Sars Of Enkephalins

Opioid Receptor Discovery and Endogenous Ligands

There was no direct evidence for the existence of specific opioid receptors until the 1970s when Goldstein et al.20 found that radiolabeled levorphanol bound stereospecifi-cally to certain mouse brain fractions. They hypothesized that this compound bound to an "opiate receptor." This prediction gained credence in 1973 when additional studies showed that opioid agonists and opioid antagonists compete for the same binding site. Building on these studies, Pert21 was able to show that the pharmacologic potencies of the opioid drugs were proportional to their ability to compete with the antagonist for opioid receptor binding.

As it seemed unlikely that these receptors evolved in response to a plant alkaloid, the search for endogenous ligands was intense. The discovery and identification of endogenous substrates for the opioid receptor by Hughes22,23 in 1975 further confirmed the existence of an opioid receptor and intensified the SAR studies of the analgesic opioids. The endogenous peptide ligands for the opioid receptors were originally isolated from pig brains but have been found in every mammal studied. The first

WHO 3-Step Analgesic Ladder - Is the pain...




WHO 3-Step Analgesic Ladder - Is the pain..."/>
Figure 24.1 • Algorithm for pain management based on the WHO three-Step Analgesic Ladder. (Source:

Met-enkephalin = Tyr1-Gly2"Gly3-Phe4-Met5(OH) Leu-enkephalin = Tyr1-Gly2-Gly3-Phe4-Leu5(OH)

-Met5 -Leu5

endogenous peptide was termed enkephalin, which was found to be a mixture of the two pentapeptides that only differ in their terminal amino acid.

Both methionine-enkephalin (Met-enkephalin) and leucine-enkephalin (Leu-enkephalin) were shown to inhibit the contraction of electrically stimulated guinea pig ileum (GPI) and mouse vas deferens (MVD). These two tests are still used as screening methods for opioid activity. Naloxone completely reversed the inhibitory effects of enkephalin, which led Hughes to infer that the peptides were acting on an opioid receptor.22 The central administration of enkephalins in rats produced short analgesic activity. The transient nature of the enkephalins' actions correlated with the rapid degradation of the enkephalin Tyr-Gly bond by aminopeptidases. Much synthetic work has been done in an attempt to increase the duration of action of the opioid peptides and maintain their analgesic effect.

SARs of Enkephalins


The first amino acid of the pentapeptide shows a distinct preference for tyrosine. Most changes to this amino acid, either by substituting with other amino acids or masking the phenolic hydroxyl (OH) or amino function, produce an inactive or weakly active peptide.


Replacing the naturally occurring l-Gly with various d-amino acids produces a peptide that is resistant to peptide cleavage by aminopeptidases. Replacement with d-Ser is the most effective replacement, and all l-amino acid analogs had low activities. Substituting d-amino acids for l-amino acids produces stable peptides, and the stereochemical change may give the peptide access to additional binding sites on the receptors; both of these actions may explain their increased potencies. Replacing the Gly2 with d-Ala2 while simultaneously replacing the l-Leu5 with d-Leu5 produces the peptide known as d-Ala2, d-Leu5 enkephalin (DADLE), which is commonly used as a selective S-agonist.


Almost all changes to this amino acid result in a drop in potency, unless they are also accompanied by another change such as replacing the Gly2 with d-Ala2 as described above.


The aromatic nature of the fourth residue is required for high activity. When combined with the d-Ala2 replacement, the addition of an electron withdrawing, lipophilic substituent (e.g.,

NO2, Cl, and Br) in the para position of Phe4 greatly increases activity. Para substitutions with electron donating, hydrophilic functional groups (e.g., NH2 and OH) abolish activity.


Position 5 appears to tolerate more residue changes than the other positions. Many amino acid substitutions at this position maintain activity (e.g., Ala, Gly, Phe, Pro). Even the loss of the fifth residue to yield the tetrapeptide Tyr:-Gly2-Gly3-Phe4 maintains weak activity in both the GPI and MVD assays. The protected peptide, Tyr:-D-Ala2-Gly3-MePhe4-Gly-ol5, known as DAMGO is highly selective for the /-receptor.

The pituitary gland, hypothalamus, and the adrenal medulla all produce various opioid peptides. Many of these materials were found to be fragments of j-lipotropin (fi-LPT) a 91 amino acid peptide. j-LPT has no opioid activity itself. The fraction containing amino acids 61-91 is designated S-endorphin (a word derived from combining endogenous and morphine). It is much more potent than the enkephalins in both in vivo and in vitro tests. The search for opioid peptides continued, and additional precursor proteins were discovered. Many additional precursor proteins synthesized in the pituitary, adrenal glands, and brain nerve terminals were found to contain sequences of amino acids that were enkephalins or other active opioid peptides. It appears that naturally occurring peptides may serve roles as both short-acting analgesics and long-term neuronal or endocrine modulators. Acupuncture, running, or other physical activity may induce the release of neuropeptides although studies exist that both confirm and refute these claims.24,25 A complete discussion on the neuropeptides is beyond the scope of this chapter. Some examples of opioid peptides are given in Figure 24.2.26-29

Opioid Receptors

The discovery of the endogenous opioid peptides paralleled the development of radiolabeling techniques. During the 1970s, researchers were able to label opioid receptors with reversible radioactive ligands that allowed pharmacological actions of specific receptors and their locations to be identified. These techniques allowed for the identification of opi-oid receptor locations in the brain. Additional pharmacological advances in gene expression studies led to the identification of peripheral opioid receptor locations.30 Opioid receptors are distributed throughout the brain, spinal cord, and peripheral tissues. The distribution of specific opi-oid receptor subtypes (/, S, and k) usually overlaps. In rats, high concentrations of all three genes for the /-, S-, and K-receptors were found in the hypothalamus and cerebral

Endogenous Precursor Proteins

Pro-opimelanocortin —» ACTH + p LPH —» ^-LPH + p endorphin Pro-enkephalin A263 —» 4Met-enkephalin + Leu-enkephalin

Pro-enkephalin B256 —» p neo-endorphin175-183 + dynorphin209-225 + Leu-enkephalin228-232

Endogenous Opioid Peptide sequences p Endorphin = Tyr-Gly-Gly-Phe-Met5-Thr-Ser-Glu-Lys-Ser10-Gln-Thr-Pro-Leu-Val15-Thr-Leu-Phe-Lys-Asn20-Ala-Ile-Ile-Lys-Asn25-Ala-Tyr-Lys-Lys-Gly-Glu31 = 8 and ^ opioid receptor ligand Endomorphin-1 = Tyr-Pro-Trp-Phe-NH2 = ^ receptor agonist Endomorphin-2 = Tyr-Pro-Phe-Phe-NH2 = ^ receptor agonist

Dynorphin = Tyr-Gly-Gly-Phe-Leu5-Arg-Arg-Ile-Arg-Pro10-Lys-Leu-Lys13 = k opioid selectivity a-Neoendorphin = Tyr-Gly-Gly-Phe-Leu5-Arg-Lys-Tyr-Pro-Lys10 p-Neoendorphin = Tyr-Gly-Gly-Phe-Leu5-Arg-Lys-Tyr-Pro9

Nociceptin = Phe-Gly-Gly-Thr-Gly5-Ala-Arg-Lys-Ser-Ala10-Lys-Ala-Asn-Gln14 = orphanin receptor Orphanin FQ = Phe-Gly-Gly-Phe-Thr5-Gly-Ala-Arg-Lys-Ser10-Ala-Arg-Lys-Leu-Ala-Asn-Gln17

Exogenous Opioid Peptide sequences "Exorphins" DADLE = Tyr-D-Ala-Gly-Phe-D-Leu = 8 selective agonist DPDPE = Tyr-D-Pen-Gly-Phe-D-Pen = 8 selective agonist DSLET = Tyr-D-Ser-Gly-Phe-Leu-Thr = 8 selective agonist Casomorphin (cow's milk ^ opioid receptor agonist) = Tyr-Pro-Phe-Pro-Gly-Pro-Ile7 Dermorphin (South American frog skin ^ opioid receptor agonist) = Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser7-NH2 Gluten exorphins (multiple peptides from wheat having opioid agonist and antagonist activity) Figure 24.2 • Endogenous and exogenous opioid peptides.

cortex. Intermediate concentrations were found in the small intestine, adrenal gland, testes, ovary, and uterus. Low concentrations of all the three gene transcripts were found in the lung and kidney. The gene for the ^-opioid receptor was not found in the stomach, heart, endothelium, or synovium of the rat.30 Opioid receptors are also found on the peripheral terminals of sensory neurons in inflamed rat paws.31 Thus, the central and peripheral distribution of opioid receptors is complex. This complicates the interpretation of pharmacological data of individual drugs that may have overlapping binding at multiple opioid receptor subtypes. In addition, opioid receptors form homodimers and heterodimers with opioid receptors and nonopioid receptors such as the a2a-adrenergic receptors resulting in different pharmacologic actions and altered coupling to second messengers.32

Genes for the four major opioid receptor subtypes have been cloned; the MOP = ^-receptor (mu for morphine), the KOP = k-receptor (kappa for ketocyclazocine), the DOP = S-receptor (delta for deferens because it was originally discovered in the MVD), and the NOP = nocicep-tion/orphanin FQ receptor (named as an orphan receptor because the endogenous/exogenous ligand was unknown at its time of discovery).33 The opioid receptor subtypes share extensive residue homology in their transmembrane (TM) domains with most of the variation found in the extracellular loops (Fig. 24.3).33 All of the opioid receptors belong to the G-protein-coupled receptor class and as such, they are composed of seven TM domains. When the receptor is activated, a portion of the G protein diffuses within the membrane and causes an inhibition of adenylate cyclase activity. The decreased enzyme activity results in a decrease in cyclic adenosine monophosphate (cAMP) formation, which regulates numerous cellular processes. One process that is inhibited is the opening of voltage-gated calcium influx channels on nociceptive C-fibers. This results in the hyperpolarazation of the nerve cell and decreased firing and release of pain neurotransmitters such as glutamate and substance P.34

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