Modeling of Human Prokineticin Receptors: Interactions with Novel Small-Molecule Binders and Potential Off- Target Drugs Anat Levit1,2, Talia Yarnitzky1, Ayana Wiener1, Rina Meidan2, Masha Y. Niv1,3* 1 Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel, 2 Department of Animal Sciences, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel, 3 The Fritz Haber Center for Molecular Dynamics, The Hebrew University of Jerusalem, Jerusalem, Israel Abstract Background and Motivation: The Prokineticin receptor (PKR) 1 and 2 subtypes are novel members of family A GPCRs, which exhibit an unusually high degree of sequence similarity. Prokineticins (PKs), their cognate ligands, are small secreted proteins of ,80 amino acids; however, non-peptidic low-molecular weight antagonists have also been identified. PKs and their receptors play important roles under various physiological conditions such as maintaining circadian rhythm and pain perception, as well as regulating angiogenesis and modulating immunity. Identifying binding sites for known antagonists and for additional potential binders will facilitate studying and regulating these novel receptors. Blocking PKRs may serve as a therapeutic tool for various diseases, including acute pain, inflammation and cancer. Methods and Results: Ligand-based pharmacophore models were derived from known antagonists, and virtual screening performed on the DrugBank dataset identified potential human PKR (hPKR) ligands with novel scaffolds. Interestingly, these included several HIV protease inhibitors for which endothelial cell dysfunction is a documented side effect. Our results suggest that the side effects might be due to inhibition of the PKR signaling pathway. Docking of known binders to a 3D homology model of hPKR1 is in agreement with the well-established canonical TM-bundle binding site of family A GPCRs. Furthermore, the docking results highlight residues that may form specific contacts with the ligands. These contacts provide structural explanation for the importance of several chemical features that were obtained from the structure-activity analysis of known binders. With the exception of a single loop residue that might be perused in the future for obtaining subtype-specific regulation, the results suggest an identical TM-bundle binding site for hPKR1 and hPKR2. In addition, analysis of the intracellular regions highlights variable regions that may provide subtype specificity. Citation: Levit A, Yarnitzky T, Wiener A, Meidan R, Niv MY (2011) Modeling of Human Prokineticin Receptors: Interactions with Novel Small-Molecule Binders and Potential Off-Target Drugs. PLoS ONE 6(11): e27990. doi:10.1371/journal.pone.0027990 Editor: Anna Tramontano, University of Rome, Italy Received July 1, 2011; Accepted October 29, 2011; Published November 21, 2011 Copyright: 2011 Levit et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was funded in part by the Niedersachsen-Israeli Research cooperation fund to MYN and the Israeli Science Foundation to RM. AL is supported by the Johanna Friedlaender memorial award for excellence from the Canadian Friends of the Hebrew University and by the Smith excellence fellowship for PhD students. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: niv@agri.huji.ac.il Introduction Prokineticins and their receptors Mammalian prokineticins 1 and 2 (PK1 and PK2) are two secreted proteins of about 80–90 residues in length, which belong to the AVIT protein family [1,2,3]. Their structure includes 10 conserved cysteine residues that create five disulphide-bridged motifs (colipase fold) and an identical (AVIT) motif in the N- terminus. PKs are expressed in a wide range of peripheral tissues, including the nervous, immune, and cardiovascular systems, as well as in the steroidogenic glands, gastrointestinal tract, and bone marrow [3,4,5,6]. PKs serve as the cognate ligands for two highly similar G- protein-coupled receptors (GPCRs) termed PKs receptor subtypes 1 and 2 (hPKR1 and hPKR2 in humans) [5,7,8]. These receptors are characterized by seven membrane-spanning a-helical segments separated by alternating intracellular and extracellular loop regions. The two subtypes are unique members of family A GPCRs in terms of subtype similarity, sharing 85% sequence identity – a particularly high value among known GPCRs. For example, the sequence identity between the b1 and b2-adrenergic receptor subtypes, which are well established drug targets, is 57%. Most sequence variation between the hPKR subtypes is concen- trated in the extracellular N terminal region, which contains a nine-residue insert in hPKR1 compared with hPKR2, as well as in the second intracellular loop (ICL2) and in the C terminal tail (Figure 1). PKR1 is mainly expressed in peripheral tissues, such as the endocrine organs and reproductive system, the gastrointestinal tract, lungs, and the circulatory system [8,9], whereas PKR2, which is also expressed in peripheral endocrine organs [8], is the main subtype in the central nervous system. Interestingly, PKR1 is expressed in endothelial cells of large vessels while PKR2 is strongly expressed in fenestrated endothelial cells of the heart and corpus luteum [10,11]. Expression analysis of PKRs in heteroge- PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27990