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Home , Casomorphin, Deltorphin I, Exorphin, Gliadorphin, Opioid food peptides, Rubiscolin

Paper No. : 10 Molecular Therapeutics

Module :29 biotherapeutics

Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resources Institute (TERI), New Delhi

Co-Principal Investigator: Prof S K Jain, Professor, of Medical Jamia Hamdard University, New Delhi

Paper Coordinator: Dr. Nirupma Trehanpati, Additional Professor - Department of Molecular and Cellular Medicine, ILBS

Content Writer: Dr. Kalpana Bhargava, Scientist F, DRDO

Content Reviwer: Dr. Nirupma Trehanpati, Additional Professor - Department of Molecular and Cellular Medicine, ILBS

Molecular Therapeutics Biotechnology biotherapeutics

Description of Module

Subject Name Biotechnology

Paper Name Molecular Therapeutics

Module Name/Title Opioid peptide biotherapeutics

Module Id 29

Pre-requisites

Objectives

Keywords

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

Table of Content: Learning Objectives Introduction to Opioid Peptide Types of Opioid and their mechanism of action Summary

Opioid Peptide Bio-therapeutics

Learning Objectives

This module aims to provide in depth knowledge about the Opioid peptides, their receptors and their therapeutic use in medical as well as in research field. This module will help students to understand about the origin of Opioid peptides, different types of opioid peptides and their receptors. The emphasis will be on the natural, semi-synthetic and synthetic form of opioid peptides and their use as bio-therapeutics.

Introduction

Opioids have been reported to act as therapeutic remedies for various ailments, ranging from mild acute nociceptive pain to unbearable chronic advanced or end-stage disease pain. produced by classical are catalyzed when it interacts with opioid receptors located in the brain or the spinal cord.

Opiates and opioids are known to mimic the function of opioid peptides which bind to opioid receptors in the brain. Brain opioid peptide systems have been reported to play an important role in motivation, emotion, attachment behaviour, stress and pain and also in appetite control. The opioid peptides can be natural (for example ), synthetic or semi- synthetic in origin. The effects of these peptides may vary but they are mostly similar to those of . Opioid-like peptides might also be acquired by absorption from partially digested food (, and ). These are usually shorter peptides consisting of only 4-8 amino acid residues in comparison to endogenous opioid peptides which are much longer.

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

Opioid peptides are products of post-translational proteolytic cleavage of precursor proteins which usually consist of: a signal sequence preceding a conserved region of approximate 50 residues; a variable-length region; and the sequence of the neuropeptides themselves. Sequence analysis has revealed that the conserved N- terminal region of the precursors comprises 6 cysteines, which probably play a role in disulfide bond formation. Therefore, this region is supposed to be important for neuropeptide processing.

Research has been widely carried out on naturally occurring and synthetic opiates in order to obtain more useful agents. Opioid peptides are more promising candidates than opiates to be used for drug development because of the following reasons:

1. These compounds are endogenous and, on metabolic degradation (unlike the opiates), break down to amino acids. Hence, the metabolites are nontoxic and do not cause kidney and liver damage (unlike the opiates). 2. Since the peptides consist of several subunits of amino acid residues, a large number of analogues can be synthesized from a few basic building blocks. Simple modifications, such as substitution of unnatural amino acids or sterically constrained amino acids, or other such residues may be attempted to develop analogues with a desired biological activity. 3. Due to flexible conformations, these peptide analogues are ideal molecules for studying structure-activity relationships. These molecules can be cyclized or may be changed to other synthetic modifications, so that analogues of various flexibilities can be synthesized. 4. These molecules are significantly polar thus, can be used for solution studies with different solvents and ions (calcium, sodium, etc.) in order to understand their effects on peptide conformations. 5. Automated solid-phase peptide synthesis methodology for the synthesis of these peptides is available, so that even complex molecules can be synthesized in larger quantities. The main purpose of synthesizing complex opioid therapeutics such as pro- (about 250 residues) and pro- has made it easy to provide substantial material to scientist for investigating the processing mechanisms. 6. To date, the compounds that are highly selective for receptors have been opioid peptides.

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

7. Due to characteristics of some of the peptide drugs, i.e., their inability to permeate through placental barrier (due to placental enzymatic deactivation of the peptides), these drugs are being preferred as analgesics in pregnant females. They are certainly superior to meperidine in this regard.

TYPES OF OPIOID PEPTIDES AND THEIR MECHANISM OF ACTION: The opioid peptides might be classified on the basis of source and site of their production:

1. Endogenous opioids

Endogenous opioid peptides are those which are produced physiologically within the body. These are classified as:

a) Endorphins b) c) d)

The human genome contains several homologous genes coding for endogenous opioid peptides. The three distinct families of opioid peptides: the endorphins, enkephalins, and dynorphins are derived from three distinct genes which form three distinct high-molecular- weight precursor proteins: (POMC) giving rise to endorphins; to enkephalins and to dynorphins. These were the first opioid peptides which was isolated from brain and during their isolation, showed high affinity and selectivity to the μ- and for this reason, were projected as endogenous μ- opioid receptor ligands.

a) Endorphins

“Endorphins” term is indicative of a pharmacological activity and not a specific chemical formulation. This term also refers to “a like substance which is produced by the human body”.

Endorphins are endogenous opioid polypeptides produced by the pituitary gland and the hypothalamus in vertebrates during strenuous exercise, pain, excitement. Endorphins work as “natural pain relievers.” Four types of endorphins are created in the human body named as alpha (α), beta (β), gamma (γ) and sigma (σ) endorphins. The four types have different

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

numbers and types of amino acids in their molecules. There are approximately 16 to 31 amino acids in each molecule. β-endorphins are the most powerful endorphins in the body. Endorphins are released in the pituitary gland at the times of pain or stress and produce an analgesic effect to diminish the pain that is inflicting our body. However, during stress, endorphins act differently. They are released in the limbic system and reduce the extent of anxiety that our body is feeling. Not only does the cause the pain to decrease; it also causes the feelings of euphoria to occur as well as many sex hormones are also released. Exercise increases the endorphin release too and for the same reason, it results in a better mood. Athletes also produce high levels of these peptides. They get a “runner high” when they have done a very hard and strenuous exercise: endorphins allow human to feel a sense of power and control over themselves in order to persist with activity for an extended time. Furthermore, endorphins may have a role in preventing obesity, diabetes and psychiatric disorders.

b. Enkephalins

Enkephalins are pentapeptides and they are known to regulate nociception in the body. Two forms of enkephalin were discovered in 1975, one having leucine (“leu”) and the other containing methionine (“met”). Both the enkephalins are products of the proenkephalin gene. Met –enkephalins have an amino acid sequence viz., Tyr-Gly-Gly-Phe–Met. These molecules are endogenous opioid peptide neurotransmitters which are found intrinsically in the brains of number of animals, including humans. On the contrary, Leu-enkephalin, has a sequence: Tyr- Gly-Gly-Phe-Leu. It acts antagonistically at both the µ- and delta-opioid receptors, with significantly preference for the latter. It also exerts little to nil effect on the k-opioid receptor.

c. Dynorphins

A class of opioid peptides originating from the precursor protein prodynorphin is called Dynorphins. When prodynorphin is cleaved during processing by proprotein convertase 2 (PC2), many active peptides are released: , , and σβ- neo endorphin. Dynorophin A, Dynorphin B consists of a high proportion of basic amino acid residues, in particular lysine and arginine as well as many hydrophobic residues and are produced liberally in brain. They are stored in large dense-core vesicles (80-120 nm diameter) which in comparison to the small synaptic vesicles demand a more intense and prolonged stimulus to discharge their contents into the synaptic cleft. Dense-core vesicle storage is typical feature of opioid peptides. Dynorphins exhibit their effects chiefly through

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

the k-opioid receptor and it act as modulators of pain response. Dynorphins are also known to control homeostasis through appetite control and circadian rhythm, weight control and regulation of body temperature.

d. Endormorphins

Endormorphins are found in the mammalian brain and they are extremely selective endogenous µ-opioid peptides. These consists of two endogenous opioid peptides namely, -1 and endomorphin-2. Endomorphin-1 is located in the nucleus of the solitary tract, the periventricular hypothalamus, and the dorsomedial hypothalamus, where it is localized in histaminergic neurons and regulates sedative and arousal behaviours. However, endomorphin-2 is more widely spread in the spinal cord. endomorphins are considered to be the cleavage products of a larger precursor, which as of now has not yet been recognized. Endormorphins play an important role in reducing pain, responses related to stress and complex functions such as reward, arousal and vigilance, as well as autonomic, cognitive, neuroendocrine and limbic homeostasis.

2.

Different food protein molecules contain certain peptide sequences that exert behaviour like opioid receptor ligands and they belong to the opioid proteins, namely ‘exorphins’.Earlier reports suggest that milk, , vegetable and meat/poultry proteins are very good sources of opioid peptides. These food-derived exogenous opioid peptides are important due to a number of advantages, e.g.: they are safe and natural alternatives due to their production using animal and plant proteins, they cause comparatively lesser side-effects, etc. The various opioid peptides derived from food are listed below:

(from casein found in milk of mammals, including cows)  Gluten (from gluten found in wheat, rye, barley)  /gluteomorphin (from gluten found in wheat, rye, barley)  Soymorphin-5 (from soybean)  (from spinach)

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

Casomorphin peptides

The casomorphines are formed by digestion of the casein protein. The most remarkable feature in these peptides is the abundance of proline. Casomorphines are known to cause addiction in severe cases of protein intolerance and are quite variable in length.  Casomorphin 1-3 Structure: H-Tyr-Pro-Phe-OH (YPF)  Casomorphin 1-4 Structure: H-Tyr-Pro-Phe-Pro-OH (YPFP)  Casomorphin 1-4, amid Structure: H-Tyr-Pro-Phe-Pro-NH2  Casomorphin 5 Structure: H-Tyr-Pro-Phe-Pro-Gly-OH (YPFPG)  Casomorphin 7 Structure: H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH (YPFPGPI)  Casomorphin 8 Structure: H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-OH (YPFPGPIP)

Gluten peptides

Gluten exorphins are peptides isolated from pepsin hydrolysates of wheat gluten. They have morphine-like opioid activity and can act like the body's own , the endorphins. Four gluten exorphins were isolated from the enzymatic digest of wheat gluten and were named gluten exorphins: A5, A4, B5 and B4. Gliadorphin is similarly formed from the gliadin component of gluten.

 Gluten exorphin A4 Structure: H-Gly-Tyr-Tyr-Pro-OH (GYYP)  Gluten exorphin A5 Structure: H-Gly-Tyr-Tyr-Pro-Thr-OH (GYYPT)  Gluten exorphin B4 Structure: H-Tyr-Gly-Gly-Trp-OH (YGGW)  Gluten exorphin B5 Structure: H-Tyr-Gly-Gly-Trp-Leu-OH (YGGWL)  Gluten exorphin C Structure: H-Tyr-Pro-Ile-Ser-Leu-OH (YPISL)  Gliadorphin Structure: H-Tyr-Pro-Gln-Pro-Gln-Pro-Phe-OH (YPQPQPF)

Spinach peptides

Rubiscolins are two naturally occurring, linear peptides: rubiscolin-5 and rubiscolin-6 (or rubixyl) isolated from the pepsin digests of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from spinach leaves. Though their structure does not resemble typical opioid peptides, rubiscolins were found to exhibit high affinity and selectivity for the delta opioid receptor and an antinociceptive effect in mice after oral

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

administration. Moreover, orally administered rubiscolin-6 was shown to possess orexigenic, anxiolytic-like, and memory-enhancing activities in mice.

 Rubiscolin-5 Structure: H-Tyr-Pro-Leu-Asp-Leu-OH (YPLDL)

 Rubiscolin-6 Structure: H-Tyr-Pro-Leu-Asp-Leu-Phe-OH (YPLDLF)

3. Amphibian opioid peptides

I and II

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2] deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, , which is highly selective for µ-opioid receptors. These peptides all have the N- terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both µ- and δ-opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.

 Dermorphin

Dermorphin is a hepta-peptide first isolated from the skin of South American frogs belonging to the genus Phyllomedusa. The peptide is a natural opioid that binds as an with high potency and selectivity to mu Opioid receptors. Dermorphin is about 30–40 times more potent than morphine but theoretically may be less likely to produce drug tolerance and addiction (due to its high potency).

4. Synthetic opioid peptides

Novel analogs of opioid peptides are synthesized for a variety of reasons. One of the major aims has been to find analogs which would have absolute selectivity for the various classes of opioid receptors. When such selectivity is achieved, further modifications might be undertaken to improve the resistance of the peptide to enzymatic degradation in order to increase potency and prolong activity in vivo and in vitro. Such selective, potent and long acting analogs could find many practical applications.

Conformational restriction has been a very valuable tool in design of selective opioid analogs. The ancestral opiate compound, morphine, and many of its analogs synthesized by medicinal chemists have led the way for more recent peptide work. Use of N-methyl amino acids in

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

peptide analogs not only restricts the conformations available to the peptide, but frequently confers enzyme resistance as well.

Large amount of opioids are either synthetic or semi-synthetic (i.e., synthesized from naturally-occurring opiates). These peptides are utilized for varied purposes which can either be beneficial or dangerous to users, if consumed in higher doses they can be fatal too.

Synthetic opioids are synthesized by pharmaceutical companies where reaction is started with a substance occurring in poppy plant to either make a semi-synthetic opioid or, to manufacture the whole drug itself, in a pursuit of creating better, quicker-acting, or longer- acting drugs.

The common synthetic and semi-synthetic opioids are:

 Synthetic opioids

Meperidine Meperidine is used to “relieve moderate to severe pain” (NLM). It is available as brand name Demerol.

Fentanyl is an effective synthetic opioid which is only used by individuals who are already tolerant to narcotics.

Methadone was formulated in the 1930s by “a team of German scientists during their search for an analgesic (pain-killing) drug that would not be as addictive as morphine” (CESAR). Methadone can still be addictive itself when consumed beyond limits.

 Semi-synthetic opioids

Buprenorphine

Buprenorphine is a semi-synthetic opioid isolated from , which is a natural opiate obtained from poppy plant. Although it is considered as an opioid, buprenorphine is only a partial opioid agonist, which means that “its maximal effects are less as compared to the complete like and methadone” (SAMHSA). This also makes it useful in treating individuals who become addicted to opioids.

Hydromorphone is generally known by the brand name Dilaudid, one of its several brand names. Abusers refer to it as dust or footballs. It is derived from morphine.

Oxymorphone

Oxymorphone is known as, ‘octagon’ because of its hexagonal shape.

Hydrocodone

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics

Hydrocodone is one of the most commonly prescribed opioid in the USA under the brand name Vicodin (also containing acetaminophen,” according to the DEA. It is also involved in more abuse and diversion than any other opioid drug, illicit or licit. It is synthesized from which occurs in the poppy plant naturally like thebaine, morphine, and .

Oxycodone Perhaps the most recognizable prescription opioid, is manufactured by modifying thebaine. It is often called oxycotton, oxy, or perc (short for Percocet which contains oxycodone and acetaminophen).

Heroin Heroin was first synthesized from morphine in 1874 by the pharmaceutical company Bayer and was publicized as a safer, non-addictive form of morphine.” However, in the present times, heroin is deemed to be one of the most addictive and dangerous illegal drug in existence.

Summary: Peptides are potential drug molecules having several advantages which include high activity, greater specificity, low toxicity and lessening of drug-drug interactions. Research in the field of opioid peptides is in a very advanced stage. In future, formulation of therapeutically useful peptide analgesics seems a possibility. However, there are few challenges which still exist, such as activity after systemic administration and their ability to cross the blood brain barrier. Stabilization of these peptides to metabolism sufficiently so as to permit their systemic administration could be accomplished through a variety of structural modifications while still retaining their desired pharmacological activities. There are several opioid peptides available in the market- natural, synthetic and semi-synthetic in origin with beneficial therapeutic effects but these should not be abused and used

Molecular Therapeutics Biotechnology Opioid peptide biotherapeutics