactor carbon atoms in magenta. (b) Family 2: Helical twist of ascending SBL branch with Ile202 shifted toward the ligand; PT70 monomer 3 after 141 ns of MD simulation. (c) Family 3: Enhanced movement of Ile202 far into the hydrophobic cavity; 6PP monomer 1 after 102 ns of MD simulation. (d) Family 4: Flip of Tyr158 toward the hydrophobic pocket; TCL monomer 3 after 119 ns of MD simulation. (e) Family 5: Ile202 movement toward the outside of the protein into the solvent; 6PP monomer 3 after 27 ns of MD simulation.
Mentions: The hierarchical cluster analysis was carried out with R [20] using the complete linkage method. This method was preferred over others not only because it tends to produce clusters with similar diameter, but primarily because it provides readily interpretable results in terms of a maximum RMSD value between members of a cluster. Here, eight clusters of recurring conformations of the InhA binding pocket were identified at an RMSD cutoff of 3.5 Å (cf. Supporting Information S2 Fig for further details). On the basis of the cluster dendrogram and the corresponding structural similarities, the clusters were further summarized to five “monophyletic” conformational families. Subsuming the clusters to monophyletic families was achieved by visual inspection instead of raising the RMSD cutoff, since mere RMSD values might overestimate the importance of minor backbone movements while concealing important side chain flips. These families are hereinafter referred to as Families 1 to 5 (cf. Fig 3):

Bottom Line: Whereas the diphenyl ether inhibitors 6PP and triclosan (TCL) do not show loop ordering and thus, no slow-binding inhibition and high koff values, the slightly modified PT70 leads to an ordered loop and a residence time of 24 minutes.The individual simulations show comparable conformational features with respect to both the binding pocket and the SBL, allowing to define five recurring conformational families.The most abundant conformation besides the stable EI* state is characterized by a shift of Ile202 and Val203 toward the hydrophobic pocket of InhA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074, Würzburg, Germany.

ABSTRACT
An important kinetic parameter for drug efficacy is the residence time of a compound at a drug target, which is related to the dissociation rate constant koff. For the essential antimycobacterial target InhA, this parameter is most likely governed by the ordering of the flexible substrate binding loop (SBL). Whereas the diphenyl ether inhibitors 6PP and triclosan (TCL) do not show loop ordering and thus, no slow-binding inhibition and high koff values, the slightly modified PT70 leads to an ordered loop and a residence time of 24 minutes. To assess the structural differences of the complexes from a dynamic point of view, molecular dynamics (MD) simulations with a total sampling time of 3.0 µs were performed for three ligand-bound and two ligand-free (perturbed) InhA systems. The individual simulations show comparable conformational features with respect to both the binding pocket and the SBL, allowing to define five recurring conformational families. Based on their different occurrence frequencies in the simulated systems, the conformational preferences could be linked to structural differences of the respective ligands to reveal important determinants of residence time. The most abundant conformation besides the stable EI* state is characterized by a shift of Ile202 and Val203 toward the hydrophobic pocket of InhA. The analyses revealed potential directions for avoiding this conformational change and, thus, hindering rapid dissociation: (1) an anchor group in 2'-position of the B-ring for scaffold stabilization, (2) proper occupation of the hydrophobic pocket, and (3) the introduction of a barricade substituent in 5'-position of the diphenyl ether B-ring.

No MeSH data available.


Related in: MedlinePlus