Modeling of Human Prokineticin Receptors: Interactions with Novel Small-Molecule Binders and Potential Off-Target Drugs

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.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

Chemogenomic Analysis of G-Protein Coupled Receptors and Their Ligands Deciphers Locks and Keys Governing Diverse Aspects of Signalling

Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs) is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI) is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic) or inhibitory (antagonistic) ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs) 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences

Residues from transmembrane helices 3 and 5 participate in leukotriene B-4 binding to BLT1

Leukotrienes are inflammatory mediators that bind to seven transmembrane, G-protein-coupled receptors (GPCRs). Here we examine residues from transmembrane helices 3 and 5 of the leukotriene B-4 (LTB4) receptor BLT1 to elucidate how these residues are involved in ligand binding. We have selected these residues on the basis of (1) amino acid sequence analysis, (2) receptor binding and activation studies with a variety of leukotriene-like ligands and recombinant BLT1 receptors, (3) previously published recombinant BLT1 mutants, and (4) a computed model of the active structure of the BLT1 receptor. We propose that LTB4 binds with the polar carboxylate group of LTB4 near the extracellular surface of BLT1 and with the hydrophobic LTB4 tail pointing into the transmembrane regions of the receptor protein. The carboxylate group and the two hydroxyls of LTB4 interact with Arg178 and Glu185 in transmembrane helix 5. Residues from transmembrane helix 3, Val 105 and Ile 108, also line the pocket deeper inside the receptor. LTB4 is becoming increasingly important as an immunomodulator during a number of pathologies, including atherosclerosis. Detailed information about the LTB4 binding mechanism, and the receptor residues involved, will hopefully aid in the design of new immunomodulatory drugs

Structure of Fab hGR-2 F6, a competitive antagonist of the glucagon receptor.

The monoclonal antibody hGR-2 F6 has been raised against the human glucagon receptor and shown to act as a competitive antagonist. As a first step in the structural characterization of the receptor, the crystal structure of the Fab fragment from this antibody is reported at 2.1 A resolution. The hGR-2 F6 Fab crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 76.14, b = 133.74, c = 37.46 A. A model generated by homology modelling was used as an aid in the chain-tracing and the Fab fragment structure was subsequently refined (final R factor = 21.7%). The structure obtained exhibits the typical immunoglobulin fold. Complementarity-determining regions (CDRs) L1, L2, L3, H1 and H2 could be superposed onto standard canonical CDR loops. The H3 loop could be classified according to recently published rules regarding loop length, sequence and conformation. This loop is 14 residues long, with an approximate beta-hairpin geometry, which is distorted somewhat by the presence of two trans proline residues at the beginning of the loop. It is expected that this H3 loop will facilitate the design of synthetic probes for the glucagon receptor that may be used to investigate receptor activity