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Orexin A (hypocretin-1) is a 33-amino-acid neuropeptide derived from prepro-orexin that functions as an endogenous agonist at the G-protein-coupled orexin OX1R and OX2R receptors.
Orexin A, which is also called hypocretin-1, is a neuropeptide consisting of 33 amino acids and is a member of the orexin/hypocretin peptide family. The adult form of the peptide is produced biologically from the precursor protein prepro-orexin, this protein also leading to the formation of the related peptide Orexin B. The peptide Orexin A was first identified in a 1998 study which looked at hypothalamic peptide signalling and previously unrecognised ligands for G protein-coupled receptors.
Orexin A has an N-terminal pyroglutamyl residue, is amidated at the C-terminus and contains two intramolecular disulfide bonds. As a result, it is a peptide with a conformationally restricted structure and a molecular weight of about 3561.1 g/mol; its primary structure is highly conserved in a number of mammalian species.
In laboratory research Orexin A is mainly known as a peptide ligand of the orexin receptor family, which includes OX1R and OX2R. Experimental studies have looked into its molecular structure, its characteristics with respect to receptor binding, the signalling associated with the GPCRs, the conformation of the peptide and the structure–activity relationships. Synthetic Orexin A also serves as a well-defined reference substance for use in receptor assays, peptide-characterisation studies and the development of analytical methods.
Orexin A is made up of 33 amino-acid residues and has a number of structural features which set it apart from the related Orexin B peptide; its N-terminus is composed of pyroglutamic acid instead of an unmodified glutamine or glutamate residue and the leucine at the C-terminus is amidated. Four cysteine residues take part in forming two intramolecular disulfide links, namely between Cys⁶ and Cys¹² and between Cys⁷ and Cys¹⁴.
These disulfide linkages constrain the N-terminal region and contribute substantially to the peptide’s three-dimensional organization. Solution-state NMR investigations have characterized a compact N-terminal region and a helix-turn-helix arrangement in the C-terminal portion. Structural studies describe helical regions extending through portions of residues 16–23 and 25–32, with both hydrophobic and hydrophilic residues distributed across the peptide surface.
Commercial synthetic preparations are available in the form of a lyophilised powder. The solubility and stability will depend on a number of factors such as the composition of the solvent, the pH, the temperature, the oxidation state and the preservation of the native disulphide connectivity.
Most experimental studies have mainly described Orexin A on the basis of its interactions with two G protein-coupled receptors: the orexin receptor type 1 (OX1R) and the orexin receptor type 2 (OX2R). These receptors are part of the class A rhodopsin-like GPCR family and offer well-established experimental systems for the study of peptide–receptor recognition and intracellular signal transduction.
It has been demonstrated that orexin A interacts with both OX1R and OX2R. The structural areas of the peptide implicated in ligand recognition, especially residues within its conserved C-terminal domain, have been investigated in receptor-binding and mutagenesis experiments. Conformational changes in the transmembrane receptor structure after ligand binding have also been investigated in studies of receptor activation.
Present laboratory research shows that orexin receptors are able to couple with several pathways of heterotrimeric G proteins, particularly those associated with Gq/11 being well understood. Experiments have looked at the activation of phospholipase C that is linked to the formation of the intracellular messengers inositol trisphosphate and diacylglycerol. The signalling events have been investigated in connection with intracellular calcium mobilisation and the pathways associated with protein kinase C.
Further investigations have analysed the signalling processes involving phospholipase A, phospholipase D, ERK1/2, p38 MAP kinase, and other downstream signalling proteins. In some experimental systems coupling with Gi/o or Gs proteins has also been studied, although receptor signalling depends on the receptor subtype, the cellular background, and the experimental conditions.
β-arrestin recruitment, receptor desensitization, receptor trafficking, and internalization have also been studied as part of the pharmacology of the orexin receptor. Such studies offer molecular models for examining how the binding of Orexin A leads to intracellular signalling, without having to make any assumptions regarding a specific physiological or therapeutic outcome.
This summary reflects findings reported in published preclinical and in vitro research. The original studies supporting this information are listed in the references.
Research in the fields of peptide chemistry and structural biology has looked at orexin A with a focus on its primary sequence, disulfide bonding, modifications at the termini, and three-dimensional conformation. Investigations based on NMR have been carried out to characterize the compact structure of its N-terminal region and its C-terminal helical arrangement.
Experimental studies have been conducted to examine the interactions of Orexin A with OX1R and OX2R. These studies have involved receptor-binding assays, investigations into ligand–receptor recognition, comparisons of the receptor subtypes and structure–activity studies concerning specific parts of the Orexin A sequence.
Orexin receptors have been studied using cell-based systems which express these receptors in order to investigate the signalling pathways associated with GPCRs. The areas of research have included G protein coupling, phospholipase signalling, intracellular calcium mobilisation, protein kinase pathways, and receptor-dependent signal transduction.
Studies involving the modification of sequences and the use of receptor assays have been carried out in order to examine the relationships between the structure of Orexin A and its interaction with receptors. The researchers have looked at the role of the C-terminal residues, the helical regions, the disulfide bonds and the terminal modifications in relation to molecular recognition.
Orexin A has also been studied by means of receptor modeling, mutagenesis, and structural methods in order to characterize the regions of the orexin receptors to which peptides bind. The various studies have yielded information regarding molecular contacts, the conformational states of the receptor, and the interactions between peptides and GPCRs.
The peptide identity, purity, molecular mass and degradation of Synthetic Orexin A can be examined using chromatographic and mass-spectrometric techniques. Furthermore, the analytical studies might look at oxidation, disulfide integrity, aggregation, and stability under specific laboratory conditions.
The 33-residue amino acid chain of Synthetic Orexin A can be assembled in a step-by-step manner by means of solid-phase peptide synthesis (SPPS). After it has been synthesised and cleaved, oxidative conditions suitable for the purpose can be applied in order to form the two intramolecular disulfide bonds which are characteristic of the mature peptide.
Preparative chromatography may then be used to separate the desired peptide from the truncated sequences, the impurities associated with synthesis and from other peptide types. Analytical high-performance liquid chromatography (HPLC) is usually employed in order to evaluate the chromatographic purity, while liquid chromatography–mass spectrometry (LC-MS) or other mass spectrometric methods can verify molecular identity by determining the molecular mass.
Where necessary, further analytical methods can be employed to investigate peptide content, disulfide bond formation or structural features.
A Certificate of Analysis (COA) specific to each product batch must be provided, this COA being intended to record the compound's identity, the lot number, the date of testing, the analytical method used and the measured purity. In cases where the Broad Payments requirements apply, independent third-party analytical documentation for the current batch must be available together with a reported purity of at least 98%.
Orexin A in lyophilized form must be stored in the laboratory under controlled storage conditions in order to prevent moisture from being taken up, oxidation, and chemical degradation. For the long-term storage of synthetic Orexin A reference material, a temperature of about −20°C is generally specified. The vial has to stay tightly sealed and must be protected from excessive light and humidity.
Repeated temperature cycling should be avoided. When materials have been prepared in solution for the purpose of analytical or experimental work, the preparation conditions should be chosen in accordance with the validated laboratory method and, where possible, freeze/thaw cycles should be avoided.
Orexin A contains two disulfide bonds, therefore handling it in a lab should also prevent situations that can change or weaken them. Maintaining the consistency of the sample throughout several analytical studies can be facilitated by careful storage and appropriate aliquoting.
Orexin A is a peptide composed of 33 amino acids and is a member of the orexin/hypocretin family of neuropeptides; it is formed biologically from the prepro-orexin precursor and is also referred to as hypocretin-1.
Yes, Orexin A and hypocretin-1 are the same mature 33-amino-acid peptide; the two names came about because separate research groups identified the orexin/hypocretin system at the same time.
Orexin A has 33 amino-acid residues and includes two intramolecular disulfide bonds, while mammalian Orexin B is shorter and consists of about 28 residues without having these disulfide bonds. The two peptides are derived from the same prepro-orexin precursor.
Orexin A acts as a ligand for both orexin receptor type 1 (OX1R) and orexin receptor type 2 (OX2R); these receptors are G protein-coupled receptors which are frequently employed in experimental investigations into peptide–receptor binding and intracellular signalling.
The peptide Orexin A has four cysteine residues which take part in forming two disulfide bonds within the molecule, specifically between Cys⁶ and Cys¹² as well as between Cys⁷ and Cys¹⁴. Investigations of its structure show that these bonds have the effect of restricting the N-terminal area and help to produce the peptide's characteristic three-dimensional conformation.
The chromatographic purity of Synthetic Orexin A can be determined by means of analytical HPLC and its molecular identity can be verified using LC-MS or other mass-spectrometric methods. The batch-specific analytical results should be recorded on the relevant Certificate of Analysis.
For research and laboratory use only. Not for human or animal consumption.
Orexin A research peptide is not intended for the prevention, diagnosis, treatment, or cure of any illness or disorder; it is solely available for use in laboratory research. This page's content should not be interpreted as recommendations for dosage, usage, or administration.
