Mechanistic Characterization of the Pharmacological Profile of HS-731, a Peripherally Acting Opioid Analgesic, at the µ-, δ-, κ-Opioid and Nociceptin Receptors

Abstract

Accumulated preclinical and clinical data show that peripheral restricted opioids provide pain relief with reduced side effects. The peripherally acting opioid analgesic HS-731 is a potent dual μ-/δ-opioid receptor (MOR/DOR) full agonist, and a weak, partial agonist at the κ-opioid receptor (KOR). However, its binding mode at the opioid receptors remains elusive. Here, we present a comprehensive in silico evaluation of HS-731 binding at all opioid receptors. We provide insights into dynamic interaction patterns explaining the different binding and activity of HS-731 on the opioid receptors. For this purpose, we conducted docking, performed molecular dynamics (MD) simulations and generated dynamic pharmacophores (dynophores). Our results highlight two residues important for HS-731 recognition at the classical opioid receptors (MOR, DOR and KOR), particular the conserved residue 5.39 (K) and the non-conserved residue 6.58 (MOR: K, DOR: W and KOR: E). Furthermore, we assume a salt bridge between the transmembrane helices (TM) 5 and 6 via K227^5.39 and E297^6.58 to be responsible for the partial agonism of HS-731 at the KOR. Additionally, we experimentally demonstrated the absence of affinity of HS-731 to the nociceptin/orphanin FQ peptide (NOP) receptor. We consider the morphinan phenol Y130^3.33 responsible for this affinity lack. Y130^3.33 points deep into the NOP receptor binding pocket preventing HS-731 binding to the orthosteric binding pocket. These findings provide significant structural insights into HS-731 interaction pattern with the opioid receptors that are important for understanding the pharmacology of this peripheral opioid analgesic.

Keywords: GPCR; HS-731; analgesia; binding; molecular docking; molecular dynamics simulations; opioid receptor; peripheral opioid agonist; selectivity.

Figure 1

Structure of HS-731 and the acid-base equilibrium under physiological conditions.

Figure 2

Binding curves of HS-731 to the human NOP receptor determined in the competitive radioligand binding assay. Concentration-dependent inhibition by HS-731 and nociceptin of [³H]nociceptin binding to membranes from CHO cells stably expressing the human NOP receptor. Values are means ± SEM (n = 3 independent experiments performed in duplicate).

Figure 3

Ramachandran plot of the NOP receptor homology model. Angles within the orange range angles are plausible (yellow spheres) and angles within the green space are optimal (green spheres).

Figure 4

Chemical structures of (A) a morphinan scaffold and co-crystallized ligands (B) BU72 in MOR (PDB-ID: 5C1M) and (C) MP1104 in KOR (PDB-ID: 6B73) under physiological pH (7.4).

Figure 5

Binding modes of HS-731 to the classical opioid receptors. (A) Global view on the MOR binding pocket with docked HS-731. (B) Binding pocket of the MOR. Residues 297–303 and 322–325 are not shown for better visualization. (C) Binding pocket of the KOR. Residues 289–294 and 311–318 are not shown for better visualization. (D) Binding pocket of the DOR. Residues 275–282 are not shown for better visualization. Blue star indicates positive ionizable interactions, red stars negative ionizable interactions, yellow spheres lipophilic contacts, green arrow hydrogen bond donors and red arrows hydrogen bond acceptors. Water molecules are depicted as red spheres.

Figure 6

Superimposition of the NOP receptor and the classical opioid receptors in complex with HS-731. Atom clash is indicated by the bold black line.

Figure 7

Ionic interaction distances. (A) Distances between K235^5.39 (Nz) or K305^6.58 (Nz) in the MOR and the carboxylate of HS-731. (B) Distances between K227^5.39 (KOR, Nz) or K214^5.39 (DOR, Nz) and the carboxylate moiety of HS-731. Dashed lines represent quantile. The width of the plot corresponds to the frequency of the measured distance.

Figure 8

TM6 translocation. Measurement between the alpha carbon atoms of 6.31 at the bottom of TM6 (MOR: R278^6.31, DOR: R257^6.31, KOR: R270^6.31) and 4.40 at the bottom of TM4 (MOR: R184^4.40, DOR: A163^4.40, KOR: L173^4.40) over the simulation time. The width of the plot corresponds to the frequency of the measured distance. Dashed black lines represent quantile. Yellow solid lines indicate the analog measured distances at the active state crystal structures (PDB-ID: 5C1M for MOR, 6PT2 for DOR and 6B73 for KOR) and inactive state crystal structures (PDB-ID: 4DKL for MOR, 4N6H for DOR and 4DJH for KOR).