Review 28 March 2024
* Author to whom correspondence should be addressed.
Pain affects one-third of the global population and is a significant public health issue. The use of opioid drugs, which are the strongest painkillers, is associated with several side effects, such as tolerance, addiction, overdose, and even death. An increasing demand for novel, safer analgesic agents is a driving force for exploring natural sources of bioactive peptides with antinociceptive activity. Since the G protein-coupled receptors (GPCRs) play a crucial role in pain modulation, the discovery of new peptide ligands for GPCRs is a significant challenge for novel drug development. The aim of this review is to present peptides of human and animal origin with antinociceptive potential and to show the possibilities of their modification, as well as the design of novel structures. The study presents the current knowledge on structure-activity relationship in the design of peptide-based biomimetic compounds, the modification strategies directed at increasing the antinociceptive activity, and improvement of metabolic stability and pharmacodynamic profile. The procedures employed in prolonged drug delivery of emerging compounds are also discussed. The work summarizes the conditions leading to the development of potential morphine replacements.
Pain treatment was one of humanity’s main problems even in ancient times. The pain-relieving properties of opium, the poppy plant (Papaver somniferum) extract, have been known for centuries. Even today, the most effective painkiller in the treatment of severe and chronic pain is morphine, an alkaloid obtained from opium. Morphine acts through opioid receptors, localized in the central nervous system (CNS) and many peripheral tissues [1]. There are three main types of opioid receptors designated µ, δ, and κ (or MOR, DOR, and KOR, respectively). These receptors, called classical opioid receptors, were discovered in the 1970s, and they all mediate analgesia in humans as well as in animal models of pain [2,3]. The strongest antinociceptive effect is associated with the activation of the MOR, which plays the main role in the signal transduction cascades responsible for pain perception. Morphine binds to all three types of opioid receptors, but its affinity to MOR is about 100 times greater than that of DOR and KOR [4]. Unfortunately, morphine treatment, especially chronic, causes serious side effects, including sedation, tolerance and dependence, constipation, respiratory depression, and hypotension [5].
The fourth member of the opioid receptor family is the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), previously referred to as opioid-receptor-like1 (ORL1), identified in 1994 [6]. On the basis of its structural similarity to the classical opioid receptors, the NOP receptor was classified as belonging to the opioid receptor family despite its unique pharmacological profile [7].
Opioid receptors are members of the G protein-coupled receptors (GPCRs) family and are coupled with G i/G o proteins. GPCR activation leads to the modulation of various intracellular signaling partners, including adenylyl cyclase, phospholipase C, ion channels, and components of the mitogen-activated protein kinase (MAPK) pathway [8]. GPCR stimulation inhibits the activity of adenylyl cyclase and cAMP production, triggering the modulation of synaptic plasticity, pain processing, and memory and reward processing [9,10]. The opening of inwardly rectifying K+ channels and inhibition of voltage-gated dependent Ca 2+ channels (VDCCs) cause intracellular hyperpolarization of the neuron and a reduction in the release of presynaptic neurotransmitters such as glutamate and substance P, which are vital in the transmission of pain. Thus, the activation of opioid receptors creates a strong analgesic effect [9].
The discovery of opioid receptors resulted in the search for their endogenous ligands, known as opioid peptides [2,11]. In the 1970s, DOR-selective enkephalins [12], KOR-selective dynorphins [13,14], and non-selective β-endorphins [15] were isolated from the mammalian brain. All these peptides have the same N-terminal amino acid sequence (Tyr-Gly-Gly-Phe) and free carboxylic group at the C-terminus. Peptides with such structure were named “typical” opioid peptides. Much later, in 1997, Zadina and co-workers discovered two tetrapeptides in the bovine brain [16] and human cerebral cortex [17], which showed high affinity and selectivity for MOR. These peptides act through the same opioid receptor as morphine and, therefore, were named endomorphin-1 and endomorphin-2 ([Table 1](https://www.frankenthalerfoundation.org Endomorphins, unlike other opioid peptides, possess a proline residue in position 2 and have amidated N-terminus. Due to these differences, they were named ”atypical” opioid peptides [2].
Table 1. Endogenous mammalian opioid peptides and their selectivities for the opioid receptors.
The endogenous ligand of the NOP receptor was identified in 1995, independently by two groups, and named nociceptin/orphanin FQ (N/OFQ) [18,19]. Nociceptin has no affinity for classical opioid receptors, and MOR and DOR ligands do not bind to the NOP receptor [20]. The main structural difference between nociceptin and other opioid peptides is the presence of Phe instead of Tyr as the N-terminal amino acid. The endogenous ligands of all four opioid receptors are presented in [Table 1](https://www.frankenthalerfoundation.org
Several “atypical” opioid peptides ([Table 2](https://www.frankenthalerfoundation.org have been obtained from body fluids such as milk and blood, including β-casomorphin [21] and its shorter form, morphiceptin [22,23], that are products of enzymatic digestion of β-casein, while hemorphins originate from the blood protein hemoglobin [24]. Another important group of opioid peptides is compounds isolated from the amphibian skin: MOR-selective dermorfin [25], DOR-selective dermenkephalin [26], and deltorphin I and II [27]. Interestingly, all these peptides from amphibians are characterized by a very high affinity for mammalian opioid receptors ([Table 2](https://www.frankenthalerfoundation.org
Table 2. Selected examples of atypical opioid peptides.
Although all three types of classic opioid receptors are involved in analgesic processes, each activation produces different specific effects. Despite strong antinociceptive activity, activation of MOR may lead to respiratory depression, inhibition of intestinal peristalsis, physical addiction, and euphoria. DOR agonists have reduced addictive potential but also lower antinociceptive efficacy. KOR ligands are responsible for strong dysphoric effects but can also be viewed as potential analgesics only for peripheral use [3,11,28]. The pharmacological profile of the NOP receptor differs from that of classic opioid receptors. The NOP receptor plays important roles in various physiological functions, most importantly in learning and memory, locomotion, and anxiety. Antinociceptive effects mediated via the NOP receptor are complex; activation via N/OFQ was shown to produce either anti- or pro-nociceptive effects, depending on the animal species, dosage, route of administration, as well as the pain model [29,30].
Since the endogenous opioid system is known to play an essential role in pain perception and modulation, opioid receptors and their ligands have been important targets in medicinal chemistry. Numerous opioid peptides and their synthetic analogs have been studied extensively in order to determine their structure–activity relationship (SAR) and also in the hope of finding structural leads for novel analgesics safer than morphine [11,31,32,33].
The opioid system, discussed in detail above, has an indisputable and long history in the treatment of pain [8]. However, in addition to the opioidergic system, there are several other systems, such as neurotensin, cannabinoid, neurokinin, and melanocortin, as well as varieties of neurotransmitters involved in the modulation of pain perception. The major types of pain-associated neurotransmitters (inflammatory mediators: prostaglandins PGE 2 and PGI 2, leukotriene B 4 (LTB 4), nerve growth factor (NGF), proton, bradykinin, ATP, adenosine, substance P, neurokinins A and B, 5-hydroxytryptamine (5-HT), histamine, glutamate, norepinephrine and NO; non-inflammatory mediators: calcitonin gene-related peptide (CGRP), GABA, glycine and cannabinoids), their cognate receptors (either pre- or post-synaptic) and eventual pharmacological effects on pain regulation are discussed in excellent review by Yam et al. [34].
Due to their evolutionary role, various natural compounds are regular ligands of receptors involved in physiological functions, including pain modulation, and, at the same time, they are inspirations for the design and development of novel bioactive compounds. Among them, nature-derived peptides, mostly GPCR ligands, are frequently studied because of their selectivity and the accumulated knowledge on peptide design and synthesis for specific purposes. The well-known metabolism and vast possibilities for derivatization make peptides one of the favorite starting points in the search for drug candidates [35].
This review aims to present natural sources of potential analgesic peptides and methods of modification, as well as the design of novel structures with improved bioavailability and pharmacodynamic properties. We will discuss antinociceptive peptides from the animal kingdom and the strategies employed in the transformation of their sequences into more drug-like molecules.
Pain affects one-third of the global population, and it is a public health issue. Currently, opioid drugs are associated with several disorders, such as tolerance, addiction, overdose, and sometimes death [36]. Problems related to the use of opioid analgesics in patients have prompted scientists to search for alternative drugs with new structures and improved properties.
The exploration of alternative therapeutic options has led researchers to delve into the world of animal peptides [37,38,39]. Many creatures in the animal kingdom produce peptides as a part of their defense mechanisms, signaling systems, or for various physiological functions. In recent years, scientists have been studying these peptides for their potential therapeutic benefits, including their ability to alleviate pain.
Animal peptides exhibit antinociceptive properties by interacting with the body’s pain-signaling pathways. They can modulate pain perception through various mechanisms, including interaction with receptors involved in pain transmission, inhibition of inflammatory processes, and modulation of neuronal excitability [39]. Understanding the specific mechanisms through which animal peptides exert their antinociceptive effects is crucial for developing targeted and effective pain management strategies.
One of the remarkable aspects of animal peptides is the diversity in their sources and structures. Venomous creatures, such as snakes, spiders [40,41], and cone snails, are well-known for producing peptides with potent bioactivities, including antinociceptive effects [42]. Additionally, peptides derived from insects or the skin secretions of amphibians have also shown promise in pain modulation.
Among the venomous animals, snails are an important source of peptides with different biological activities. Several peptides with antinociceptive activity are characterized by the venoms of various snails [43,44,45]. The very interesting molecules with antinociceptive activity are conotoxins ([Table 3](https://www.frankenthalerfoundation.org These peptides from cone snails are potent and highly selective blockers or modulators of ion channel functions [46].
Table 3. Selected peptides from sea snails.
For example, α-conotoxin RgIA, the peptide containing 13 amino acid residues with two intramolecular disulfide bonds ([Table 3](https://www.frankenthalerfoundation.org was isolated from Conus regius [46]. It has been reported that in rat models of neuropathy, RgIA is a potent pain-relieving compound. Its effects are associated with modulating N-type Ca v 2.2 VGCCs. Furthermore, the structure–activity relationship (SAR) studies led to the development of a new analog of RgIA (RgIA4—H-Gly-Cys-Cys-Thr-Asp-Pro-Arg-Cys-Cit-Tyr(3-I)-Gln-Cys-Tyr-OH) with high potency in humans and rodents [46].
Liu et al., described the strong antinociceptive activity of α-conopeptide, Eu1.6 from Conus eburneus. Eu1.6 exhibited antinociceptive activity in rat partial sciatic nerve injury and chronic constriction injury models, and its activity was more potent than that of a combination of morphine and gabapentin [47].
Other important classes of conotoxin peptides are the ω-conotoxins that block calcium channels [43]. The ω-conotoxin GVIA (27 amino acids peptide with three disulfide bonds) is the first peptide isolated from C. geographus [43]. The ω-conotoxins have also been isolated from many other species, such as C. magus and C. catus, forming a family of peptides 24–30 residues long and containing three intramolecular disulfide bonds ([Table 3](https://www.frankenthalerfoundation.org The ω-conotoxin GVIA is a selective blocker of N-type voltage gated Ca v (VGCCs) channels.
The most extensively analyzed ω-conotoxin is ω-conotoxin MVIIA ([Table 3](https://www.frankenthalerfoundation.org from C. magus [43]. This peptide specifically blocks N-type Ca v channels (Ca v 2.2) and inhibits K+-induced [3 H]-γ-aminobutyric acid (GABA) release in the hippocampus in vivo. This conotoxin has been approved by the Food and Drug Administration (FDA) as a non-opioid analgesic peptide against long-term neuropathic pain in humans under the commercial name of ziconotide or Pria
