Modeling of human prokineticin receptors: interactions with novel small-molecule binders and potential off-target drugs

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.

Figure 1. Snake plot of hPKR1.

The secondary structure is according to hPKR1 protein annotation in the UniProtKB database (entry Q8TCW9). Positions in the hPKR1 sequence differing from hPKR2 (entry Q8NFJ6) are shaded black. Conserved positions between the two subtypes are shaded white. A nine-residue hPKR1-unique insert in the N terminus is shaded gray with dashed lines. The seven transmembrane domains are denoted by roman numerals. Extracellular and intracellular sides of the membrane are labeled, as well as the N terminus (NH₂) and C terminus (COOH) ends of the protein.

Figure 2. SAR analysis of small-molecule PKR antagonists identifies activity-determining chemical groups.

The four variable positions in the scaffold – A1, D, L2, and Q, were compared in a dataset composed of 56 active compounds (IC₅₀<0.05 µM) and 51 inactive compounds (IC₅₀>1 µM) to determine the required chemical features at each position that elicit activity (a representative set is shown). These features are indicated in dashed boxes for each position. HB - hydrogen bond.

Figure 3. Ligand-based pharmacophore models recapture the known binders.

(A) ligand-based four-feature pharmacophores used for virtual screening, with mapping of a known active small-molecule antagonist used for constructing the pharmacophores. The pharmacophores are represented as tolerance spheres with directional vectors where applicable. Green spheres represent hydrogen bond acceptors, red - positive ionizable, light blue – hydrophobic, and orange - aromatic ring. (B) ROC curve demonstrating the enrichment achieved following ligand-based pharmacophore mapping of 56 known active PKR antagonists and 5909 random molecules obtained from the ZINC database. Known actives are significantly enriched by both pharmacophore hypotheses.

Figure 4. Final hits retrieved from virtual screening.

Figure 5. Homology model of hPKR1.

The model is viewed perpendicular to the plasma membrane, with the extracellular side of the receptor shown on top, and the intracellular side shown on the bottom of the figure. The structure is colored from the N (blue) to the C (orange) terminal amino acid sequence. The insert shows the 7TM-bundle allosteric small-molecule binding site, predicted by the QSite Finder server. The binding site is located among TMs 3,4,5,6, and 7.

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 µM), and two inactive compounds (C, D, IC₅₀>1 µM) 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 π-cation or π-π interactions are denoted by orange lines.

Figure 7. Clustering of hydrogen bond, charged, and π 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 π interactions (either π-cation or π- π 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.

Figure 8. The essential activity-determining groups of the known binders form specific interactions with the receptor.

(A) An example of the ligand's chemical properties, which are important for its activity, identified in the SAR analysis, and how they interact with receptor residues. An active molecule is shown docked into the hPKR1 binding site. The activity-related chemical groups are indicated in red. The ligand is denoted by yellow sticks. Interacting receptor residues are shown as green sticks and labeled. Hydrogen bonds are denoted by dashed green lines, π-cation interactions are denoted by orange lines. (B) Schematic 2D representation of ligand-receptor interactions. The residues shown have at least one atom within 4Å of the ligand. Blue lines indicate hydrogen bonds and orange lines indicate hydrophobic interactions. Residues shaded in green are involved in van der Waals interactions. Residues involved in hydrogen bonds, charge, or polar interactions are shaded in cyan.

Figure 9. Docking of potential PKR binders identified through VLS, to the hPKR1 binding site.

The proposed docked conformations 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 π-cation interactions are denoted by orange lines.