Representative structures of ApoE isoforms.

<p>(A) ApoE3 representative structure (<i>i</i>.<i>e</i>., centroid of the most populated cluster) from clustering analysis of the protein conformations extracted from the free energy basin at T1 (~275 K, see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.g002" target="_blank">Fig 2B</a>). The same compact, native state of the N-terminal helices is observed in all three ApoE isoforms (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.s002" target="_blank">S2A–S2C Fig</a>). At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively) the representative structure of the intermediate state for: (B) ApoE2 exhibits an expanded volume of the N-terminal domain due to an increase of the average inter-helical distances; (C) ApoE3 exhibits a pairing of N-terminal helix–1 and helix–4 which separate from helix–2 and helix–3; (D) ApoE4 exhibits a separation of helix–1 from the other three helices. Such conformation represents the identified isoform-specific misfolded intermediate state (inter-residue contacts shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.s006" target="_blank">S6A Fig</a>) The size of the most populated cluster is reported in each panel. For all structures, helix–1 (H1), helix–2 (H2), helix–3 (H3), and helix–4 (H4) are represented in purple, green, blue, and red, cartoon respectively. The rest of the protein is represented in grey cartoon. (The sequence numbers for helices is reported in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.s001" target="_blank">S1 Fig</a>).</p

Free energy landscapes of ApoE isoforms.

<p>ApoE isoforms’ conformational landscapes derived from PMF as a function of RMSD and Rg of ApoE variants’ N-terminal domains. C-terminal domains are excluded from the analysis to reduce the degeneracy of protein conformational states. (A-C) The free energy landscapes from REX/DMD simulations at T1 (~275 K for all three ApoE isoforms) are isolated in the lowest range of RMSD and Rg suggesting the majority of conformations are close to the native N-terminal domain state. (D-F) At T2 (~321 K, ~318 K, and ~309 K for ApoE2, ApoE3 and ApoE4 respectively) all three variants explore a larger area of the conformational landscape as denoted by the larger RMSD and Rg values. (G-I) At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively) the isoforms transition to their intermediate states. ApoE3 is characterized by both the native and alternate N-terminal domain conformations, while ApoE2 visits only the latter. ApoE4 exhibits a unique, more compact intermediate conformational state as denoted by the smaller range of RMSD and Rg values compared to the two other variants. (J-L) At T4 (~355 K, ~365 K, and ~342 K for ApoE2, ApoE3, and ApoE4, respectively) all three isoforms undergo complete unfolding as inferred by their extended landscapes in the high range of RMSD and Rg values, although ApoE4 also visits the previous conformational states identified at temperature T3. (Note the different scale on x- and y-axes; representative structures are reported in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004359#pcbi.1004359.s002" target="_blank">S2 Fig</a>). The color bar represents the relative Helmholtz free energy in kcal/mol.</p

ApoE4-specific Misfolded Intermediate Identified by Molecular Dynamics Simulations

<div><p>The increased risk of developing Alzheimer’s disease (AD) is associated with the <i>APOE</i> gene, which encodes for three variants of Apolipoprotein E, namely E2, E3, E4, differing only by two amino acids at positions 112 and 158. ApoE4 is known to be the strongest risk factor for AD onset, while ApoE3 and ApoE2 are considered to be the AD-neutral and AD-protective isoforms, respectively. It has been hypothesized that the ApoE isoforms may contribute to the development of AD by modifying the homeostasis of ApoE physiological partners and AD-related proteins in an isoform-specific fashion. Here we find that, despite the high sequence similarity among the three ApoE variants, only ApoE4 exhibits a misfolded intermediate state characterized by isoform-specific domain-domain interactions in molecular dynamics simulations. The existence of an ApoE4-specific intermediate state can contribute to the onset of AD by altering multiple cellular pathways involved in ApoE-dependent lipid transport efficiency or in AD-related protein aggregation and clearance. We present what we believe to be the first structural model of an ApoE4 misfolded intermediate state, which may serve to elucidate the molecular mechanism underlying the role of ApoE4 in AD pathogenesis. The knowledge of the structure for the ApoE4 folding intermediate provides a new platform for the rational design of alternative therapeutic strategies to fight AD.</p></div

Temperature-dependent pair-wise inter-residue distances of ApoE isoforms.

<p>(A-C) At T1 (~275 K for all three ApoE isoforms) and (D-F) at T2 (~321 K, ~318 K, and ~309 K for ApoE2, ApoE3 and ApoE4 respectively), all three isoforms exhibits the highest level of inter-residue contacts observed in the REX/DMD simulations, with ApoE4 having the highest density contacts. (G-I) At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively), all three isoforms exhibit a dramatic decrease in density of inter-residue contacts. ApoE4 displays a unique series of contacts (outlined in red) mediating the domain-domain interaction as discussed in the main text. (J-L) At T4 (~355 K, ~365 K, and ~342 K for ApoE2, ApoE3, and ApoE4, respectively), the majority of inter-residue contacts have been lost besides some transient contacts involving the N-terminal helix–4. The upper and lower triangular matrices represent respectively the average and the standard deviation of the pair-wise inter-residue distance in Å. The color bar represents the distance between the centroid computed over the residues’ side chains in Å.</p

ApoE isoform-specific mutations.

<p>ApoE isoform-specific mutations.</p

Heat capacity curves of ApoE isoforms.

<p>(A) The heat capacity (Cv) curves computed using WHAM on REX/DMD trajectories for ApoE2 (black), ApoE3 (red) and ApoE4 (blue) in the range of 275 to 400 K show intermediates states that appear at different temperatures for each isoform. The position of the first peak (<i>i</i>.<i>e</i>., unfolding of the hydrophobic core of the protein) suggests that ApoE4 is less thermally stable than ApoE2 and ApoE3. (B-D) Cv curves of individual ApoE isoforms including the error bars (shaded grey area). The shaded grey area in panels B-D represents the statistical uncertainty (<i>i</i>.<i>e</i>., the square root of the variance of the specific heat) in the WHAM estimation of heat capacity. Local minima in the curves at temperatures T1, T2, T3, and T4 represent different conformational states of the protein for each ApoE variant.</p

Docking and Scoring with Target-Specific Pose Classifier Succeeds in Native-Like Pose Identification But Not Binding Affinity Prediction in the CSAR 2014 Benchmark Exercise

The CSAR 2014 exercise provided an important benchmark for testing current approaches for pose identification and ligand ranking using three X-ray characterized proteins: Factor Xa (FXa), Spleen Tyrosine Kinase (SYK), and tRNA Methyltransferase (TRMD). In Phase 1 of the exercise, we employed Glide and MedusaDock docking software, both individually and in combination, with the special target-specific pose classifier trained to discriminate native-like from decoy poses. All approaches succeeded in the accurate detection of native and native-like poses. We then used Glide SP and MedusaScore scoring functions individually and in combination with the pose-scoring approach to predict relative binding affinities of the congeneric series of ligands in Phase 2 of the exercise. Similar to other participants in the CSAR 2014 exercise, we found that our models showed modest prediction accuracy. Quantitative structure–activity relationship (QSAR) models developed for the FXa ligands using available bioactivity data from ChEMBL showed relatively low prediction accuracy for the CSAR 2014 ligands of the same target. Interestingly, QSAR models built with CSAR data only yielded Spearman correlation coefficients as high as ρ = 0.69 for FXa and ρ = 0.79 for SYK based on 5-fold cross-validation. Virtual screening of the DUD library using the FXa structure was successful in discriminating between active compounds and decoys in spite of poor ranking accuracy of the underlying scoring functions. Our results suggest that two of the three common tasks associated with molecular docking, i.e., native-like pose identification and virtual screening, but not binding affinity prediction, could be accomplished successfully for the CSAR 2014 challenge data set

Methylations of Tryptophan-Modified Naphthoquinone Affect Its Inhibitory Potential toward Aβ Aggregation

Aggregation of amyloid beta (Aβ) is the hallmark of Alzheimer’s disease (AD). Small molecules inhibiting Aβ can be valuable therapeutics for AD. We have previously reported that 1,4-naphthoquinon-2-yl-l-tryptophan (NQTrp), reduces aggregation and oligomerization of Aβ in vitro and in vivo. In silico analysis further showed that certain functional groups of NQTrp, not in the aromatic rings, are also involved in binding and inhibiting Aβ. To better understand the exact mode of action and identify the groups crucial for NQTrp inhibitory activity, we conducted structure–activity analysis. Four derivatives of NQTrp were studied in silico: a <i>D</i>-isomer, two single-methylated and one double-methylated derivative. In silico results showed that the NQTrp groups involved in hydrogen bonds are the anilinic NH (i.e., the NH linker between the quinone and tryptophan moieties), the quinonic carbonyls, and the carboxylic acid. These predictions were supported by in vitro results. Our results should aid in designing improved small-molecule inhibitors of Aβ aggregation for treating AD

Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for “soft” treatment of various diseases. Crystal structures of subtypes A_2A and A₁ARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of A_2AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson–Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting A_2AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A_2A/A₃, ARs, or A₃AR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A_2A/A₃, A₁/A₃, A₁/A_2A/A₃, and A₃). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A₃ receptor

6TM-mOR transfected Be2C cells show positive Ca²⁺ response to the stimulation with morphine.

<p><b>(A)</b> The percentage of Ca²⁺-responding cells upon 10 μM morphine treatment. Be2C cells transfected with 6TM-mOR, 7TM-mOR, or empty vector plasmids. *p<0.03, one-way ANOVA and Fisher’s LSD. Perfusion conditions: 1 min PBS; 59 min 10 μM morphine. <b>(B)</b> Time lapse images of Be2C cells transfected with 6TM-MOR at baseline (left; perfused with PBS) and after morphine treatment (right; perfused with 10 μM morphine). Ca²⁺ responses are imaged using Fura-2AM ratio-metric dye. Color scale shows the F340/380 fluorescence intensity ratio increase. Scale bar: 100 μm. <b>(C)</b> Ca²⁺ responses of individual 6TM-mOR-transfected cells, presented as increases in F340/380 fluorescence intensity; 10 μM morphine was perfused between 60–3600 s (blue bar).</p