Small-Molecule Binders of Prokineticin Receptors Figure 6. Interaction of receptor residues with active and inactive antagonists in the allosteric hPKR1 binding site. Representative docked poses of two known active compounds (A, B, IC50,0.05 mM), and two inactive compounds (C, D, IC50.1 mM) to the hPKR1 binding site. The active compounds are denoted by yellow sticks and the inactive ones as orange sticks. Interacting receptor residues are denoted by cyan sticks and labeled. Hydrogen bonds are denoted by dashed green lines and p-cation or p-p interactions are denoted by orange lines. doi:10.1371/journal.pone.0027990.g006 For the active compounds, the most prevalent interaction is observed between the ligand and residues Arg1443.32 and Arg3076.58, either through a hydrogen bond or a p-cation interaction. The active ligands interact with at least one of these two residues. In addition, an electrostatic interaction was observed between the active ligands and Glu1192.61 (as seen from figure 6A, B). To quantify this observation, the specific interactions formed (HB, charged, p-p and p-cation) were monitored across all the best scoring poses of the docked ligands (active and inactive), and the results, which represent the number of specific contacts formed between each ligand and all polar/hydrophobic binding site residues, were clustered (figure 7). As shown, the hierarchical structure obtained from the clustering procedure of receptor-ligand contacts only, clearly separates the compounds into sub-trees that correspond to the experimental active/inactive distinction. In the active sub-tree, the ligands form a charged interaction with Glu1192.61, and interact mainly with Cys1373.25, Arg1443.32, and Arg3076.58. In contrast, in the inactive sub-tree, the molecules still form interactions with Arg1443.32 to some extent, but the interactions with Glu1192.61, Cys1373.25, and Arg3076.58 are drastically reduced, and instead some of the ligands interact with Thr1453.33 and Met3327.47. In addition, some of the active ligands form either specific interactions or van der Waals contacts with Asn1413.29, Phe3006.51, and Phe3247.39. All of these positions have been shown experimentally to be important for ligand binding in different family A GPCRs members, ranging from aminergic (such as the b2-adrenergic receptor) to peptide receptors (such as chemokine receptors) [25]. In general, the functional groups in the scaffold, which were identified in our SAR analysis as being important for antagonist activity, form specific interactions within the binding site (figure 8). Namely, the main triazine ring of the scaffold forms hydrogen bonds through its O and N atoms and p-cation interactions. The two aromatic rings form p-cation interactions and hydrogen bonds through the O/F/Cl atoms at position 4 of the ring, and the positive charge at position Q and hydrogen bond donors interact with residues from helices 2, 3, and 6, predominantly, Glu1192.61 and Arg1443.32, and Arg3076.58, as described above. The compatibility of the SAR data with the docking results supports the predicted binding site and modes, and provides a molecular explanation of the importance of particular pharmacophores in the ligand. The positions predicted to specifically bind essential functional groups in the ligands (mainly Glu1192.61, Arg1443.32, and Arg3076.58) can be mutated in future studies, to confirm their role in ligand binding inside the predicted TM-bundle cavity, as recently applied to other GPCRs [58] and summarized in [25]. Docking of virtual hits to the hPKR1 model suggests potential binders Next, the 10 molecules identified through ligand-based virtual screening of the DrugBank database were docked to the hPKR1 homology model. All docking experiments were performed using LigandFit, as described in the previous section. However, here the analysis was more strict: the resulting docked poses of each molecule were post-processed using structure-based filters derived PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27990