Biochimica et Biophysica Acta (BBA) - General Subjects
A novel synthetic luteinizing hormone-releasing hormone (LHRH) analogue coupled with modified β-cyclodextrin: Insight into its intramolecular interactions
Graphical abstract
Introduction
Peptides and proteins are well known weaponry against serious diseases. Thanks to advances in biotechnology, the production of these molecules on a large-scale has been accelerated. However, this huge impetus given in: a) the synthesis and development of novel synthetic peptides and b) the progress in molecular biology, has not found a parallel in the development of delivery systems for this class of molecules [1]. It is well known that peptides suffer from chemical and enzymatic instability, poor absorption through biological membranes, rapid plasma clearance, peculiar dose response curves and immunogenicity [2].
A great effort is made by the scientific community in order to eliminate these drawbacks of peptides and proteins. An interesting approach is to administer peptides and proteins in combination with cyclodextrins (CDs). Actually, this approach is not limited only to the class of peptide molecules but also to other smaller size bioactive molecules which suffer from similar problems [3], [4], [5]. Partial encapsulation of a bioactive molecule inside the CD cavity is the basis of the approach. Thus, it is reported in the literature that CDs can protect peptides and proteins against enzymatic as well as chemical degradation [6].
CDs are a family of cyclic oligosaccharides, and the most common which are commercially available consisting of six (α-CD), seven (β-CD) or eight (γ-CD) α-1,4-linked glucosidic bonds. Their shape is toroidal or cone-shaped, due to the absence of free rotation of the bonds linking the glucose units. Each glucose unit has three hydroxyl groups, two secondary connected at carbons 2 and 3 of the glucose unit and one primary connected at carbon 6. Thus, a total number of twenty one hydroxyl groups are present in β-CD and their presence is mainly responsible for CD water solubility. The primary hydroxyl groups are located in the narrow rim of the cone, while secondary hydroxyl groups are located in the wide rim. On the other hand, the interior of CDs is relatively hydrophobic due to the presence of ether oxygens at the C4 and hydrogens attached at carbons C3 and C5. This allows CDs to entrap hydrophobic molecules [7]. α-CD may typically complex low molecular weight molecules or compounds with aliphatic side chains, β-CD can complex aromatic and heterocyclic molecules, and γ-CD can form inclusion complexes with molecules as large as macrocycles, steroids and C60 [2], [8].
In peptides, encapsulation of the whole molecule inside any type of CD is not possible, since peptides are usually much larger compared to CDs. However, as with other types of drugs, inclusion of a small part of the peptide can take place and this process in certain cases can reduce the above mentioned problems like enzymatic and chemical degradation [2]. A carefully designed attachment of the CD unit could leave parts of the peptide, important to the binding process, free of encapsulation and as a result would permit the drug to retain its bioactivity. Also, the use of modified CDs can greatly alter drug lipophilicity and enhance transport through membranes.
CDs and CDs based systems have tested for the carriage of many anticancer drugs [9]. The gonadotropin-releasing hormone (GnRH) known as luteinizing-hormone-releasing hormone (LHRH) stimulates the secretion of gonadotropins via binding to receptors on the surface of gonadotrophin cells of the anterior pituitary. It is a decapeptide, amide (pGlu–His–Trp–Ser–Tyr–Gly–Leu–Arg–Pro–Gly–NH2) produced in the hypothalamus and adjusts the steroidogenesis and gametogenesis. The gonadotropins (LH, luteinizing hormone and FSH, follicle-stimulating hormone) regulate the production of gametes and steroid sex hormones from the gonads (ovaries and testes) [10], [11].
Over the last years the synthesis of various LHRH peptide analogues (agonists or antagonists) introduced new perspectives in the treatment of hormone dependent cancer and fertility. Leuprolide (pGlu–His–Trp–Ser–Tyr–DLeu–Leu–Arg–Pro–NHEt) is a synthetic altered peptide ligand (APL) of LHRH. Leuprolide has an amino acid skeleton and thus is sensitive to proteolysis. Sensitivity is enhanced further by the fact that Leuprolide has low intestinal absorption and bioavailability (less than 1%). In general, three mainly peptide bonds of the LHRH analogues are more sensitive to hydrolysis, namely Trp3–Ser4, Ser4–Tyr5 and Tyr5–Gly6 [12], [13].
Aiming the reduction of the proteolysis sensitivity, we report here, for the first time, the synthesis and conformational study of a LHRH analogue coupled with 3-monoamino-β-CD. The synthesized LHRH analogue is based on the Leuprolide sequence (DLeu instead of Gly at position 6) and the intramolecular interactions between the peptide and the CD moiety were explored via a high field 2D NMR.
Section snippets
Materials
2-Chlorotrityl chloride polystyrene resin (1% DVB, 200–400 mesh), Fmoc–εAhx–OH, Fmoc–Arg(Pbf)–OH, Fmoc–Gly–OH, Fmoc–Leu–OH, Fmoc–DLeu–OH, Fmoc–Ser(tBu)–OH, Fmoc–Tyr–OH, Fmoc–Trp–OH, Fmoc–His(Trt)–OH, pGlu–OH and Fmoc–Pro–OH were purchased from Chemical and Biopharmaceutical Laboratories of Patras, Patras, Greece. 3-Monoamino-β-CD hydrate was purchased from TCI Europe. All solvents and other reagents were purchased from Merck, Sigma-Aldrich and Fluka companies. DC-Alufolien Kieselgel 60 F254
Molecular design
The synthesized LHRH analogue has DLeu at position 6, pyroglutamic amino acid as amino-terminal and glycine amino acid as carboxy terminal. The carboxyl group of glycine is an attractive candidate for the attachment of the CD unit. Unmodified CD, as a polyhydric alcohol, can react with carboxylic acid groups to produce esters as products. However, this reaction is complicated by the fact that β-CD has 21 hydroxyl groups. Thus, a commercially available, modified CD was used instead, where one of
Conclusions
An LHRH analogue elongated with εAhx serving as a flexible spacer and conjugated with 3-monoamino-β-CD was successfully synthesized for the first time. The conformational properties of this molecule were studied using a combination of 2D NOESY experiments and MD simulations. The results obtained clearly show that aromatic segments of Tyr and Trp can approach with a different affinity the interior core of CD. The encapsulation of the two aromatic segments into the CD cavity might favor the drug
Acknowledgements
The NMR experiments were accomplished at Slovenian NMR centre,National Institute of Chemistry, Ljubljana, Slovenia. Financial support was provided by the Bio-NMR (EU FP7) program (Project number: BIO-NMR-00102) and by the Slovenian Research Agency program grant P1-0010.
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