Figure 7. Clustering of hydrogen bond, charged, and p interaction patterns in the docked compounds correspond to activity level. Receptor residues forming the interactions are specified on the bottom of the clustergram. Residues denoted by p form p interactions (either p-cation or p- p stacking). Yellow represents a ligand-receptor contact. The sub-tree formed predominantly by the active ligands is in green, and the one formed predominantly by inactive ligands is in red. The ‘‘A’’ on the right side denotes active compounds. The specific pattern formed by the active compounds is boxed in orange. The black subtrees are mixed. doi:10.1371/journal.pone.0027990.g007 from the analysis of ligand-receptor interactions formed between the known small-molecule antagonists and receptor residues (see Materials and Methods for details) and were not only selected based on the highest docking score. The underlying hypothesis is that the same interactions are perused by the potential ligands as by the known antagonists. Selected poses of all 10 molecules successfully passed this procedure. All poses were visually examined by checking that they adequately fill the binding site and form the desired specific interactions. All 10 molecules successfully passed this analysis and were considered as candidate compounds that may serve as potential hPKR binders. Next, we focused on a representative of the three FDA- approved hits, which we identified as potential ligands for hPKRs, namely, Indinavir, Argatroban, and Lapatinib. Figure 9 shows representative examples of docking of Indivavir, Argatroban, and Lapatinib to the hPKR1 binding site. As shown, the compounds adequately fill the binding site and are predicted to form specific interactions with residues found to be important for binding of the known hPKR antagonists, namely, charged interaction with Glu1192.61, and hydrogen bonds and/or stacking interactions with Arg1443.32 and Arg3076.58. These compounds also form interactions with additional binding site residues, which interact with the known binders (see figure 7). Each of the compounds is widely used in the clinic, and provides well-tested and safe compounds that may also exert their actions via hPKRs. The potential cross-reactivity of one such candidate drug, Indinavir, is further addressed in the Discussion. Discussion Prokineticin receptor (PKR) subtypes 1 and 2 are novel members of family A GPCRs. Prokineticins and their receptors Small-Molecule Binders of Prokineticin Receptors play important roles under various physiological conditions, and blocking PKRs may serve as a therapeutic tool for various pathologies, including acute pain, circadian rhythm disturbances, inflammation, and cancer. In this study, we extracted essential functional groups from small-molecule PKR antagonists that were previously reported, using structure-activity relationship analysis, and we used them in a virtual screening procedure. Consequently, we were able to identify several potential PKR ligands with novel scaffolds. Interestingly, the virtual hits included several HIV protease inhibitors that are discussed next in terms of known side effects and potential new indications of these drugs. Computational docking of known ligands to the multiple-template 3D model of a PKR’s structure enabled us to predict ligand-receptor contacts and provided a structural explanation of the importance of the chemical features we obtained from the analysis of known PKR binders. Homology modeling of the hPKR subtypes and docking of known small-molecule antagonists In this study we modeled the 3D structure of the hPKR subtypes and explored the interactions formed between hPKR1 and small-molecule binders. Our computational analysis revealed that hPKR1 is predicted to possess a TM-bundle binding site, capable of binding small-molecule ligands, similarly to other GPCR family A members, such as the aminergic receptors. This occurs despite the fact that the receptors’ endogenous ligands are relatively large proteins, which most likely bind the extracellular surface of the receptors. The latter is demonstrated in experimen- tal data on Kallmann syndrome mutations. Kallmann syndrome is a human disease characterized by the association of hypogonad- otropic hypogonadism and anosmia. Several loss-of-function mutations in the human PKR2 gene have been found in Kallmann patients [45]. Among them is the p.Q210R mutation in ECL2 (corresponding to Q219 in hPKR1), which completely abolishes native ligand binding and has no affinity for the orthologue ligand MIT1 (Mamba intestinal toxin 1, which shares 60% sequence identity with PK2, and contains the essential N- terminal motif AVITGA) [59]. Existence of both an orthosteric extracellular binding site capable of binding small proteins and an allosteric TM binding site was already shown in family A GPCRs. For example, the melanin-concentrating hormone receptor (MCHR), for which the endogenous ligand is a peptide, also binds small-molecule antagonists in its TM-bundle cavity [60,61]. The predicted TM-bundle site is identical between the two hPKR subtypes, except for one residue in ECL2 (Val207 in PKR1 corresponding to Phe198 in PKR2). Since this is a hydrophobic residue in both receptors, its side chain will probably face the TM cavity and not the solvent. Indeed, the residue was modeled to face the TM cavity and was predicted by the energy-based methods to be part of the TM-bundle binding site. If specific binders are pursued in the future, this, albeit minor, difference between two hydrophobic amino acids might be targeted. Through docking experiments of the known hPKR antagonists, we have identified important residues that interact at this site, namely, Glu1192.61, Arg1443.32, and Arg3076.58. These residues form specific interactions with the chemical features of the ligand that we found in our SAR analysis to be essential for the molecules’ antagonistic activity. Specifically, Arg1443.32 is analo- gous to Asp1133.32 of the b2-adrenergic receptor, which is an experimentally established receptor interaction site for both agonists and antagonists [62]. This position has also been shown to be important for ligand binding in many other family A GPCRs as well as in other branches of the GPCR super-family, such as the PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27990