Illustration to show the interactions between the designed inhibitor A5 (Ad―(2―ethanol)―(3―amino)) and the M2 proton channel.

<p>The designed adamantane-based inhibitor A5 has two pharmacophore groups. The amino group of inhibitor A5 binds at the 1-Asp44 of Chain-1 through two hydrogen bonds (green dotted lines), while the second pharmacophore hydroxyl group forms two hydrogen bonds with the 2-Arg45 of Chain-2. It is through the two binding sites that the inhibitor A5 holds the Chain-1 tightly with its adjacent Chain-2 of the tetrameric M2 proton channel.</p

Scaffold-Based Pan-Agonist Design for the PPARα, PPARβ and PPARγ Receptors

<div><p>As important members of nuclear receptor superfamily, Peroxisome proliferator-activated receptors (PPAR) play essential roles in regulating cellular differentiation, development, metabolism, and tumorigenesis of higher organisms. The PPAR receptors have 3 identified subtypes: PPARα, PPARβ and PPARγ, all of which have been treated as attractive targets for developing drugs to treat type 2 diabetes. Due to the undesirable side-effects, many PPAR agonists including PPARα/γ and PPARβ/γ dual agonists are stopped by US FDA in the clinical trials. An alternative strategy is to design novel pan-agonist that can simultaneously activate PPARα, PPARβ and PPARγ. Under such an idea, in the current study we adopted the core hopping algorithm and glide docking procedure to generate 7 novel compounds based on a typical PPAR pan-agonist LY465608. It was observed by the docking procedures and molecular dynamics simulations that the compounds generated by the core hopping and glide docking not only possessed the similar functions as the original LY465608 compound to activate PPARα, PPARβ and PPARγ receptors, but also had more favorable conformation for binding to the PPAR receptors. The additional absorption, distribution, metabolism and excretion (ADME) predictions showed that the 7 compounds (especially Cpd#1) hold high potential to be novel lead compounds for the PPAR pan-agonist. Our findings can provide a new strategy or useful insights for designing the effective pan-agonists against the type 2 diabetes.</p> </div

The sequence alignments between the M2 protein isolated from H1N1 influenza A virus (NCBI code: GQ385383) and the M2 channel isolated from the Udorn strain of human influenza virus.

<p>The latter 3D structure has been determined by NMR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009388#pone.0009388-Schnell1" target="_blank">[16]</a> with PDB code of 2RLF, and can be used to serve as a template to model the former. The GQ385383 is a complete M2 sequence consisting of 97 residues, while 2RLF only contains a segment of 43 structure-defined residues (18–60). The red codes highlight the functional residues: pH sensor His37, channel gate Trp41, and channel lock Asp44. Those residues, which are different between the two M2 sequences, are framed by a box drawn in green line, while those framed with a box drawn in light-blue line indicate the possible binding sites (Thr43, Asp44, and Arg45) of inhibitors. The functional residue Asp44 is the first binding site, and the Arg45 or Thr43 is the possible second binding site. Thr43 is a natural mutation Leu43Thr in the H1N1-M2 proton channel.</p

Illustration to show the interactions between the designed inhibitor A4 (Ad―3―aminoethanol) and the M2 proton channel.

<p>The designed adamantane-based inhibitor A4 has two pharmacophore groups. The amino group binds at the 1-Asp44 of Chain-1 through two hydrogen bonds (green dotted lines), while the second pharmacophore hydroxyl group forms two hydrogen bonds with the 2-Arg45 of Chain-2. It is through the two binding sites that the inhibitor A4 holds the Chain 1 tightly with its adjacent Chain 2 of the tetrameric M2 proton channel.</p

The computational three-dimensional structure of 2009-H1N1 M2 proton channel (NCBI code: GQ385303) and the template 2RLF with ligand rimantadine.

<p>(<b>A</b>) Superposition of the homology model of 2009-H1N1 M2 proton channel with its template, the NMR structure with the PDB code of 2RLF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009388#pone.0009388-Schnell1" target="_blank">[16]</a>. The two M2 proteins are highly homologous, and no differences are found on their backbones. The three functional residues of the M2 channel are rendered in ball-and-stick drawing with chemical element color; they are located at the middle of the channel. The different residues between two M2 proteins are show in green for 2RLF and blue for GQ385383. (<b>B</b>) A close-up view from the bottom of the M2 channel. The three functional residues are rendered in green for pH sensor His37, blue for channel gate Trp41, and chemical element color for channel lock Asp44. The ligand rimantadine binds at four equivalent sites near the channel lock Asp44 on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. (<b>C</b>) The complex structure between the inhibitor rimantadine and the receptor M2 proton channel. The channel lock Asp44 holds the channel gate Trp41 through a hydrogen bond keeping it in the closed conformation. The ligand rimantadine forms two hydrogen bonds (green dotted lines) with the Asp44 of M2 proton channel.</p

The RMS deviations for the backbone structures of the apo, LY465608-bound and Cpd#1-bound states of PPARα, PPARβ and PPARγ receptors.

<p>Both the fluctuations of total RMS deviations and final RMS deviations for all the simulations systems are no more than 1 Å during our molecular dynamics simulations, indicating that all the simulation systems are in the equilibrium states.</p

The ligand-binding domains of PPARα, PPARβ and PPARγ receptors.

<p>The ligand-binding domains for PPARα (red), PPARβ (blue) and PPARγ (yellow) receptors share some common features: 1) composed of 12 α-helices arranged in an antiparallel helix sandwich, and a 4-stranded antiparallel β sheet; 2) Y-shaped hydrophobic ligand binding pocket with a volume of ∼1300 cubic angstroms; and 3) a C-terminal helix (Helix 12 or AF2 helix) showing widely conformational variations in different crystals and playing essential roles in activation of PPAR receptors.</p

The top 7 hits in the core hopping and glide docking. For comparison, the typical PPAR pan-agonists bezafibrate, LY465608 and GW677954 are also involved.

<p>The top 7 hits in the core hopping and glide docking. For comparison, the typical PPAR pan-agonists bezafibrate, LY465608 and GW677954 are also involved.</p

Illustration to show the conformation obtained by docking GW409544 and Comp#1, respectively, to (A) PPARα (1k7l) and (B) PPARγ (1k74).

<p>The binding pocket is defined by those residues that have at least one heavy atom with a distance of 5 Å from the ligand <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038546#pone.0038546-Chou8" target="_blank">[45]</a>. The ligand GW409544 (in grey color) was extracted from the crystal structure while the ligand Comp#1 (rendered by three colors: grey for Core A; red for Core B; and blue for Core C) was generated by the “core-hopping” method. The hydrophobic surface of the receptor is colored in green. The blue dotted lines indicate the H-bond interactions of the receptor with its ligand. The red helix is a part of AF-2 function domain. See the text for further explanation.</p

Illustration to show the outcomes of molecular dynamics simulations for Comp#1 ranked number 1 in <b>Table 1</b>.

<p>(<b>A</b>) The RMSD (root mean square deviation) of all backbone atoms for the receptor PPARα. (<b>B</b>) The RMSF (root mean square fluctuation) of the side-chain atoms for the receptor PPARα. (<b>C</b>) The RMSD (root mean square deviation) of all backbone atoms for the receptor PPARγ. (<b>D</b>) The RMSF (root mean square fluctuation) of the side-chain atoms for the receptor PPARγ. The green line indicates the outcome for the system of the receptor alone without any ligand, the red line for that of the receptor with the ligand GW409544, and the black line for that of the receptor with the ligand Comp#1. The curves involved with the AF2 helix region are framed with grey line. See the text for further explanation.</p