Hemorphins are short atypical opioid peptide fragments embedded in the β-chain of hemoglobin. They have received considerable attention recently due to their interaction with opioid receptors. The affinity of hemorphins to opioid receptors μ-opioid receptor (MOR), δ-opioid receptor (DOR), and κ-opioid receptor (KOR) has been well established. However, the underlying binding mode and molecular interactions of hemorphins in opioid receptors remain largely unknown. Here, we report the pattern of interaction of camel and other mammalian hemorphins with DOR. Extensive in silico docking and molecular dynamics simulations were employed to identify intermolecular interactions and binding energies were calculated to determine the affinity of these peptides for DOR. Longer forms of hemorphins - hemorphin-7, hemorphin-6, camel hemorphin-7, and camel hemorphin-6 had strong interactions with DOR. However, camel hemorphin-7 and camel hemorphin-6 had high binding affinity towards DOR. Thus, the findings of this study provide molecular insights into how hemorphins, particularly camel hemorphin variants, could be a therapeutic agent for pain regulation, stress management, and analgesia.
Hemorphins are a group of endogenous opioid peptides derived from the β-chain of hemoglobin. These bioactive peptides have received considerable attention due to their potential therapeutic effects on spatial learning, analgesia, inflammation and antihypertension. Opioid receptors are G protein-coupled receptors (GPCR) characterized by the presence of the archetypical seven transmembrane helices. The μ-opioid receptor (MOR) is the primary target of opioid analgesics while δ-opioid receptor (DOR) and κ-opioid receptor (KOR) are also involved in the regulation of pain and analgesia. The affinity of hemorphins to opioid receptors plays a crucial role in regulating pain perception.
Hemorphins peptides vary in size from 4 to 10 amino acids. Hemorphin peptides share a core tetrapeptide sequence of tyrosine-proline-tryptophan-threonine (YPWT). Hemorphin-4 (hem-4) was the first hemorphin characterized from bovine blood. Variations of N-terminal and C-terminal extensions of the core sequence have been isolated from human and bovine tissues. Various forms of hemorphin peptides including, hemorphin-4, hemorphin-6, hemorphin-7, LVV-hemorphin-6, LVV-hemorphin-7, and VV-hemorphin-7 showed partial to full binding in a competitive manner with the endogenous opioid-related peptides such as enkephalins and dynorphins in radioligand experiments. Different forms of hemorphins including synthetic and purified hemorphin peptides from human and bovine blood and brain tissues show varying affinities for opioid receptors.
In humans, MOR, DOR, and KOR share around 60% sequence identity indicating a conserved structure supporting its functional roles. Hence, a hypothesis worth testing is whether hemorphins could bind to DOR and KOR in a manner similar to MOR. The interaction of hemorphins with μ-opioid receptor (MOR) has been well studied. Hemorphin-4 and hemorphin-5 have been shown to inhibit nociception by acting on MOR. Furthermore, VV-hemorphin-7 and LVV-hemorphin-7 were also shown to bind with the same potency as hemorphin-4 and hemorphin-5 but with less potency than hemorphin-6 and hemorphin-7. We previously reported the interaction of LVV-hemorphin-7 with MOR. Identification of a similar mode of interaction would suggest that hemorphins could assist with antinociceptive, antidepressant and sedative effects via its action on DOR, which could then be investigated further. The δ-opioid receptor mediates its effects through Gi/o protein signaling, reducing cAMP levels, inhibiting calcium channels, and activating potassium channels, resulting in neuronal inhibition and analgesia. Additional pathways, including PLC activation and β-arrestin signaling, further contribute to its antinociceptive, antidepressant, and sedative effects by modulating neurotransmitter release and synaptic plasticity. As potential DOR agonists, hemorphins can utilize these pathways to produce therapeutic effects, offering a promising route for the development of safer and more effective analgesics and antidepressants.
The hemorphin sequence is well-conserved among mammals, except camels, which uniquely feature a Q > R variation following the shared YPWT sequence. We also studied and reported this single amino acid disparity in camels on several targets using in silico and in vitro techniques. The camel forms of these peptides were reported to exhibit a greater affinity for all the protein targets tested. The primary objective of this study was to identify and understand the molecular mechanisms underlying the interaction of hemorphins of camels and that of other mammals with DOR using in silico docking and molecular dynamics simulations.
The crystal structure of DOR (PDB ID: 6PT2) was obtained from the Protein Data Bank (PDB). The structure was prepared for in silico docking using the protein preparation wizard of Schrödinger suite 2022–2. The protein structure was preprocessed by adding hydrogens, removing unwanted water molecules, assigning proper bond orders, adjusting the ionization state, disoriented group orientation, disulfide bond addition, partial charge assignment, and fixing residues with missing atoms and side chains. The unwanted ligands and chains were removed while the tautomeric states were generated at pH 7.0. Finally, the structure of the proteins were further optimized and minimized by OPLS_2005 force field in order to preserve geometric structural stability.
A receptor grid was generated for site-specific docking by identifying the location of the co-crystallized ligand in the protein structure. OPLS 2001 force field was used to represent the protein. Default parameters were used and the van der Waals radii of the receptor atom were scaled to 1.0 and the partial charge cutoff value was set to 0.25.
Peptide docking was performed to determine the most likely binding orientation of hemorphins within DOR, and to analyze the resulting intermolecular interactions and the binding free energy. Standard precision flexible docking was performed for docking the peptide using Schrödinger Glide version 2022–2 with default parameters. The docked poses were ranked in using the GlideScore (GScore) scoring function. The poses with lowest GScore value were selected for further analysis.
After docking, the best docked poses were taken to analyze the various types of contacts including hydrogen bonds, hydrophobic interactions, salt bridges, π–π stacking and π-cation interactions. The best-docked poses were subjected to molecular mechanics generalized Born surface area (MM-GBSA) calculations to evaluate the binding free energy in an implicit solvent model. MM-GBSA binding energy was calculated using Schrödinger Prime employing the OPLS 2005 force field and the VSGB 2.0 implicit solvent model.
MD simulations of the best docked pose of the top ranked non-camel and camel hemorphins were performed to evaluate the stability and dynamics of the binding conformation of the peptides. MD simulations were carried out using the Desmond simulation package of Schrödinger LLC with the OPLS 2005 force field. 250 nanoseconds (ns) MD simulations were performed in triplicate for the best docked pose of camel and non-camel hemorphins with DOR. The three runs of each of the peptide bound complexes were performed with different initial velocities. The complexes of receptors with the best docked non-camel and camel hemorphins were embedded into a pre-equilibrated DPPC membrane in an orthorhombic box. All systems were solvated with a water box, using SPC water model with a buffer distance of 10 Å. The simulation models were neutralized with the required number of counterions, and the salt concentration was set at 0.15 M NaCl.
The systems were subjected to steepest descent minimization with Desmond’s default protocol prior to performing the MD simulations. All systems were first relaxed using the default relaxation protocol for membrane proteins which consists of eight stages. After relaxation, 250 ns simulation run was performed for each system using the default simulation protocol. The simulations were performed under NPT ensemble and the systems were maintained at a constant temperature of 300 K using the Nose-Hoover thermostat. Isotropic Martyna–Tobias–Klein barostat was used to maintain a pressure of 1 atm.
