Ranking docking poses by graph matching of protein–ligand interactions: lessons learned from the D3R Grand Challenge 2

International audienceA novel docking challenge has been set by the Drug Design Data Resource (D3R) in order to predict the pose and affinity ranking of a set of Farnesoid X receptor (FXR) agonists, prior to the public release of their bound X-ray structures and potencies. In a first phase, 36 agonists were docked to 26 Protein Data Bank (PDB) structures of the FXR receptor, and next rescored using the in-house developed GRIM method. GRIM aligns protein–ligand interaction patterns of docked poses to those of available PDB templates for the target protein, and rescore poses by a graph matching method. In agreement with results obtained during the previous 2015 docking challenge, we clearly show that GRIM rescoring improves the overall quality of top-ranked poses by prioritizing interaction patterns already visited in the PDB. Importantly, this challenge enables us to refine the applicability domain of the method by better defining the conditions of its success. We notably show that rescoring apolar ligands in hydrophobic pockets leads to frequent GRIM failures. In the second phase, 102 FXR agonists were ranked by decreasing affinity according to the Gibbs free energy of the corresponding GRIM-selected poses, computed by the HYDE scoring function. Interestingly, this fast and simple rescoring scheme provided the third most accurate ranking method among 57 contributions. Although the obtained ranking is still unsuitable for hit to lead optimization, the GRIM–HYDE scoring scheme is accurate and fast enough to post-process virtual screening dat

Targeting the Central Pocket of the Pseudomonas aeruginosa Lectin LecA

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i. e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moieties targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms

Targeting the Central Pocket of the Pseudomonas aeruginosa Lectin LecA

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i. e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moieties targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms

Targeting undruggable carbohydrate recognition sites through focused fragment library design

Carbohydrate-protein interactions are key for cell-cell and host-pathogen recognition and thus, emerged as viable therapeutic targets. However, their hydrophilic nature poses major limitations to the conventional development of drug-like inhibitors. To address this shortcoming, four fragment libraries were screened to identify metal-binding pharmacophores (MBPs) as novel scaffolds for inhibition of Ca2+-dependent carbohydrate-protein interactions. Here, we show the effect of MBPs on the clinically relevant lectins DC-SIGN, Langerin, LecA and LecB. Detailed structural and biochemical investigations revealed the specificity of MBPs for different Ca2+-dependent lectins. Exploring the structure-activity relationships of several fragments uncovered the functional groups in the MBPs suitable for modification to further improve lectin binding and selectivity. Selected inhibitors bound efficiently to DCSIGN-expressing cells. Altogether, the discovery of MBPs as a promising class of Ca2+- dependent lectin inhibitors creates a foundation for fragment-based ligand design for future drug discovery campaigns

Differential Effects of CSF-1R D802V and KIT D816V Homologous Mutations on Receptor Tertiary Structure and Allosteric Communication

International audienc

Targeting the Central Pocket of the Pseudomonas aeruginosa lectin LecA

International audiencePseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i.e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moities targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib

MD simulations of the CSF-1R cytoplasmic domain in the inactive state.

<p>Two forms of receptor, the native (CSF-1R^WT and CSF-1R^MU (D802V) were simulated twice during 50 ns. (<b>A</b>) The Root Mean Square Deviation (RMSD) values were calculated for backbone atoms from trajectories 1 and 2 of MD simulations of CSF-1R^WT (black and blue) and CSF-1R ^MU (red and orange). RMSDs (in <i>nm</i>) plotted versus simulation time (<i>ns</i>) and showed separately for N- and C-lobes, JMR and A-loop regions. (<b>B</b>) The Root Mean Square Fluctuations (RMSF) computed on the backbone atoms over the total production simulation time of CSF-1R^MU (red) were compared to those in CSF-1R^WT (black). The RMSFs of the A-loop is zoomed in the insert. The average conformations for CSF-1R^WT (<b>C</b>) and CSF-1R^MU (<b>D</b>) are presented as tubes. The size of the tube is proportional to the by-residue atomic fluctuations computed on the backbone atoms. The high fluctuation region found in proteins, are specified by red color and numerated from 1 to 10 in B–D. The size of numbers in <b>D</b> is proportional to RMSFs.</p

H-bonds stabilized the inactive conformation in CSF-1R^WT and CSF1R^MU.

<p>Residues involved in H-bonding and the H-bond occurrences (in %) are computed over MD simulations. Occurrences showed a major difference are denoted by asterisk.</p

Top. Structure of CSF-1R cytoplasmic region.

<p>Crystallographic structures of the native receptor (A) in the inactive (2OGV<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097519#pone.0097519-Walter1" target="_blank">[22]</a>) and (B) the active forms (3LCD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097519#pone.0097519-Meyers1" target="_blank">[85]</a> are presented as cartoon. The different domains of CSF-1R and key structural fragments are highlighted in color. The N-terminal proximal lobe (N-lobe) is in blue, the C-terminal distal lobe (C-lobe) is in green, the Cα-helix is in cyan, the activation loop (A-loop) is in red, the juxtamembrane region (JMR) is in orange. The DFG motif (Asp796, Phe797, Gly798) and position of D802V mutation (insert) are represented in sticks. Bottom. The JMR sequence in RTKs of III family. The sequence alignment shows a poor sequence conversation of the JMR among the receptors TK of type III. Identical residues and similar residues are shown in red and in green, respectively. The three strongly conserved tyrosine residues are contoured. Except CSF-1R, the other RTKs from III family possess a second functional phosphotyrosine (green, contoured) in JM-Switch.</p