Towards Automated Binding Affinity Prediction Using an Iterative Linear Interaction Energy Approach

Binding affinity prediction of potential drugs to target and off-target proteins is an essential asset in drug development. These predictions require the calculation of binding free energies. In such calculations, it is a major challenge to properly account for both the dynamic nature of the protein and the possible variety of ligand-binding orientations, while keeping computational costs tractable. Recently, an iterative Linear Interaction Energy (LIE) approach was introduced, in which results from multiple simulations of a protein-ligand complex are combined into a single binding free energy using a Boltzmann weighting-based scheme. This method was shown to reach experimental accuracy for flexible proteins while retaining the computational efficiency of the general LIE approach. Here, we show that the iterative LIE approach can be used to predict binding affinities in an automated way. A workflow was designed using preselected protein conformations, automated ligand docking and clustering, and a (semi-)automated molecular dynamics simulation setup. We show that using this workflow, binding affinities of aryloxypropanolamines to the malleable Cytochrome P450 2D6 enzyme can be predicted without a priori knowledge of dominant protein-ligand conformations. In addition, we provide an outlook for an approach to assess the quality of the LIE predictions, based on simulation outcomes only

Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors

The absence of an adequate reversal strategy to prevent and stop potential life-threatening bleeding complications is a major drawback to the clinical use of the direct oral inhibitors of blood coagulation factor Xa. Here we show that specific modifications of the substrate-binding aromatic S4 subpocket within the factor Xa active site disrupt high-affinity engagement of the direct factor Xa inhibitors. These modifications either entail amino-acid substitution of S4 subsite residues Tyr99 and/or Phe174 (chymotrypsinogen numbering), or extension of the 99-loop that borders the S4 subsite. The latter modifications led to the engineering of a factor Xa variant that is able to support coagulation in human plasma spiked with (supra-)physiological concentrations of direct factor Xa inhibitors. As such, this factor Xa variant has the potential to be employed to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation.A major drawback in the clinical use of the oral anticoagulants that directly inhibit factor Xa in order to prevent blood clot formation is the potential for life threatening bleeding events. Here the authors describe factor Xa variants that are refractory to inhibition by these anticoagulants and could serve as rescue agents in treated patients

Mutation in KERA Identified by Linkage Analysis and Targeted Resequencing in a Pedigree with Premature Atherosclerosis

Genetic factors explain a proportion of the inter-individual variation in the risk for atherosclerotic events, but the genetic basis of atherosclerosis and atherothrombosis in families with Mendelian forms of premature atherosclerosis is incompletely understood. We set out to unravel the molecular pathology in a large kindred with an autosomal dominant inherited form of premature atherosclerosis.Parametric linkage analysis was performed in a pedigree comprising 4 generations, of which a total of 11 members suffered from premature vascular events. A parametric LOD-score of 3.31 was observed for a 4.4 Mb interval on chromosome 12. Upon sequencing, a non-synonymous variant in KERA (c.920C>G; p.Ser307Cys) was identified. The variant was absent from nearly 28,000 individuals, including 2,571 patients with premature atherosclerosis. KERA, a proteoglycan protein, was expressed in lipid-rich areas of human atherosclerotic lesions, but not in healthy arterial specimens. Moreover, KERA expression in plaques was significantly associated with plaque size in a carotid-collar Apoe-/- mice (r2 = 0.69; p<0.0001).A rare variant in KERA was identified in a large kindred with premature atherosclerosis. The identification of KERA in atherosclerotic plaque specimen in humans and mice lends support to its potential role in atherosclerosis

Characteristics of the core pedigree included in linkage analysis.

<p>Age is expressed as mean ± standard deviation and data are expressed as number (N). Lipid values are expressed as median with interquartile range (IQR). Pedigree members with an event use lipid lowering medication. LDL = low density lipoprotein; HDL = high density lipoprotein.</p

Expression of KERA in atherosclerotic tissue in <i>Apoe^−/−</i> mice after induction of atherosclerosis by collar placement.

<p><b>A–B</b>, Early (week = 2, A) and advanced (week = 8, B) atherosclerotic tissue from murine carotid arteries were stained for KERA (brown) and hematoxylin (blue). While present mainly near endothelial cells in early lesion, KERA is predominantly present in the matrix of the plaque at more advanced lesions. <b>C–D</b>, KERA expression overtime in <i>Apoe^−/−</i> mice with collar placement show significant correlation with plaque size (r² = 0.69, P<0.0001).</p

KERA is expressed in atherosclerotic but not in non-diseased arteries.

<p><b>A</b>, KERA is not expressed in tonsil tissue; <b>B</b>: KERA is expressed in corneal tissue <b>C</b>: KERA is not expressed in mammary artery. <b>D</b>: KERA is expressed in arterial segments obtained from a patient with the <i>KERA</i> mutation.</p

Structures obtained from molecular dynamic simulations of the mutant p.Ser307Cys KERA protein.

<p>Molecular dynamic simulations were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098289#pone.0098289.s001" target="_blank">File S1</a>. The residue Cys303 is highlighted in yellow, Cys343 in green, Ser307 in cyan and Cys307 residue in blue. <b>A</b>, Structure of wild-type KERA. <b>C</b> Structure of the KERA mutant p.Ser307Cys. <b>B</b>, A detailed view of the C-terminal part of wild type KERA highlighting the Cys303–Cys343 disulphide bond. <b>D</b>, Possible structural effects of the substitution of a serine for a cysteine at residue 307 showing a favourable Cys303–Cys307 disulphide bond. Consequently, Cys343 is available for binding with cysteine residues of other proteins.</p

Co-localization of KERA with CXCL1 and CD3 positive type I helper T lymphocytes was assessed in human plaque segments.

<p><b>A</b>: triple stain with KERA (blue), CXCL1 (red) and CD3 positive cells (brown). <b>B</b>: Spectral Imaging of triple staining. <b>C</b>: The yellow staining demonstrates that these cells are positive for all three components.</p

Clinical characteristics of the core pedigree included in linkage analysis.

<p>AMI = Acute Myocardial Infarction; AP = Angina Pectoris; PTCA = Percutaneous Transluminal Coronary Angioplasty; CABG = Coronary Artery Bypass Graft; CVA = Cerebrovascular Accident. Simva = Simvastatin, Rosuva = Rosuvastatin, Prava = Pravastatin and Atorva = Atorvastatin. BMI = body mass index (kg/cm²).</p>†<p> = index case. Subjects were considered smokers if they were current smokers or when they quitted smoking within the last 5 years.</p