Figure 9. Docking of potential PKR binders identified through VLS, to the hPKR1 binding site. The proposed docked conforma- tions of (A) Indinavir, (B) Argatroban, and (C) Lapatinib are shown. The ligands are denoted by yellow sticks. Interacting receptor residues are denoted by gray sticks and labeled. Hydrogen bonds are denoted by dashed green lines, and p-cation interactions are denoted by orange lines. doi:10.1371/journal.pone.0027990.g009 ligands in structure-based virtual screening campaigns [57,70], and to rationalize specificity profiles of GPCR antagonists [71] and thus was the approach taken here. In several non-GPCR cases, good docking results have been reported using multiple receptor conformations [72]. Such an Small-Molecule Binders of Prokineticin Receptors approach was successful for a sequence identity range of 30–60% between models and available templates [73]. Though GPCR homology models typically have a lower sequence identity to their potential templates, using ensembles of multiple homology models or of a perturbed X-ray structure may nevertheless be a viable approach, as was recently reported [74,75,76]. Current breakthroughs in X-ray structure determina- tion of GPCRs will enable systematic testing of the most appropriate receptor structure representation and of docking performance, against the benchmark of experimental structures. Identification of potential novel hPKR binders Our study used SAR of known hPKR binders to identify novel potential binders of hPKR1, and highlighted possible ’off-target’ effects of FDA-approved drugs. Interestingly, the novel candidates share little structural chemical similarity with the known hPKR binders but share the same pharmacophores and similar putative interactions within the TM-bundle binding site. Such a "scaffold hopping" result is common and is often sought after in drug discovery. The term is based on the assumption that the same desired biological activity may be achieved by different molecules that maintain some of the essential chemical features as the template molecule, i.e., the molecule possesses the desired biological activity on the target, but is structurally dissimilar otherwise. Scaffold hopping is required, for instance, when the central scaffold is involved in specific interactions with the target, and changing it may lead to improved binding affinity. One example of successful scaffold hopping, resulting in a structurally diverse structure, is the selective D2 and D3 dopamine receptor agonist Quinpirole [77]. The newly identified potential cross-reactivity may have two implications – it might explain the side effects of these drugs (as discussed next), and it might also suggest novel roles for these drugs as potential hPKR inhibitors. One such example of potential cross-reactivity identified through our VLS procedure is Indinavir. Indinavir sulfate is a hydroxyaminopentane amide and a potent and specific FDA-approved inhibitor of the HIV protease. Indinavir acts as a competitive inhibitor, binding to the active site of the enzyme, since it contains a hydroxyethylene scaffold that mimics the normal peptide linkage (cleaved by the HIV protease) but which itself cannot be cleaved. Thus, the HIV protease cannot perform its normal function - proteolytic processing of precursor viral proteins into mature viral proteins. Specific adverse effects associated with Indinavir include hyperbilirubinaemia and cuta- neous toxicities [78,79], accelerated atherosclerosis, and an increased rate of cardiovascular disease [80]. Protease inhibitors may cause cardiovascular disease by inducing insulin resistance, dyslipidemia, or by endothelial dysfunction. A study of the effects of HIV protease inhibitors on endothelial function showed that in healthy HIV-negative subjects, Indinavir induced impaired endothelium-dependent vasodilation after 4 weeks of treatment owing to reduced nitric oxide (NO) production/release by the endothelial cells or reduced NO bioavailability [81]. HIV patients treated with Indinavir presented lower urinary excretion of the NO metabolite NO3 [82]. Wang et al. demonstrated that Indinavir, at a clinical plasma concen- tration, can cause endothelial dysfunction through eNOS (endothelial nitric oxide synthase) down-regulation in porcine pulmonary artery rings and HPAECs (human pulmonary arterial endothelial cells), and that endothelium-dependent relaxation of the vessel rings was also reduced following Indinavir treatment [83]. Endothelium-derived NO is the principal vasoactive factor that is produced by eNOS. Lin et al. showed that PK1 induced eNOS PLoS ONE | www.plosone.org 13 November 2011 | Volume 6 | Issue 11 | e27990