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Definition
The enkephalins are two pentapeptides with pharmacological properties similar to narcotic drugs. These peptides are widely distributed in the central nervous system (CNS). There are two fully processed peptides: Met-enkephalin (YGGFM) and Leu-enkephalin (YGGFL), with methionine and leucine as C-terminal amino acid. Other cleavage products such as YGGFMRF, YGGFMRGL and YGGFMRRV-amide (metorphamide) have been described. Met- and Leu-enkephalin have relative preference for the d-opioid receptor, while the C-terminally extended enkephalins have relatively high affinity for all opioid receptors 1.

Related Peptides
The endogenous opioid peptides all contain the enkephalin sequence Tyr-Gly-Gly-Phe-Met and Tyr-Gly-Gly-Phe-Leu at their amino terminus. Three distinct families of these peptides (endorphins, enkephalins and dynorphins) are present in different neuronal pathways within the CNS. Molecular genetics have shown that these three families of opioid peptides are derived from three distinct precursors. Pro-opiomelanocortin gives rise to the endorphins, as well as adrenocorticotropic hormone (ACTH) and the melanotropic hormones (MSH's). Met-enkephalin, Leu-enkephalin and the related heptapeptide Met-enkephalin-Arg6-Phe7 and octapeptide Met- enkephalin-Arg6-Gly7-Leu8 are derived from proenkephalin. The third family is derived from prodynorphin and includes dynorphin A, dynorphin B (also known as rimorphin) and alpha- and beta-neo-endorphin. The structure of the genes coding for these precursors are similar, suggesting the possibility of one common ancestral gene. The most common scheme for enzymatic maturation of precursors proposes the action of a trypsin-like endopeptidase followed by a carboxypeptidase B-like exopeptidase. Endo-oligopeptidase A, an enzyme, known to hydrolyze the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin, has also been shown to produce, by a single cleavage, Leu-enkephalin or Met-enkephalin from small enkephalin-containing peptides 1.


Discovery In 1974 John Hughes and Hans Kosterlitz of Scotland discovered that enkephalins are produced in the brains of vertebrates, serving as a kind of opioid receptor and regulating pain pathways leading from peripheral nerves to the brain 2.

Structural characteristics
The mass spectrometric studies revealed that the natural enkephalin was a mixture of two pentapeptides, i. e. Met-enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) and Leu-enkephalin (H-Try-Gly-Gly-Phe-Leu-OH) in the ratio of 3 or 4 to 1 2. The structures of Met-enkephalin (Tyr-Gly-Gly-Phe-Met) and Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) have been determined from single crystal x-ray diffraction studies. The Met-enkephalin structure consists of dimers forming antiparallel-sheets extending in the monoclinic ac plane with 10.6 water molecules per dimer. The two molecules, related by pseudo two-fold axes, have similar backbone conformations and similar tyrosine and phenylalanine side-chain conformations. Leu-enkephalin crystallizes as a monohydrate that is isomorphous with the Met-enkephalin structure with respect to the ß-sheet but different with respect to the tyrosine and phenylalanine side-chain conformations and water content. The peptide chains in both structures are fully extended and more nearly planar than pleated. The detailed three-dimensional information on three different forms of Leu-enkephalin and one form of Met-enkephalin provide data on minimum energy conformations of these important peptides, which serve as models for the receptor-bound conformation3.

Mode of Action
Enkephalin regulates nociception through opioid receptor mediated signaling pathways. Short ProEnk peptides, such as Leu- and Met-enkephalin are selective for d-opioid receptors, but C-terminally extended peptides such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe have a high affinity to µ, d, ? receptors 4.

Receptor Specific Enkephalins:

1. (D-Ala2,D-Leu5)-Enkephalin / DADLE : H-Tyr-D-Ala-Gly-Phe-D-Leu-OH : DADLE has generally served as the putative, prototypical d-opioid receptor ligand. It is more potent and selective than Leu-enkephalin or Met-enkephalin in the MVD versus GPI bioassay systems and more potent and approximately as selective as Leu-enkephalin in binding assays. It shows roughly the same antinociceptive activity as ß-endorphin. [5-8]

2. (D-Ala2,N-Me-Phe4,methionin(O)-ol5)-Enkephalin / DAMME: H-Tyr-D-Ala-Gly-N-Me-Phe-methionin(O)-ol: DAMME shows only slight preference for µ-receptors over d-receptors and has no significant affinity for K-receptors. DAMME exerts a potent analgesic effect after systemic administration. It is one of the best-known analogs that has undergone fairly extensive clinical testing. [9-13]
3. (D-Ala2,N-Me-Phe4,glycinol5)-Enkephalin / DAMGO or DAGO: H-Tyr-D-Ala-Gly-N-Me-Phe-glycinol : µ-Receptor-specific enkephalin analog.[14, 15]
4. (D-Pen2,Pen5)-Enkephalin / DPLPE: H-Tyr-D-Pen-Gly-Phe-Pen-OH: This enkephalin analog exhibits an increase in d-receptor selectivity by an order of magnitude. [16]
5. (D-Ser2)-Leu-Enkephalin-Thr: H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH: This hexapeptide exhibits a very high specificity for the d-receptor. [17-18]
6. (D-Thr2)-Leu-Enkephalin-Thr: H-Tyr-D-Thr-Gly-Phe-Leu-Thr-OH and (D-Cys(tBu)2,Thr(tBu)6)-Leu-Enkephalin-Thr: H-Tyr-D-Cys(tBu)-Gly-Phe-Leu-Thr(tBu)-OH: Very potent d-receptor-specific enkephalin analogs. [19]

Functions
Enkephalins are opioid peptides that bridge the neuroendocrine and immune systems.
Enkephalins, pentapeptides containing the consensus Tyr-Gly-Gly-Phe-Xaa sequence, are the smallest of the molecules with pain killing or opiate activity. Enkephalins are found in the thalamus of the brain and in some parts of the spinal cord that transmit pain impulses. In the spinal cord, enkephalins inhibit painful sensations by reacting with specific receptor sites on the sensory nerve endings. Nerve endings of the central nervous system (CNS) and the adrenal medulla release these naturally occurring morphine-like substances. Enkephalins bind to opiate receptors and release controlled levels of pain. [1]
Leu-enkephalin is an endogenous agonist for the receptors that are stimulated by opiate alkaloids. It has multiple effects on the CNS, including the neuroendocrine hypothalamus. Leu-enkephalin also controls gonadal function.[20]
Met-enkephalin is involved in phenomena associated with modulated pain perception, regulation of memory and emotional conditions, food and liquid consumption and regulation of immunological system. It also has an impact on the digestive system motility, gastric as well as in pancreatic secretion and metabolism of carbohydrates.[21, 22]

References

1. Rossier J. 1988. [Biosynthesis of opioid peptides]. Ann Endocrinol (Paris). 1988;49(4-5):371-3.
2. S.K. KULKARNI . 1977. Enkephalins : Possible Neurotransmitters? Ind.J. Pharmac. 9 (2) 125-129 (1977).
3. J. F. Griffin, D. A. Langs, G. D. Smith, T. L. Blundell, I. J. Tickle, and S. Bedarkar. 1986. The crystal structures of [Met5]enkephalin and a third form of [Leu5]enkephalin: Observations of a novel pleated 18-sheet (enkephalin conformation/opioid peptides/anti-parallel planar ß-sheet)  Proc. Natl. Acad. Sci, Vol. 83, pp. 3272-3276, May 1986.
4. Mansour A, Hoversten MT, Taylor LP, Watson SJ, Akil H. 1995. The cloned mu, delta and kappa receptors and their endogenous ligands: evidence for two opioid peptide recognition cores. Brain Res. 1995 Nov 27; 700(1-2):89-98.
5. Wei, E. T., Tseng, L. F., Loh, H. H., and Li, C. H .1977. Comparison of the behavioral effects of ß-endorphin and enkephalin analogs. Life Sci., 21: 321-328.
6. Pfeiffer A, Herz A.1982. Different type of opiate agonists inter- act distinguishability with mu, delta and kappa opiates binding sites. Life Sci 31:1355- 1358
7. A.H. Mulder, G. Wardeh, F. Hogenboom, A.L. Frankhuyzen. 1984. Kappa and delta opioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release, Nature 308 (1984) 278– 280.
8. N.A.Sharif and J.Hughes, 1989. Discrete mapping of brain Mu and delta opioid receptors using selective peptides: quantitative autoradiography, species differences and comparison with kappa receptors. Peptides. 1989 May-Jun;10(3):499-522.
9. D.Roemer et al., 1977. A synthetic enkephalin analogue with prolonged parenteral and oral analgesic activity. Nature 268, 547 - 549 (11 August 1977)
10. W.A.Stubbs et al., 1978. Hormonal and metabolic responses to an enkephalin analogue in normal man. Lancet ii (1978), pp. 1225–1227.
11. von Graffenried B, del Pozo E, Roubicek J, Krebs E, Poldinger W, Burmeister P, Kerp L. 1978. Effects of the synthetic enkephalin analogue FK 33-824 in man. Nature. 1978 Apr 20; 272(5655):729–730.
12. B.Allolio et al., 1986. FK 33-824, a Met-Enkephalin Analog, Blocks Corticotropin-Releasing Hormone-Induced Adrenocorticotropin Secretion in Normal Subjects but not in Patients with Cushing’s Disease. J Clin Endocrinol Metab 1986 63: 1427-1431.
13. P.W.Schiller, 1991. Development of receptor-specific opioid peptide analogs. Prog. Med. Chem. 28 (1991), pp. 301–340.
14. Handa, B.K.; Lane, A.C.; Lord, J.A.H.; Morgan, B.A.; Ranee, M.J.; Smith, C.F.C. 1981. Analog of ß-LPH 61–64 possessing selective agonist activity of mu opiate receptors. Eur. J. Pharmacol. 70:531–540(1981).
15. J. Kowalski. 1998. Immunomodulatory action of class µ-, d- and ?-opioid receptor agonists in mice. Neuropeptides 32 (1998), pp. 301–306.
16. H I Mosberg, R Hurst, V J Hruby, K Gee, H I Yamamura, J J Galligan, and T F Burks. 1983. Bis-penicillamine enkephalins possess highly improved specificity toward delta opioid receptors. PNAS October 1, 1983 vol. 80 no. 19 5871-5874.
17. Gacel G, Fournie-Zaluski MC, Roques BP (1980) D-tyr-ser-gly-phe-leu- thr, a highly preferential ligand for 6-opiate receptors. FEBS Letters. 118:245-247.
18. David M, Moisand C, Meunier JC, Morgat JL, Gacel G, Roques BP. 1982.[3H]Tyr-D-Ser-Gly-Phe-Leu-Thr: a specific probe for the delta-opiate receptor subtype in brain membranes. Eur J Pharmacol. 1982 Mar 12;78(3):385–387
19. Zajac JM, Gacel G, Petit F, Dodey P, Rossignol P, Roques BP. 1983. Deltakephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr: a new highly potent and fully specific agonist for opiate delta-receptors. Biochem Biophys Res Commun. 1983 Mar 16;111(2):390–397.
20. Dudás, B. and I. Merchenthaler et al. 2003. Close Juxtapositions between LHRH Immunoreactive Neurons and Substance P Immunoreactive Axons in the Human Diencephalon .J. Clin. Endo.Met. 88, 1842 (2003).
21. Ning Zhang, Dave Hodge, Thomas J. Rogers, and Joost J. Oppenheim (2003); Ca2+-independent Protein Kinase Cs Mediate Heterologous Desensitization of Leukocyte Chemokine Receptors by Opioid Receptors. J. Biol. Chem., Vol. 278, Issue 15, 12729-12736, April 11, 2003.
22. Owczarek D, Garlicka M, Pierzchala-Koziec K, Skulina D, Szulewski P. 2003. Met-enkephalin plasma concentration and content in liver tissue in patients with primary biliary cirrhosis. Przegl Lek 2003; 60: 461-466.

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